Entanglement between thermoregulation and nociception in the rat: the case of morphine
Abstract
In thermoneutral conditions, rats display cyclic variations of the vasomotion of the tail and paws, the most widely used target organs in current acute or chronic animal models of pain. Systemic morphine elicits their vasoconstriction followed by hyperthermia in a naloxone-reversible and dose-dependent fashion. The dose-response curves were steep with ED50 in the 0.5–1 mg/kg range. Given the pivotal functional role of the rostral ventromedial medulla (RVM) in nociception and the rostral medullary raphe (rMR) in thermoregulation, two largely overlapping brain regions, the RVM/rMR was blocked by muscimol: it suppressed the effects of morphine. “On-” and “off-” neurons recorded in the RVM/rMR are activated and inhibited by thermal nociceptive stimuli, respectively. They are also implicated in regulating the cyclic variations of the vasomotion of the tail and paws seen in thermoneutral conditions. Morphine elicited abrupt inhibition and activation of the firing of on- and off-cells recorded in the RVM/rMR. By using a model that takes into account the power of the radiant heat source, initial skin temperature, core body temperature, and peripheral nerve conduction distance, one can argue that the morphine-induced increase of reaction time is mainly related to the morphine-induced vasoconstriction. This statement was confirmed by analyzing in psychophysical terms the tail-flick response to random variations of noxious radiant heat. Although the increase of a reaction time to radiant heat is generally interpreted in terms of analgesia, the present data question the validity of using such an approach to build a pain index.
NEW & NOTEWORTHY
In rats, morphine-induced hyperthermia results from vasoconstriction of the tail and paws that in turn increases the reaction time to a thermal stimulus; this is generally interpreted in terms of analgesia. In addition, the rostral ventromedial medulla is involved in these processes, possibly through the blockade of on-cells and the activation of off-cells, both involved in pain modulation. These findings stress the potential pitfalls generated by the convoluted interactions between pain and thermoregulation.
in anesthetized rats maintained in thermal neutrality, large cyclic variations of the sympathetic drive of the vasomotion of the tail and paws maintain the core body temperature within a narrow range (El Bitar et al. 2014a). These thermoregulatory changes of skin temperature exert a heavy influence on the behavioral responses to noxious heat commonly used to study nociception in rodents, such as the tail flick (El Bitar et al. 2014a). This constitutes a first, peripheral track regarding the possible interaction between thermoregulation and nociception, well illustrated by the negative correlation between tail skin temperature and tail-flick latency (Benoist et al. 2008; Berge et al. 1988; Hole and Tjølsen 1993; Milne and Gamble, 1989; Ren and Han 1979; Roane et al. 1998; Sawamura et al. 2002; Tjølsen et al. 1988, 1989a, 1989b).
The premotor afferents to the sympathetic preganglionic neurons in the spinal intermediolateral cell column that control the vasomotor tone of the tail are located in the midline of the brain stem in a region designated the rostral medullary raphe (rMR), which includes the raphe pallidus, the raphe magnus, and the laterally extending parapyramidal nucleus (Blessing and Nalivaiko 2001; Cerri et al. 2010; Korsak and Gilbey 2004; Morrison 2001; Ootsuka and McAllen 2005; Ootsuka et al. 2004; Rathner and McAllen 1999; Rathner et al. 2008; Vianna et al. 2008). This region overlaps the rostral ventromedial medulla (RVM), which includes the nucleus raphe magnus and the reticular formation that extends under the gigantocellular reticular nucleus; the RVM is believed to control the spinal transmission of nociceptive messages (Basbaum et al. 2009; Fields and Basbaum 1999; Fields et al. 2006; Heinricher and Ingram 2009). We recently delimited in the rat the location of the premotor sympathetic neurons producing the vasoconstriction of both the tail and the hind paws to the parts of the rMR and RVM adjacent to the facial nucleus (El Bitar et al. 2016). This matches very closely the brain regions often described as specifically devoted to the control of nociception (e.g., Vanegas et al. 1984). Following the proposition by Mason (2001, 2005a, 2005b, 2006, 2011, 2012), we therefore assembled in a single entity (RVM/rMR) the raphe pallidus, raphe magnus, parapyramidal nucleus, and gigantocellular reticular nucleus pars alpha.
Two important categories of neurons designated “on-” and “off-” cells have been recorded in RVM/rMR; they are respectively activated or inhibited by nociceptive stimulation (Fields et al. 1983a, 1988; Vanegas et al. 1984). They are also implicated in regulating the cyclic variations of the vasomotion of the tail and paws seen in thermoneutral conditions (El Bitar et al. 2014b). When, during a cycle, a relative low core body temperature is reached, the on-cells stop firing, and within half a minute, the off-cells are activated, followed within 3–5 min by vasoconstriction of the tail and hind paws that causes a rise in core body temperature. When the increase of core temperature achieves a few tenths of a degree, sympathetic activation switches off and converse variations occur, providing cycles of 3–7 periods/h. In other words, on- and off-cell activities are correlated with inhibition and activation of the sympathetic system, respectively. This is a second (central) point for interaction between thermoregulation and nociception in rodents, centered on the functional role of RVM/rMR neurons.
One must stress the enormous conceptual difficulty in understanding functions (in this case, thermoregulation and nociception) that interact at several levels (in this case, at a peripheral and a central level), knowing that the peripheral and central effects of any manipulation are entangled. To further explore this issue, we thought of using, as a dissecting tool, the cardinal painkiller morphine, which is known to impact on both pain and thermoregulation (see references cited in discussion). In a first series of experiments, we investigated the possibility that the well-documented morphine-induced hyperthermia could result from vasoconstriction of the tail and paws. In a second series, we investigated the possible involvement of the RVM/rMR in morphine-induced hyperthermia by blocking the neuronal activities with muscimol. We then recorded the firing of on- and off-cells in the RVM/rMR together with the vasomotor tone of the tail and paws during morphine-induced hyperthermia. In short, morphine inhibited on-cells and activated off-cells in the RVM/rMR with a resultant vasoconstriction of the tail and paws that produced hyperthermia. We used the experimental data of the present experiments for simulating the tail-flick latency (TFL) with a model that takes into account the power of the radiant heat source, the initial skin temperature, the core body temperature, and the peripheral nerve conduction distance (Benoist et al. 2008). We chose the tail-flick test (d'Amour and Smith 1941) because it is representative of frequently used heat tests for assessing nociception in rodents on the basis of the measurement of a reaction time to heating the extremities of the animal body, e.g., the “paw-withdrawal test” (Hargreaves et al. 1988) or the “hot-plate test” (Woolfe and MacDonald 1944). Finally, we investigated in psychophysical terms the effects of morphine on the behavioral tail-flick reaction while the tail temperature remained constant between trials.
MATERIALS AND METHODS
Ethical Statement
Animal experiments were performed with permission of the Board of the Veterinarian Services of the French Ministry of Agriculture (Permit No. 75-151) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, the European Communities Council Directive 86/609/EEC regulating animal research, and the Ethics Committee of the International Association for the Study of Pain (Covino et al. 1980; Zimmermann 1983). The Ethics Committee in Animal Experimentation Charles Darwin approved the procedures.
Animals
A total of 86 adult male Sprague-Dawley rats weighing 320–370 g (Janvier Labs, Saint-Berthevin, France) were used in the experiments. They were housed in groups of three to four per cage, allowed free access to food and water with a 12-h alternating light-dark cycle, and acclimatized to the laboratory for at least 1 wk before the experiment. The experiments were conducted between 9:00 AM and 5:00 PM.
General Experimental Procedures
Animals were deeply anesthetized with 2.5% halothane in 100% oxygen. A tracheal cannula was inserted, and the ventilation was controlled mechanically with an open-circuit respirator equipped with a scavenging system at a rate of 50 breaths/min. The expiratory halothane level and end-tidal CO2 (ETCO2) were assessed with a capnometer (Capnomac II; Datex Instruments, Helsinki, Finland), sampled every 10 s and under control of alarms throughout the experiment. A catheter was inserted into the common carotid artery to monitor arterial blood pressure and heart rate (HR). A catheter was inserted into the internal jugular vein for intravenous drug injection. After surgery, halothane was optimized to maintain anesthesia without troubling thermoregulation and tail vasomotor fluctuations. The mean level was 0.89% (0.86–0.91%). Oxygen was kept at 100%. Tidal volume was adjusted to keep the ETCO2 at 3.9% (3.8–4.1%). Experimental measures started at least 30 min after surgery. At the end of the experiment, the rat was euthanized with an intraperitoneal thiopental injection.
To maintain the anesthetized rat in a thermoneutral condition, the body was wrapped up with a water-warming pad connected to an extra-capacity warm water circulator (TP 220; Kent Scientific, Torrington, CT) sparing the head, paws, and tail. The heating blanket was covered with an isothermic metalized polyester film to stabilize the temperature around the body. The warming temperature (Twarm) was adjusted to 0–0.3°C above or below the core body temperature after stabilization [38.1°C (37.9–38.4°C)] to achieve a thermoneutral condition or a vasoconstriction of the hind paws and tail, respectively (El Bitar et al. 2014a), depending on the objective of experimental conditions. An air conditioner was used to maintain a stable ambient room temperature [25.4°C (25.0–25.8°C)].
Thermographic Measures of Skin Temperature
Skin temperature is an accurate correlate of the vasomotor tone status (El Bitar et al. 2014a). For this purpose, an infrared camera (Jade MWIR; CEDIP Infrared Systems, Croissy-Beaubourg, France) with a 3- to 5-μm optical bandpass and a 500-μs integration time was used to obtain images of 320 × 240 pixels at a 1-Hz sampling rate, with a sensitivity of 0.02°C at 25°C. The camera was placed 1.5 m above the scene and was controlled by Cirrus software (CEDIP Infrared Systems). The camera was calibrated using a black body as previously described (Benoist et al. 2008). Altair software (CEDIP Infrared Systems) was used to explore the spatial and temporal dynamics of the skin temperature at the level of the tail and paws, as follows.
First, several regions of interest (ROI) were defined in the recorded scene, each comprising 10 pixels. Three ROIs were located on the tail. A proximal ROI (tail-prox) was placed 3 cm from the root of the tail, an intermediate ROI (tail-mid) was placed at the middle of the tail, and a distal ROI (tail-distal) was placed 3 cm from the tip of the tail. Two additional ROIs were located on the plantar aspect of the left and right hind paws of the anesthetized rats (paw-left and paw-right). Finally, a ROI was located over a piece of wood placed in the scene to monitor the ambient temperature (Tamb). For each time point, the mean of the 10 pixels defining each ROI was computed to obtain one single-temperature time course for each ROI, namely, Ttail-prox, Ttail-mid, Ttail-dist, Tpaw-left, Tpaw-right, and Tamb.
Arterial Blood Pressure and Heart Rate
The arterial blood pressure and heart rate were monitored continuously, via a catheter inserted into the common carotid artery, using a transducer connected to a computer. Mean arterial blood pressure (MAP) and heart rate (HR) were calculated and recorded using LabChart data acquisition software (AD instruments, Dunedin, New Zealand). Each variable was sampled every seconds.
Core and Heating Temperature
A two-channel OMEGA HH506RA digital thermometer and two VIP-T-CT25515 probes (0.1°C resolution) were used to measure the core body temperature (Tcore). The probe was inserted 6 cm in the rectum (Lomax 1966). The second probe was placed around the trunk of the rat to estimate the heating provided by the isothermal warming blanket. Each measurement was sampled every 60 s.
Specific Procedures for Experiment 1
The aim of the first series of experiments was to evaluate the outcome of intravenous injection of morphine at different doses on the vasomotor tone of the hind paws and tail of rats hold in thermoneutral conditions (El Bitar et al. 2014a). After surgery, halothane was decreased to ∼0.8% with oxygen being kept at 100%, the tidal volume was adjusted to keep the ETCO2 at ∼3.5%, and there was a wait of at least 30 min before the experimental procedure was started. The rats were maintained in thermoneutral conditions by preserving a stable, homogeneous, and constant surrounding temperature at 0.3°C above Tcore.
Rats were divided into four groups; each group received a single dose following at least 15 min of a stable control period. The first group (n = 12) received intravenous injection of 6 mg/kg morphine chloride; in 5 cases the time evolution of morphine effect was followed till the end of its effect, whereas in 7 cases, intravenous naloxone (0.4 mg/kg) was injected at least 30 min after morphine, to check the specificity of the effects of morphine. In the remaining groups, 0.1 (n = 5), 0.5 (n = 5), or 1 (n = 12) mg/kg morphine was administered and recordings were monitored thereafter during at least 30 min.
The decreasing skin temperature following morphine injection were fitted with a three-parameter exponential decay model of the form T(t) = A·exp(−B·t) + C, with A expressing the vertical expansion of the curve, B representing the inverted time constant (min−1) of cooling, and C being the asymptote toward which the exponential series converges.
Specific Procedures for Experiment 2
The aim of the second series of experiments was to assess the role of the RVM/rMR (El Bitar et al. 2016) regarding the effect of intravenous 6 mg/kg morphine on the vasomotor tone of the hind paws and tail. While deeply anesthetized, 9 rats were mounted on a Horsley-Clarke stereotaxic frame. Xylocaine (2%, 0.5 ml) was injected subcutaneously in the scalp, followed by a 2-cm midline incision. After trepanation, a small incision of the dura mater was made to introduce the tip of a microinjection glass needle. After surgery, halothane was decreased to ∼0.8% with oxygen being kept at 100%, the tidal volume was adjusted to keep the ETCO2 at ∼3.5%, and there was a wait of at least 30 min before the experimental procedure was started. The paws and tail of the rat were maintained in vasoconstriction during the control period by preserving a stable, homogeneous, and constant surrounding temperature at 0.3°C below Tcore.
We prepared muscimol solution in a concentration of 0.01 nmol/nl, added Pontamine sky blue to identify the site of injection, fractioned the solution in 2-μl aliquots for single use, and preserved them at −20°C. Muscimol produces a rapid and persistent hyperpolarization of neurons (Hikosaka and Wurtz 1985; Martin and Ghez 1999) on the basis of its high affinity and selectivity for the GABAA receptor (Beaumont et al. 1978; Enna and Snyder 1975; Gallagher et al. 1983; Krogsgaard-Larsen et al. 1977; Nicholson et al. 1979). The day of the experiment, we filled the circuit of the glass needle with paraffin to wash out air bubbles, aspirated 0.5 μl of muscimol solution at the tip of the needle using a 1-μl Hamilton syringe, and introduced the needle in the brain at the end of the surgery.
After 15 min of control period in steady vasoconstriction, 57 ng (50 nl) of muscimol were injected in the RVM/rMR close to midline (target zone between −1.8 and −3.0 rostral to interaural line) over 60 s. When the skin temperature increase began within 15 min post-muscimol microinjection, the sympathetic drive was considered as sufficiently blocked and the experiment was selected for morphine injection. The location of injection sites was a direct consequence of this choice given the purpose was to have a vasomotor reaction within less than 15 min (El Bitar et al. 2016). At least 15 min after a stable period of vasodilation, 6 mg/kg morphine chloride were then injected intravenously. Intravenous naloxone (0.4 mg/kg) was injected at least 30 min after morphine, to check the specificity of the effects of morphine. Experiment duration was between 75 and 105 min post-muscimol microinjection.
Specific Procedures for Experiment 3
The aim of the third series of experiments was to assess the possible involvement of on- and off-cells recorded in RVM/rMR (El Bitar et al. 2014b) on the effect of intravenous morphine on the vasomotor tone of the hind paws and tail. While deeply anesthetized, 13 rats were mounted on a Horsley-Clarke stereotaxic frame. Xylocaine (2%, 0.5 ml) was injected subcutaneously in the scalp, followed by a midline incision. After trepanation, a small incision of the dura mater was made to introduce a recording electrode. The electrodes were glass micropipettes with filaments filled with a solution of KCl (0.75 M) colored with Pontamine sky blue. The glass micropipettes were made from glass capillary tubes fixed by the chuck of the drill of a Narashige vertical puller (model PE-2). Both the gravitational force of their own weight and the force of the magnet stretched the micropipettes. The diameter of the tip of the electrodes was ∼1 μm. Their impedance was 8–12 MΩ. The pipette was connected to an amplifier and then to LabChart data acquisition software (AD instruments). The tip of the electrode was inserted to the RVM/rMR. After surgery, halothane was decreased to ∼0.8% with oxygen being kept at 100%, the tidal volume was adjusted to keep the ETCO2 at ∼3.5%, and there was a wait of at least 30 min before the experimental procedure was started. The rats were maintained in thermoneutral conditions by preserving a stable, homogeneous, and constant surrounding temperature of 0.3°C above Tcore. After the vasomotor fluctuations were checked, the tip of the glass micropipette was inserted in the RVM.
Recorded extracellularly, cells candidates for analysis were located in the RVM/rMR mainly at −1.7 to −2.5 mm caudal to the interaural line (El Bitar et al. 2014b). On- and off-cells were identified on the basis of the criteria described by Fields et al. (1983a, 1988) during a tail-flick response to radiant heat: on- and off-cells exhibit a sudden increase and an abrupt pause in firing rate, respectively, before the occurrence of the tail flick. The tail-flick tests were performed using an Ugo Basile apparatus (7370) with the stimulus being placed halfway between intermediate and distal ROIs, as defined above. The infrared source is lodged into a metal frame containing a halogen bulb (Halogen “Bellaphot,” model 64607 OSRAM, 8 V, 50 W) coupled to an infrared filter, which cuts off most of the visible part of the spectrum, cooled by an operating fan. The Ugo Basile controller (7371) records the stimulation time from the moment the start key is activated to the moment the animal withdraws the tail with a 0.1-s accuracy (see details at http://ugobasile.com/support/documentation/viewcategory/3.html). It was connected through an amplifier to LabChart data acquisition software (AD Instruments) together with the electrophysiological signal. The operator started the stimulus, and a sensor detected the tail withdrawal, stopped the timer, and switched off the bulb. We used a 10-s cutoff time.
Six on- and seven off-cells were identified. After 5 min of control period, 6 mg/kg morphine chloride were injected intravenously, followed by intravenous naloxone (0.4 mg/kg) at least 30 min after morphine.
Procedures for Experiment 4
We used the experimental paradigm developed to analyze in psychophysical terms the tail-flick responses of rats or mice to random variations of noxious radiant (Benoist et al. 2008; Pincedé et al. 2012). The stimulus was elicited by a CO2 laser to avoid the drawbacks associated with standard methods of thermal stimulation (Plaghki and Mouraux, 2005). Only a brief account of this approach is given below.
Each animal was placed in an openwork Plexiglas device allowing an effective ventilation of the body of the animal. The tail was depilated by means of a cream (Vichy). The animal was then habituated to this environment for 1 h before the experiment. Monitoring of the basal temperature of the tail was operated by an external device, consisting of two low-power infrared lamps (50 W) coupled to a rheostat, to stabilize the temperature at ∼33.5°C. They were switched off during the application of the thermal test stimulus, produced by a CO2 laser (LSD; SIFEC, Ferrière, Belgium). With a 3.4-mm beam diameter and within the 150- to 350-mW power range, the responses were elicited without damaging the skin within less than 2.5 s. The heating process was recorded with the infrared camera described above with a higher time resolution (172 Hz). The beam was oriented at 45° with respect to the vertical, to elicit a contralateral withdrawal movement. Four sites on the left and the right side, 3.5 and 4.5 cm from the tip, were stimulated with the power of the CO2 laser being adjusted in a pseudorandom order. The left and the right side were stimulated alternatively, and a minimum of 4 min passed between stimulation of a given site and the next stimulation at the same site. Such an experimental procedure allows the determination of the two latent descriptors of the behavioral response to heat, namely, the behavioral threshold (Tβ) and the behavioral latency (Lβ). The obvious inconvenience of this approach lives in its duration, superior to half an hour to determine Tβ and Lβ, at least in our hands.
For convenience and to minimize stress reaction, we choose intraperitoneal administration. The duration of the test imposes a stable state throughout the manipulation. Cerletti et al. (1980) provided evidence for an acceptable compromise in this respect. In short, following the control procedure, the same protocol was repeated 15–45 min after injection of saline or morphine. The dose of 4 mg/kg was chosen with respect to its position in the dose-response curves observed in our study (Fig. 3) and the literature regarding its effects on both the TFL and the central temperature.
Histological Identification of Microinjection Sites (Experiments 2 and 3)
At the end of the experiment, the rat was deeply anesthetized with 3% halothane and the brain was perfused through the heart with 0.9% NaCl followed by 10% formaldehyde and then removed. The brain was frozen, cut in serial 100-μm-thick sections, and Nissl-stained with cresyl violet or carmin. Sites of microinjections were determined from microscopic visualization of the serial sections and reported on schemas of frontal sections of the brain (Paxinos and Watson 1982). Coordinates were then expressed relative to the interaural line: x-axis = latero-lateral, y-axis = ventrodorsal, and z-axis = rostrocaudal.
Data Processing and Statistical Analysis
Only recordings lasting more than 60 min were considered. The recordings were analyzed using MATLAB R2006A (The MathWorks) to compute the following.
Descriptive statistics.
Data are represented as means with their 95% confidence interval.
Skin temperatures and vital signs.
Skin temperature results are demonstrated as differences from the mean of the control period with their 95% confidence intervals. To be accurate in analysis, each period representing an injection was calculated with a new timescale, centering the time of injection on a new zero-time. In Fig. 2B, the maximal time gap between data in the left and right panels is 5 min. In Fig. 5, the maximal time gap between data in the left and middle panels is 25 min, and that between the middle and right panels is 15 min. In Fig. 7, the maximal time gap between data in the left and right panels is 2 min.
Dose-response curve (experiment 1).
Morphine achieved the maximal effect on the vasomotor tone within 25 min. Therefore, data recorded in the 26- to 30-min time period are expressed in terms of difference from the mean of the control period and averaged. Mean Tskin, Tcore, MAP, and HR are represented on scatter plots, which best fitted to sigmoid curves (3 parameters): y = A/[1 + exp−(x − x0)/B], where A represents temperature values at minimal doses of morphine (°C), B represents temperature values at maximal doses of morphine (°C), and x0 is the ED50 value that represents the inflection point of the sigmoid curve (mg/kg).
Analysis of cell firing.
The cell activity is expressed in terms of spike counts per second and then synchronized with Tskin, Tcore, MAP, and HR. Results are expressed as percentages of variation to the difference from the mean of the control period. To be accurate in analysis, for each period representative of the injection of a drug (morphine, naloxone, or muscimol), the timescale is centered on the time of injection.
Data representation and statistical analysis.
Scatter plots were drawn using Excel and Canvas. Sigmoid curve fitting and statistical analyses were performed with SigmaPlot software. The comparisons were done using the Mann-Whitney U-test and Kruskal-Wallis one-way analysis of variance on ranks. Results were considered significant at P < 0.05.
For experiment 4, least-squares linear regressions and t-tests were used for statistical purposes. Calculations were performed with the statistical software StatView 5.0 (Abacus). Results were considered significant at P < 0.05. Data are expressed as means ± 95% confidence interval.
RESULTS
Effects of Morphine on Vasomotor Tone of the Hind Paws and Tail
Illustrative examples of the effects of 6 and 0.1 mg/kg morphine on Tskin, Tcore, MAP, and HR are shown in Fig. 1, A and B, respectively. Rats were maintained in thermoneutral conditions (El Bitar et al. 2014a). Morphine chloride was delivered following a stable control period of at least 15 min. Naloxone (0.4 mg/kg iv) was used to reverse the effects of the opioid. Figure 1, Aa and Ba, shows the temporal evolutions of the temperature of the skin measured at the left and right paws (Tpaw-left and Tpaw-right), at three sites of the tail (Ttail-prox, Ttail-mid, and Ttail-dist), and in the environment (Tamb). During the control period, the temperature of the skin exhibited marked and synchronous fluctuations. Tcore (Fig. 1, Ab and Bb) presented corresponding fluctuations, opposite to the skin variations but maintained within small limits. MAP and HR (Fig. 1, Ac and Bc) exhibited similar periodic fluctuations. They preceded skin temperature changes in opposition of phase: vasodilatations and vasoconstrictions were associated with drops and rises of MAP and HR, respectively, as previously described (El Bitar et al. 2014a). Shortly after intravenous injection of 6 mg/kg morphine, the skin temperatures decreased progressively to reach a plateau within 15–30 min (Fig. 1Aa). Note that Ttail-dist joined Tamb within 15 min. Tcore increased concomitantly, reaching a 0.6°C rise at 30 min (Fig. 1Ab). Small fluctuations of MAP and HR were first observed, followed by a minimal progressive decrease and increase, respectively; however, both remained within the range observed during the control period (Fig. 1Ac). Naloxone reversed these phenomena immediately by producing an abrupt increase of the temperatures (maximum = Δ9°C for the hind paws). Shortly after the vasodilatation of the skin, Tcore dropped by 0.6°C. The relative stabilities of MAP and HR were disrupted by steep increases. Minimal perturbations were seen following 0.1 mg/kg morphine in the second individual example (Fig. 1B): mainly, a transitional perturbation of the fluctuation pattern, including a tiny drop in MAP and HR.
Fig. 1.Individual examples of the effects of morphine. Two individual examples are shown from rats maintained in thermoneutral conditions: temporal evolution (abscissa: time in minutes) of the variables during a 30-min control period, a 30-min period following 6 mg/kg morphine injection, and a 15-min period following naloxone injection (A) and during a 30-min control period and a 45-min period following 0.1 mg/kg morphine injection (B). The 5 analyzed regions of interest for the skin temperatures (Tskin) are shown on the thermographic image at right with the corresponding temperature color scale: proximal tail (Ttail-prox; dark blue), middle tail (Ttail-mid; intermediate blue), distal tail (Ttail-dist; light blue), left hind paw (Tpaw-left; green), right hind paw (Tpaw-right; yellow), and ambient temperature measured from a small piece of wood (Tamb; brown). Aa and Ba: spontaneous variations of Tskin at a ∼5–6 cycles/h frequency. Note the cycles of synchronized fluctuations of Tskin during the control period. Ab and Bb: corresponding evolution of the core temperature (Tcore; black) recorded through conventional means. During the control period, the variations of Tcore of ∼0.2°C were in opposition of phase with Tskin variations, but Tcore was overall maintained within small limits (horizontal dotted lines). Ac and Bc: corresponding evolution of mean arterial blood pressure (MAP; red) and heart rate (HR; violet; bpm, beats/min). Note the fluctuations of MAP and HR during the control period, in opposition of phase to Tskin variations in the 30-mmHg and 60-bpm range, respectively. A: 6 mg/kg morphine (see Supplemental Video S1; supplemental material for this article is available online at the Journal of Neurophysiology website). After morphine injection, one sees a decline of Tskin, attaining a nadir at 20 min (a); an increase of Tcore, achieving 0.6°C at 30 min (b), and disruption of the regular fluctuations of MAP and HR (c), which however remain within upper and lower limits of the control period. All effects were abruptly blocked by naloxone. B: effects of 0.1 mg/kg morphine were minimal, if any.
Figure 2A shows the mean temporal evolution of the recorded variables. Shortly after morphine injection, the skin temperatures dropped, decreasing by 5–8°C (Fig. 2, Aa–Ae). Interestingly, the skin temperature decayed exponentially, following Newton's law of cooling (Fig. 2C) with time constants in the 5- to 15-min range. One hour after morphine injection, the temperatures started to go back up, with mean full recovery being achieved 1 h later. During the first 60-min postinjection, the core temperature (Fig. 2Af) increased progressively by 1.8°C. Overall, mean MAP (Fig. 2Ag) and HR (Fig. 2Ah) showed no major mean change after morphine injection. Figure 2B shows a second group of experiments where naloxone was administered at least 30 min after morphine injection. The left panels of Fig. 2B show the control period (15 min) and the 30-min following morphine injection. The right panels illustrate the rebound effect elicited by naloxone: an abrupt increase of the skin temperatures (Fig. 2, Ba–Be), achieving a maximum within ∼7–9 min. Tcore (Fig. 2Bf) rise was totally blocked by naloxone injection, with an abrupt drop of ∼1°C. MAP (Fig. 2Bg) and HR (Fig. 2Bh) exhibited no major changes.
Fig. 2.Mean effects of morphine. Data are represented as differences (Δ) from the mean of the control period with their 95% confidence intervals. Color coding is as shown in Fig. 1. A and B, top to bottom: a, ΔTpaw-right; b, ΔTpaw-left; c, ΔTtail-prox; d, ΔTtail-mid; e, ΔTtail-dist; f, ΔTcore; g, ΔMAP; h, ΔHR. A: time course (n = 5). Morphine (6 mg/kg) elicited a decrease of skin temperatures of both the hind paws and the tail (a–e), associated with an increase of Tcore (f) but overall sparing MAP (g) and HR (h). The effect of morphine on temperatures was mitigated after 1 h and disappeared after 2 h. B: effects of naloxone (n = 7) on skin temperatures (a–e), Tcore (f), MAP (g), and HR (h). Left panels show a 15-min period of control followed by a 30-min postmorphine period. Right panels show a 5-min postmorphine period followed by a 15-min postnaloxone period. All effects of morphine were reversed by naloxone. Note the rebounded skin and core temperatures. MAP and HR were spared. C: decreasing skin temperatures following morphine injection (6 mg/kg). Graph shows data from A regarding mean skin, ambient, and core temperatures observed during 1 h following morphine injections. Temperatures from right and left paws were averaged for clarity of presentation (Tpaws; orange). The skin temperature decay was fitted (R2 > 0.990 in all cases) with a 3-parameter exponential decay model of the form T(t) = A·exp(−B·t) + C (black lines). The skin temperature declines exponentially, suggesting that skin cooling was passive because it obeyed Newton's law of cooling. Model values “A” express the vertical expansion of the curve: Ttail-dist ∼ Ttail-mid > Tpaws > Ttail-prox. Model values “B” represent the inverted time constant (min−1) of cooling. In terms of time constant (min; black points): Ttail-dist < Ttail-mid < Tpaws < Ttail-prox (6.7, 10, 12.5 and 16.7 min, respectively). Model values “C” are the asymptote toward which the exponential series converges: Ttail-dist < Ttail-mid < Tpaws < Ttail-prox. In the case of Ttail-dist, the temperature reached Tamb within <30 min.
The dose-response relationships of the effects of morphine were investigated in the 0.1- to 6-mg/kg range. Overall, skin temperatures changes reached a nadir 25 min after morphine injection. Results between 26 and 30 min were thus averaged and are represented in Fig. 3. Results were best fitted by a sigmoid function, and the inflection points (ED50) were between 0.55 and 0.98 mg/kg for the skin temperatures and 0.58 mg/kg for Tcore. Mean MAP and HR showed no significant difference in evolution after morphine injection for the four groups.
Fig. 3.Dose-responses relationships. Values on the ordinate are expressed in terms of the difference from the mean of the control period, observed during the period 26–30 min postmorphine, averaged and represented with their 95% confidence intervals (see Fig. 1 for color code). The abscissa is a logarithmic scale of morphine chloride doses (in mg/kg). The dose-responses curves followed a sigmoid curve of 3 parameters: y = A/[1 + exp−(x−x0)/B], where A is the minimal value on the ordinate, B is the maximal value on the ordinate, and x0 is the inflection point of the sigmoid curve (ED50 for a given effect of morphine). A: ΔTpaw-right and ΔTpaw-left. B: ΔTtail-prox, ΔTtail-mid, and ΔTtail-dist. C: ΔTcore. D: ΔMAP. E: ΔHR. ED50 = 0.98, 0.77, 0.55, 0.71, 0.57, and 0.58 mg/kg for Tpaw-left, Ttail-prox, Ttail-mid, Ttail-dist, and Tcore, respectively, but there were no statistically significant differences among the 4 groups for MAP (P = 0.72) and HR (P = 0.06). Total number of experiments was 34 (n = 5, 5, 12, and 12 for 0.1, 0.5, 1, and 6 mg/kg morphine, respectively).
Role of RVM/rMR in the Sympathetic Activation Elicited by Morphine
We aimed at assessing the role of the RVM/rMR regarding the effect of morphine on the vasomotor tone by blocking the neuronal activities with muscimol. An illustrative example of the effects of muscimol (500 pmol, 50 nl) microinjection within the RVM/rMR, followed by intravenous injection of 6 mg/kg morphine and then 0.4 mg/kg naloxone, is shown in Fig. 4. Figure 4A shows four thermographic images in a steady state of the four experimental periods. Rats were initially maintained in vasoconstriction by adjusting Twarm slightly below the Tcore (El Bitar et al. 2014a; 2014b). Fifteen minutes after stabilization of Tskin, muscimol was microinjected within the RVM/rMR (Fig. 4B). Figure 4C shows the temporal evolution of Tskin and Tamb. During the control period, Tamb (∼23.5°C) and Tcore (∼37.4°C) were stable while the skin was vasoconstricted. Muscimol induced a vasodilatation of the tail and the hind paws: Tskin increased and stabilized within ∼15 and 19 min for hind paws and tail, respectively. Morphine did not cause any changes in Tskin, except a transitory negligible drop concomitant with a transitory hemodynamic perturbation following injection. Naloxone also produced transitory tiny perturbations. Figure 4D shows the temporal evolution of Tcore: shortly after vasodilation of the tail and hind-paws, Tcore decreased. A 1.5°C loss was observed before morphine injection despite stable Twarm over the experiment. After morphine injection, Tcore continued to decrease (compare with Figs. 1Ab, 2Af, and 2Bf). A 2.5°C loss was achieved within 90 min (end of the experiment). MAP (Fig. 4E) and HR (Fig. 4F) were also affected by muscimol: slight drops were observed before stabilization within 15–20 min (∼15 mmHg and 50 beats/min, respectively). Once morphine was injected, a transitional small drop of MAP and HR was observed. Afterwards, MAP remained relatively stable while HR increased slightly over 30 min, both remaining close to values seen during the postmuscimol period. Naloxone did not change the outcome of morphine. From such an example, one can infer that a microinjection of muscimol centered on the raphe pallidus, as determined following histological examination (Fig. 4B), was able to block the sympathetic activation of the vasomotor tone of the hind paws and the tail induced by systemic morphine. The skin and core temperatures, remaining at highest level and declining, respectively, tended to merge over the 90-min experimental period.
Fig. 4.Example of the effect of morphine following functional blockade of RVM/rMR. Recordings are from a rat that received muscimol microinjection in the RVM/rMR after a 15-min control period. The effects of morphine, injected 37 min later, were followed for 35 min, and then the effects of naloxone were observed for 15 min. A: example images from a thermographic movie, recorded with 320 × 240-pixel resolution, representative of each stationary episode of the experiment. The false rainbow color scale for temperature is shown at far left. From left to right, thermographic images were taken during control and after injections of muscimol, morphine, and naloxone, successively. Note that the temperatures of the tail and hind paws were low during the control period, evidence of vasoconstriction, and warmer following muscimol administration, evidence of vasodilation. B: localization of the site of injection drawn on a frontal section of the brain in plane −2.8 mm caudal to the interaural line, based on the stereotaxic atlas by Paxinos and Watson (1982). The center of the injection site is midline and 0.8 mm below the interaural line. 7, facial nucleus; Gi, gigantocellular reticular nucleus; GiA, gigantocellular reticular nucleus pars alpha; ml, medial lemniscus; RMg, raphe magnus nucleus; RPa, raphe pallidus nucleus; Py, pyramidal tracts; Ppy, parapyramidal nucleus. C: time evolution of the skin temperatures (see Fig. 1 for color code). Note the stability of the control period in vasoconstriction with skin temperatures near Tamb. After muscimol microinjection, the skin temperatures increased, and the stability of the vasodilation state was not perturbed by the injections. D: time evolution of Tcore. Note the early decrease of Tcore, starting shortly after the rise of tail skin temperature. Morphine did not disturb this decline. E: temporal evolution of MAP. F: temporal evolution of HR. Note the slow drop of both MAP and HR after muscimol administration and then a progressive stabilization within 13 min. Transient perturbations were observed after morphine and naloxone injection.
Figure 5 provides a summary of results, expressed in terms of difference from the mean of the control period. Each column of Fig. 5, A–H, corresponds to a period of experiment with time 0 centered on the injection time of muscimol (left), morphine (middle), and naloxone (right), respectively.
Fig. 5.Mean effects of morphine following functional blockade of RVM/rMR. Data are represented as differences from the mean of the control period with their 95% confidence intervals (n = 9). Left panels show 15 min of control period and 30 min of postmuscimol period; middle panels show 5 min of postmuscimol period followed by 30 min of postmorphine period; and right panels show 5 min of postmorphine period and 15 min of postnaloxone period. Color code is as shown in Fig. 1. A: ΔTpaw-right. B: ΔTpaw-left. C: ΔTtail-prox. D: ΔTtail-mid. E: ΔTtail-dist. F: ΔTcore. G: ΔMAP. H: ΔHR. The microinjection of muscimol in the RVM/rMR blocked the somatic sympathetic activation elicited by morphine. I: schema of frontal sections of the brain from interaural −2.0 and −3.0 mm, based on the stereotaxic atlas by Paxinos and Watson (1982). The black circles indicate the center of the corresponding injection sites, with diameter scaled to a 50-nl sphere.
When the skin temperature increase began quickly (see materials and methods), the sympathetic drive was considered sufficiently blocked and the experiment was selected for morphine injection at least 30 min later (Fig. 5, A–E). At least 5 min after a steady temperature was reached, 6 mg/kg morphine was injected, followed by 0.4 mg/kg naloxone 30 min later. No changes were observed for skin temperature following these injections. Tcore decreased regularly following muscimol injection (Fig. 5F). In contrast with the control animals, morphine did not reverse the decline in Tcore, which achieved in most cases more than 2°C at the end of experiments (over 75–105 min). Muscimol induced a slow mean decline of MAP (Fig. 5G) and HR (Fig. 5H), starting within 5 min. The kinetics of vasodilatation and bradycardia were clearly different, which makes the slow diffusion of muscimol to the rostral ventrolateral medulla a very likely explanation of the latter. Neither morphine nor naloxone changed these outcomes. However, the transitional small drop of MAP and HR was observed when morphine was injected, as described above in the absence of RVM/rMR blockade.
Figure 5I shows the corresponding location in the brain of the muscimol microinjection sites. They were located between planes −1.8 and −3.0 mm with reference to the interaural line, close to midline, mainly in raphe pallidus and raphe magnus. Note that the decision to inject morphine was based on the effectiveness of muscimol acting on vasomotor tone within less than 15 min. The location of injection sites was de facto a direct consequence of this choice (see El Bitar et al. 2014b).
Involvement of On- and Off-Cells in the Sympathetic Activation Elicited by Morphine
The rats were maintained in thermoneutral conditions (El Bitar et al. 2014a). On- and off-cells were recorded within the RVM/rMR, 2.0–3.0 mm caudal to the interaural line. They were identified on the basis of the criteria described by Fields et al. (1983a, 1988) during a tail-flick response to radiant heat: on- and off-cells exhibit a sudden increase and an abrupt pause in firing rate, respectively, before the occurrence of the tail flick (see Fig. 1 in El Bitar et al. 2014b). During the control period, on- and off-cell activities oscillated spontaneously. MAP, HR, and Tcore exhibited synchronous fluctuations in opposition of phase to the on-cell firing and in phase to the off-cell firing, whereas skin temperatures were in phase and in opposite phase, respectively (El Bitar et al. 2014b). Morphine chloride (6 mg/kg iv) was delivered at least 15 min after a stable control period was achieved. At least 30 min later, 0.4 mg/kg naloxone was delivered to reverse the effect of morphine. Figure 6Aa shows the temporal evolution of the individual firing of an on-cell. Once morphine was injected, a blockade of the on-cell activity was observed. When injected 30 min later, naloxone reversed this phenomenon, including a rebound effect. All other measured variables, namely, Tskin (Fig. 6Ab), Tcore (Fig. 6Ac), and MAP/HR (Fig. 6Ad), exhibited variations similar to those described above. Figure 6Ba shows the temporal evolution of the individual firing of an off-cell. After morphine injection, a burst activation of the off-cell neuron was observed. Intravenous naloxone reversed totally this phenomenon with a transitory silent period. All other measured variables were similar to those described above (Fig. 6, Bb–Bd).
Fig. 6.Two individual examples of the effects of morphine on RVM neurons and other variables in rats maintained in thermoneutral conditions. In both cases, morphine (6 mg/kg) was injected after a 5-min control period, and naloxone (0.4 mg/kg) was injected 30 min later. A: example on-cell. Immediately after morphine injection, a complete silencing of the cell was obvious. Naloxone reversed this effect with an immediate burst of activation. The effects of morphine and naloxone on the other variables are akin to those described in Figs. 1 and 2. B: example off-cell. Immediately after morphine injection, a bursting activation was seen, before stabilization above control 10 min later. Naloxone injection reversed the effect of morphine. Again, the effects of morphine and naloxone on the other variables a re akin to those described in Figs. 1 and 2.
Figure 7A summarizes the global effect of morphine on the on-cells: immediately after morphine injection, the neuronal activities were blocked until the administration of naloxone, which produced a burst activation that achieved 2.7 (1.3–4.1) times the mean activity observed during the control period. Figure 7B summarizes the global effect of morphine on the off-cells: immediately after morphine injection, off-cells showed a burst of activity [mean rate between = 6.06 (1.46–3.76) times the mean control, as measured at 25–30 min]. Once naloxone was injected, the cell activity achieved a level largely below the control period (nearby silent; P = 0.0003). The following graphs show the corresponding variations of Tpaw-right (Fig. 7C), Tpaw-left (Fig. 7D), Ttail-prox (Fig. 7E), Ttail-mid (Fig. 7F), Ttail-dist (Fig. 7G), Tcore (Fig. 7H), MAP (Fig. 7I), and HR (Fig. 7J), similar to those previously described. Figure 7K shows the corresponding location in the brain of the recording sites of the on- and off-cells.
Fig. 7.Mean effects of morphine on RVM neurons and other variables in rats maintained in thermoneutral conditions. Data (±95% confidence intervals) in A (n = 6) and B (n = 7) are mean percentages of variation with reference to the mean firing during the control period (bin width 1 min), and data in C–J (n = 13) are differences from the mean of the control period. Left panels show 15 min of control period followed by 30 min after morphine injection (6 mg/kg), and right panels show 5 min before and 15 min after naloxone administration (0.4 mg/kg). A: on-cells (n = 6). B: off-cells. C: ΔTpaw-right. D: ΔTpaw-left. E: ΔTtail-prox. F: ΔTtail-mid. G: ΔTtail-dist. H: ΔTcore. I: ΔMAP. J: ΔHR. K: schematic representation of the localization of the recording sites for the on-cells (red circles) and off- cells (blue circles) on a frontal section of the brain (interaural −2.3 mm), based on the stereotaxic atlas by Paxinos and Watson (1982).
Modeling and Simulating the Effect of Morphine on the Tail-Flick Test
We proposed and verified experimentally in the rat a simple model for simulating by computer the response time (e.g., tail-flick latency, TFL) to radiant heat stimuli, taking into account the power of the radiant heat source, the initial skin temperature, the core body temperature, and the peripheral distance of stimulation site (Benoist et al. 2008). The model has been applied successfully to reconstruct the predictable variations of the TFL following 1) a conditioned stress response (Carrive et al. 2011), 2) the spontaneous variations of the skin and core body temperatures seen in thermoneutral conditions (El Bitar et al. 2014b), and 3) following the functional blockade of the RVM/rMR (El Bitar et al. 2016). The model provides the following equation for the expected tail-flick latency elicited: TFL = [(T0 − 0.73·Tskin)2/α + 90/(0.041·Tcore − 0.47) + (D − 90)/(0.041·Tskin − 0.47) + 138]/1,000, where α is the slope of the squared temperature variation generated by the power of the radiant heat source (expressed in °C2/ms) and D is the distance between the stimulation site and the dorsal horn entry zone (in mm). The variations of the TFL are inversely correlated to the variations of the temperature of the skin. In Fig. 8A the model was used to compute the predictable variations of TFL introduced by morphine when the heat stimulus is applied to the distal (D = 250 mm) or proximal (D = 150 mm) part of the tail. The mean numerical data for Ttail-dist and Ttail-prox are from Fig. 2, Ac and Ae, and the mean numerical data for Tcore are from Fig. 2Af: in the control situation, Ttail-dist was lower than Ttail-prox, and then morphine produced a larger decrease of the former (∼8°C) than the latter (∼5°C). In both cases, the effect of morphine on the calculated TFL increases when the power of radiant heat decreases; for example, the TFL from the distal part of the tail increases by ∼2 and 3 s for α = 0.08 and 0.05, respectively. The same numerical data are normalized in terms of percent increase with reference to the mean control period in Fig. 8B. Whether the proximal or distal part of the tail is stimulated, the calculated morphine-induced percent changes of TFL are close for all α values in the 0.05–0.08 range. This is easily explained by the fact that the equation TFL = f(Tskin) provides curves that are almost linear in the presently concerned range of skin variation, namely, 25–35°C. Figure 8Bb confirms that the effects of morphine are lesser when the proximal part of the tail is stimulated.
Fig. 8.Temporal evolution of the predictable tail-flick latency (TFL). We proposed and verified experimentally a simple model for computing TFL in the rat, taking into account the power of the radiant heat source, initial temperature of the skin, core body temperature, and the site of stimulation on the tail (Benoist et al. 2008). This model was used to compute the predictable variations of the TFL introduced by the variations of the skin and core body temperatures after morphine injection (6 mg/kg). The model provides the following equation: TFL (s) = [(36.8 − 0.73·Tskin)2/α + 90/(0.041·Tcore − 0.47) + (D − 90)/(0.041·Tskin − 0.47) + 138]/1,000, where α is the slope of the squared temperature variation (in °C2/ms), indicating the power of the radiant heat source, and D is the distance between the stimulation site and the dorsal horn entry zone (in mm). A: the model was used to compute the predictable variations of TFL introduced by morphine when the heat stimulus is applied to the distal (a; D = 250 mm) or proximal part of the tail (b; D = 150 mm). The mean numerical values for Ttail-dist and Ttail-prox are from Fig. 2, Ac and Ae, and the mean numerical values for Tcore are from Fig. 2Af. A–D: abscissa indicates time following morphine injection (min). Left ordinates are temperature (°C); right ordinates (red) are calculated TFL (A; in s), TFL normalized in terms of percent increase with reference to the mean control period in A (B), TFL normalized in terms of %MPE [= (TFL after treatment − control TFL)/(cutoff time − control TFL) × 100; calculation is for α = 0.05; C], and TFL normalized in terms of %MPE but calculated for a cutoff time of 8 s (D). See text for further details.
It is very common in pharmacological studies that the investigator defines a limit duration of the stimulus (the “cutoff time”) and calculates the effects of a drug in terms of the percentage of maximum possible effect: %MPE = (TFL after treatment − control TFL)/(cutoff time − control TFL) × 100. Such a calculation made for α = 0.05 in Fig. 8C reveals that the efficacy of morphine should be increased by reducing the cutoff time. An identical calculation made for cutoff time = 8 s in Fig. 8D reveals that the model forecasts an increased efficacy of morphine by lowering the power of the radiant heat source. Figure 9 provides the predictable variations of TFL introduced by morphine following functional blockade of the RVM/rMR by muscimol.
Fig. 9.Predictable variations of TFL elicited by morphine following functional blockade of the RVM/rMR by muscimol. The simulation displayed in Fig. 8 was performed to compute the predictable variations of TFL introduced by morphine with the use of numerical values of Tmid-tail and Tcore (left abscissas) from Fig. 2Ad for the control situation and from Fig. 5D following functional blockade of the RVM/rMR by muscimol. Considering a site of stimulation on the mid-tail, far from the dorsal horn entry zone (D = 200 mm), the model provides the following equation: TFL (s) = [(36.8 − 0.73·Ttail)2/α + 90/(0.041·Tcore − 0.47) + (D − 90)/(0.041·Ttail − 0.47) + 138]/1,000, where α is the slope of the squared temperature variation (in °C2/ms), indicating the power of the radiant heat source. The corresponding computed TFL (right abscissas) would vary in the ranges 2–4 s and 3–5 s in the control situation and in the muscimol-treated rats, respectively, for powers of the radiant heat source representative of the most commonly used procedures (α = 0.45–0.7°C2/ms). The temporal evolution of the simulated TFL is shown in the control situation (A) or following functional blockade of the RVM/rMR by muscimol (B). The systemic administration of morphine increases the computed TFL (∼100% at 30 min) in the control animals but is ineffective in the muscimol-treated rats. Whatever the effects of morphine on the nociceptive system, its only effect on thermoregulation is sufficient to explain a doubling of the latency of the TFL in the control situation. Correlatively, such effects disappear completely following functional blockade of the RVM/rMR. In other words, the morphine-induced drop of skin temperature is translated into an increase of the tail withdrawal reaction time and falsely interpreted as a sign of hypoalgesia elicited from the RVM/rMR.
Effects of Morphine in Clamped-Tail Temperature Conditions
The results described above point out that the morphine-induced sympathetic drop of skin temperature predicts the rise of a behavioral reaction time elicited by heat. However, they do not exclude the possibility that morphine contributes to some extent in the response. We thought to use the paradigm we introduced recently to analyze in psychophysical terms the tail flick of rats or mice in response to random variations of noxious radiant heat (Benoist et al. 2008; Pincedé et al. 2012). The aim was to hold tail temperature constant between trials (∼33.5°C) and test whether morphine produced changes of the two latent psychophysical descriptors of the behavioral response, namely, the behavioral threshold (Tβ) and the behavioral latency (Lβ). As shown in Fig. 10, the temperature increases proportionally with the square root of time during the CO2 laser stimulation, according to the law of radiant heat transfer: T = T0 + a·t0.5; the constant term a (a2 = α slope of the straight lines in Fig. 10C) is proportional to the laser power density. According to the paradigm illustrated in Fig. 10, we determined both the Tβ and Lβ of the responses elicited by stimulation of the tail (∼4 cm from the tip). The overall results summarized in Table 1 indicate that saline did not modify significantly the measured variables (despite a 0.5°C increase of Tβ). The effects of morphine (4 mg/kg ip) were as follows. Tβ increased by 1.5°C compared with the baseline control period (but this effect was not significant with regard to the saline group). The most reproducible observation was a statistically highly significant but very small (∼40 ms) increase of Lβ compared with the control period of the experimental group or the saline group. Since the paradigm enforces varying stimulus intensities, it is difficult to reason in terms of the usual TFL. However, by expressing the reaction time tR as a function of the slope α [tR = f(α)], one generates highly significant hyperbolas (Fig. 10E) as previously described (Benoist et al. 2008; Pincedé et al. 2012). After morphine injection, the horizontal asymptote shifted upward by 71 ms in this example. The overall results (Table 1) indicate that saline did not modify tR, whereas morphine increased tR by ∼60 ms, significant compared with both the baseline control period and the saline group. Note that the coefficient of the α−1 term was not significantly modified by morphine (paired t-test: t28 = 1.62, not significant).
Fig. 10.Analysis of the behavioral responses elicited by stimulation of the tail (∼4 cm from the tip) in a single rat. The stimulus (150–350 mW) was applied from time 0 until movement of the tail. A: temporal evolution of the temperature of the skin (38 trials), T = f(t). In each individual case, the reaction time (tR) and the apparent threshold (AT) were determined (blue symbols). B: identical data following adjustment of the origin of the timescale for each individual curve to the actual moment of the reaction, T = f(t − tR). Such a change of origin allows one to visualize the back-timing of events and to note the clear tendency of these curves to cross each other in a privileged zone (open circle), the coordinates of which materialize the behavioral latency Lβ and the behavioral threshold Tβ. C: identical data expressed in terms of the square of the difference from initial temperature (T − T0)2 = ΔT2 = f(t − TR). As expected from the law of radiant heat transfer, T = T0 + α·t0.5, the temperature increases linearly with the square root of time. All these linear relationships were highly significant, and their slopes α could therefore be computed confidently. D: relationship between ΔAT2 and the slope α, ΔAT2 = f(α). Such a plot gives rise to a highly significant linear relationship (dotted lines = 95% confidence intervals). From the slope and intercept on the ordinate, one can infer Lβ = 294 ms and ΔTβ2 = 155.6°C2 and calculate Tβ = √ΔTβ2 + T0 = 46.2°C. After intraperitoneal injection of morphine 4 mg/kg (red symbols), Lβ and Tβ increased by 62 ms and 1.1°C, respectively. E: relationship between tR and slope α, tR = f(α). Such a plot gives rise to a highly significant hyperbolic relationship. The horizontal asymptote of the hyperbola is shifted upward by 71 ms after morphine administration. F: relation tR = f(α−1), which transforms the hyperbolas into linear relationships.
| Morphine vs. Control | ||||
|---|---|---|---|---|
| Saline | Morphine | t19 | P | |
| Control baseline | ||||
| T0, °C | 33.6 (33.4−33.7) | 33.3 (33.0−33.5 | ||
| Tβ, °C | 44.4 (43.7−45.2) | 45.4 (44.9−46.0) | ||
| Lβ, ms | 353 (341−364) | 367 (350−383) | ||
| tR, ms | 450 (403−496) | 412 (390−434) | ||
| Variation following treatment | ||||
| ΔT0, °C | −0,1 | −0.02 | 0.84 | ns |
| ΔTβ, °C | 0.4 (−0.6−+1.5) | 1.6 (0.4−2.7) | 2.67 | <0.05 |
| ΔLβ, ms | −5 (−22−+11) | 33 (14−52)† | 5.77 | <0.001 |
| ΔtR, ms | 1 (−22−+24) | 62 (44−79)* | 7.17 | <0.001 |
DISCUSSION
In the first series of experiments with rats maintained in thermoneutral conditions, we monitored the vasoconstrictive effects of morphine recorded from the tail and hind paws and the concomitant hyperthermia. These effects were naloxone reversible and dose dependent. The dose-response curves were steep with ED50 in the 0.5–1 mg/kg range. The periodic fluctuations of blood pressure and heart rate were abolished by the higher doses of morphine, but their mean values remained within the range observed during the baseline period. In the second series of experiments, we observed the disappearance of these effects of morphine when the RVM/rMR was functionally blocked by the high-affinity and selective GABAA receptor agonist muscimol. In the third series of experiments, we observed that morphine elicited abrupt inhibition and activation of the firing of on- and off-cells recorded in the RVM/rMR, together with the progressive vasoconstriction and hyperthermia described in the first series. In the last series of experiments, we examined the weak increase elicited by morphine of the behavioral threshold and the behavioral latency of the tail-flick reaction while the tail temperature was kept up constant between trials.
Hyperthermia Elicited by Systemic Morphine
It was long ago observed in the rat (Ary and Lomax 1979; Appelbaum and Holtzman 1984; Basilico et al. 1992; Chahovitch and Vichnjitch 1928; Cox et al. 1979; French 1979: Geller et al. 1983; Gunne 1960; Hermann 1942; Jorenby et al. 1988, 1989; Martin and Papp 1979; Martin et al. 1977; McDougal et al. 1983; Numan and Lal 1981; Paolino and Bernard 1968; Quock et al. 1985; Rudy and Yaksh 1977; Sharkawi 1972; Slater and Dickinson, 1982; Sloan et al. 1962; Stewart and Eikelboom 1981; Szikszay et al. 1983; Tanaka et al. 1985; Thornhill and Saunders 1985; Tjølsen and Hole, 1992; Ushijima et al. 1985; Wallenstein et al. 1982; Zelman et al. 1985) and other species (e.g., mice, primate) that low doses of morphine elicit hyperthermia, whereas higher doses elicit hypothermia. The ambient temperature was noticed as a major source of variation in determining the morphine-induced core temperature changes (Adler and Geller 1993; Adler et al. 1988; Clark and Clark 1980; Clark and Lipton 1985). Since then, these observations have been regularly reproduced (e.g., recently: Bhalla et al. 2011; Nikolov 2010; Rawls et al. 2007; Savić Vujović et al. 2013). In brief, although influenced by other factors (e.g., species, strain, gender, age, etc.), the induction of hyperthermia by systemic morphine is 1) favored by low doses, the absence of animal constraint, and high ambient temperatures and 2) blocked by naloxone. There is support for the hypothesis that the hyperthermic response to low doses of morphine is due to an upward setting of the hypothalamic set point; in particular, morphine-treated rats display a delayed escape time from a heat lamp (Adler and Geller 1993; Cox et al. 1976; Spencer et al. 1990). Note, however, that the parsimonious explanation for the morphine-induced hyperthermia is a direct activation of the sympathetic-mediated vasoconstriction (see below). Since one is confronted by a huge amount of literature on the subject, we are only referring to experiments made in the rat, but similar observations have been made in mice and sometimes in cats or monkeys.
We presently showed in the halothane-anesthetized rat that the hyperthermia elicited by systemic morphine is associated with peripheral vasoconstriction. To the best of our knowledge, this is a surprisingly original observation, although some hints can be found in the literature (Cox et al. 1976). We observed in the rat maintained in thermoneutral conditions that the temperature of the tip of the tail could decline by 8°C after injection of 6 mg/kg morphine. In these cases, the temperature of the skin was close to the ambient temperature (∼25°C). Since the vasculature of the tail is not endowed with an active vasodilator system (O'Leary et al. 1985), such effects were exclusively mediated through changes in the sympathetic constrictor tone (Johnson and Gilbey 1994, 1996, 1998; Richardson et al. 1991).
Although not formally demonstrated, one can hypothesize similar mechanisms for the control of the sympathetic drive of the palmar aspect of the paws. Interestingly, whether recorded from any part of the tail or the sole of the paws, the skin temperature decayed exponentially after morphine, suggesting a passive phenomenon obeying Newton's law of cooling (although elicited by an active physiological process). The time constants were short (range 5–15 min), notably so regarding the tip of the tail. The steepness of the dose-response curves fit with the bistable character of the regulation of blood flow in the arterial-venous anastomoses of the tail (Dawson and Keber 1979; Gemmell and Hales 1977; Rand et al. 1965; Thorington 1966; Young and Dawson 1982). The bimodal (dilated/constricted) status of skin vasculature also fit with the reported “quantal” blocking of the tail flick by morphine: for a given dose, most rats either do or do not respond to the stimulus, with few intermediate values of the TFL. The proportion of animals whose tail-flick reaction time reaches the cutoff time increases when the dose of morphine increases (Carstens and Wilson, 1993; D'Amour and Smith 1941; Levine et al. 1980; Yoburn 1985).
Note that the morphine-induced sharp transient fall in blood pressure and heart rate in rats is known to result from parasympathetic vagal activation (Delle et al. 1990; Evans et al. 1952; Fennessy and Rattray, 1971; Kiang et al. 1983; Randich et al. 1991, 1992; Stein 1976; Thornhill et al. 1989; Tung et al. 2015; Willette and Sapru 1982). In our hands, this slight fall was not affected by the RVM/rMR functional blockade.
Since the skin temperature of the tail is determined by local blood flow, within the extremes of ambient and core temperature that correspond to full vasoconstriction and vasodilatation, respectively (Dawson and Keber 1979; El Bitar et al. 2014a; Grant 1963; O'Leary et al. 1985; Rand et al. 1965; Young and Dawson 1982), one can conclude that the morphine-induced vasoconstriction is largely dependent on the ambient temperature. For example, a minimal increase of the ambient temperature can produce a large vasodilatation according to the “on-off” trait of the blood flow in the arterial-venous anastomoses of the tail and paws (see references in El Bitar et al. 2014a); such basal changes would increase “physically” the pharmacological effects of morphine described in this article. By contrast, a decrease of the ambient temperature will produce a large vasoconstriction that will erase such effects.
It follows that the morphine-induced peripheral vasoconstriction provides a parsimonious explanation for the concomitant hyperthermia. Apart from that, Thornhill and Desautels (1984) discarded the activation of brown adipose tissue thermogenesis as a basis for the hyperthermia elicited by systemic morphine, at least in the unrestrained rat. In chloralose/urethane-anesthetized rats, however, fentanyl elicited brown adipose tissue (BAT) thermogenesis (Cao and Morrison 2005). Although halothane anesthesia reduces the activity of BAT (Dicker et al. 1995; Ohlson et al. 2003), we cannot exclude an additional participation of BAT to the morphine-induced hyperthermia reported presently. Incidentally, the mean effects of 100 μg/kg intravenous fentanyl reported by Cao and Morrison (2005) regarding blood pressure and heart rate are very similar to those described in the present study with 6 mg/kg morphine.
The application of thermal radiation to the tail of rats or mice provokes its withdrawal characterized by a brief vigorous movement designed as the tail flick (d'Amour and Smith, 1941). It is the reaction time of this movement that is recorded and often referred to as the tail-flick latency (TFL). The TFL increases following the administration of opioids with very few effects for partial agonist compounds and no effect for mild antalgics (Chau 1989; Dewey and Harris 1975; Taber 1974). Before the discovery of opioid receptors, there was a consensus that this test was adequate for predicting the analgesic efficacy of opiates in man (Archer and Harris 1964; Grumbach 1964).
There are several hints in the literature suggesting possible interactions between morphine-induced hyperthermia and antinociception. For example, the ranked orders of potency of a series of morphine doses and a series of morphine-like compounds including fentanyl derivatives in producing hyperthermia are very similar to the rank of their respective antinociceptive potencies, as measured with the tail-flick test (Bhargava et al. 1991; Savić Vujović et al. 2013).
Hyperthermia and Analgesia Elicited by Intracerebral Morphine
Morphine or the selective μ-agonist [d-Ala2, N-MePhe4, Gly-ol5]-enkephalin (DAMGO) produces a dose-related, naloxone-sensitive rise in core temperature after intracerebroventricular (ICV) injection in unrestrained rats (Adler and Geller 1993; Appelbaum and Holtzman 1985, 1986; Geller et al. 1986; Prakash and Dey 1980; Rawls et al. 2003; Spencer et al, 1988; Watanabe 1971; Wilson and Howard 1996), whereas the selective κ-agonist U50488H elicits hypothermia (Adler and Geller 1993; Spencer et al. 1988). In addition, several lines of evidence suggest that low systemic doses of morphine increase core temperature primarily via brain μ-opioid receptors, whereas high doses elicit a decrease primarily through κ-opioid receptors (Chen et al. 1996). The involvement of δ-opioid receptor in temperature regulation appears to be minor (Adler and Geller 1993; Handler et al. 1992, 1994; but see Salmi et al. 2003; Rawls and Benamar 2011). Interestingly, the hyperthermia elicited by ICV morphine in conscious rabbits is accompanied by obvious ear vasoconstriction (Konecka et al. 1982).
ICV morphine also displays antinociceptive potencies in the rat, as measured with the tail-flick and hot-plate tests (Brady and Holtzman, 1982; DeLander and Hopkins 1986; Ding et al. 1990; Galligan et al. 1984; Hu et al. 1984; Lee et al. 1979; Luger et al. 1995; Miyamoto et al. 1991; Ray and Dey 1980; Sawynok and Reid 1989; Suh and Tseng 1990; Suh et al. 1996; Tseng and Fujimoto, 1984, 1985; Tseng et al. 1979; van Ree 1977; Yeung and Rudy 1980a; Zonta et al. 1981). Interestingly, ICV naloxone reverses the effects of systemic morphine (Yeung and Rudy 1980b).
Morphine or μ-opioid agonists directly injected into the preoptic-anterior hypothalamus (POAH) elicit hyperthermia (Cox et al. 1976; Martin and Morrison 1978; Martin and Papp 1979; Thornhill and Saunders 1985; Trzcinka et al. 1977; Xin et al. 1997; Zhukov et al. 1988), associated with tail and hind paw vasoconstriction (Lin 1982). Note that the iontophoretic application of morphine to POAH neurons resulted in excitation of the majority of cold-responsive cells and inhibition of the majority of warm-responsive cells, whereas most of the thermally nonresponsive cells were unaffected (Lin et al. 1984). Interestingly, microinjection of morphine within POAH elicited both hyperthermia and antinociception as measured with the tail-flick and hot-plate tests (Zhukov et al. 1988).
Morphine injection into the periaqueductal gray (PAG) elicited a dose-dependent, naloxone-reversible increase in body temperature (Widdowson et al. 1983; Zhukov et al. 1988). Injection sites producing hyperthermia were distributed mostly in the caudal ventral PAG, many of them also increasing TFL (Shen et al. 1986), as already described for morphine (Guo and Tang 1990; Iwamoto et al. 1978; Jensen and Yaksh 1986, 1989; Kasman and Rosenfeld 1986; Levy and Proudfit 1979; Lewis and Gebhart 1977; Tortorici and Morgan 2002; Urban and Smith 1994; Yaksh et al. 1976, 1988; Yeung et al. 1977, 1978) and for DAMGO (Fang et al. 1989, Smith et al. 1988). Identical effects were reported with the hot-plate test (Bobeck et al. 2012; Iwamoto et al. 1978; Jensen and Yaksh 1986, 1989; Lane et al. 2005; Morgan et al. 2006, 2014; Yaksh et al. 1976). Interestingly, the effects of systemic morphine on TFL are reduced by lesion of the PAG (Abbott et al. 1982; Deakin and Dostrovsky 1978; Dostrovsky and Deakin 1977) or microinjection of naloxone or muscimol within the PAG (Iwamoto et al. 1978; Romandini and Samanin 1984; Zambotti et al. 1982). These observations impute an important role of the PAG in the morphine-induced tail-flick blockade.
Effects of Morphine on RVM/rMR
The role of RVM/rMR in thermoregulation is well documented, notably regarding the vasomotion of the tail in the rat. Microinjection of glutamate or bicuculline increases the activity of vasomotor sympathetic nerves of the tail, thus decreasing the blood flow, without affecting the mesenteric vascular bed (Blessing and Nalivaiko 2001; Morrison 2001; Rathner and McAllen 1999). The functional blockade of the RVM/rMR by microinjection of glycine, GABA, or muscimol blocks the activity of the sympathetic fibers innervating the tail (Blessing and Nalivaiko 2001; Cerri et al. 2010; Korsak and Gilbey 2004; Ootsuka and McAllen 2005; Ootsuka et al. 2004; Rathner et al. 2008; Vianna et al. 2008). We recently described an extensive increase of the temperature of both the paws and the tail, associated with a reduction of the central temperature following muscimol microinjection within the RVM/rMR (El Bitar et al. 2016). The effective zones were circumscribed to the parts of the RVM/rMR facing the facial nucleus. They matched very exactly the brain regions often described as specifically devoted to the control of nociception.
The role of RVM/rMR in pain processes was suggested on the basis of behavioral observations. For example, bicuculline or glutamate microinjection within RVM/rMR increases TFL (Drower and Hammond 1988; Heinricher and Kaplan 1991; Heinricher and Tortorici 1994; McGowan and Hammond 1993; Nason and Mason 2004). Conversely, electrolytic lesions of the RVM/rMR or microinjection of local anesthetic or muscimol within the RVM/rMR resulted in significant decrease in TFL (Gilbert and Franklin 2001; Heinricher and Kaplan 1991; Nason and Mason 2004; Proudfit, 1980a, 1980b, 1981; Proudfit and Anderson, 1975; Yaksh et al. 1977). Similar results were reported with paw withdrawal and hot-plate tests (Gilbert and Franklin 2001; Martenson et al. 2009).
Such RVM/rMR blockade also decreased the antinociceptive effects of morphine assessed by the tail-flick or hot-plate assays (Abbott and Melzack 1982; Abbott et al. 1982; Azami et al. 1982; Chance et al. 1978; Cannon et al. 1983; Gilbert and Franklin 2002; Guo and Tang 1990; Mitchell et al. 1998; Proudfit 1980a, 1980b, 1981; Proudfit and Anderson 1975; Young et al. 1984).
Microinjection of morphine or DAMGO within the RVM/rMR increases TFL (Azami et al. 1982; Boyer et al. 1998; Conroy et al. 2013; Fang et al. 1989; Guo and Tang 1990; Heinricher et al. 1994; Hurley et al. 2003; Jensen and Yaksh 1986, 1989; Kaplan and Fields 1991; Llewelyn et al. 1983, 1986; Mitchell et al. 1998; Paul and Phillips 1986; Prado and Roberts 1984; Rosenfeld and Stocco 1980; Tseng et al. 1990). Interestingly, the effects of systemic morphine on TFL are reduced by microinjection of naloxone (Azami et al. 1982; Iwamoto et al. 1978) or muscimol within the RVM/rMR (Phillips et al. 2012).
Unfortunately, there are few studies regarding the role of RVM/rMR in opioid-induced hyperthermia. Cao and Morrison (2005) reported that microinjection of glycine in RVM/rMR selectively reversed the intracerebroventricular fentanyl-evoked increase of BAT thermogenesis. We presently have shown that muscimol microinjected within the RVM/rMR did completely block both the morphine-induced peripheral vasoconstriction and the accompanying hyperthermia. This result does not suggest that the RVM/rMR was the main site of morphine action but does demonstrate its critical involvement in the final sympathoexcitatory effect. The dynamics of such effects were identical on all regions of interest. This suggests a global effect of morphine on all sympathoexcitatory RVM/rMR cells whether or not they are organized into functional subgroups controlling the tail and the paws vascular beds as suggested (El Bitar et al. 2014a).
Effects of Morphine on On- and Off-Cells Recorded from the RVM/rMR
The on- and off-cells were identified on the basis of the criteria described by Fields et al. (1983a, 1988) during a nociceptive response to radiant heat applied to the tail: on- and off-cells exhibit a sudden increase and an abrupt pause in firing rate, respectively, before the occurrence of the tail-flick. Interestingly, these are nonserotonergic cells, numerous in the raphe nuclei (Auerbach et al. 1985; Gao and Mason 2000; Gao et al. 1998; Mason 1997).
After others, we confirmed the effects elicited by morphine on the firing of on- and off-cells: complete blockade of the activity of on-cells and strong activation of off-cells, with naloxone restoring the initial firing in both cases, as initially described (Barbaro et al. 1986; Fields et al. 1983b, 1988) and reviewed (Basbaum et al. 2009; Fields and Basbaum 1999; Fields et al. 2006; Heinricher and Ingram 2009). Although the effects of morphine differ in nonanesthetized animals (Martin et al. 1992; Hellman and Mason 2012), these observations were interpreted as the most compelling evidence in support of the hypothesis that “the integrative output of these opioid effects on on- and off-cells would, of course, be enhanced inhibitory controls arising from the medulla, i.e., greater pain control” (Basbaum et al. 2009).
Nason and Mason (2006) investigated the possibility of interaction between thermoregulation and μ-opioids at the level of such neurons. Although these cells did not respond to thermoregulatory challenges that evoked an increase in BAT temperature, a noxious stimulation suppressed the BAT activation. DAMGO microinjection blocked noxious stimulation-evoked suppression of BAT temperature increases.
However, as illustrated in the present study, morphine also induced 1) vasoconstriction of the tail and the hind paws, 2) an increase in core temperature, and 3) damping of fluctuations of arterial blood pressure and heart rate. All effects were reversed by naloxone and thwarted by a functional blockade of the RVM/rMR. We must stress that RVM/rMR neurons project not only toward the dorsal horn (Aicher et al. 2012; Basbaum and Fields 1979; Fields and Basbaum 1999) but also to the intermediolateral cell column of the spinal cord (Allen and Cechetto 1994; Bacon et al. 1990; Basbaum et al. 1978; Hossaini et al. 2012; Lefler et al. 2008; Loewy 1981; Morrison and Gebber 1985). Most importantly, the sympathetic premotor neurons in the RVM/rMR project to the intermediolateral column through the dorsolateral funiculus (Bacon et al. 1990; Loewy 1981; Morrison and Gebber 1985; Nalivaiko and Blessing 2002; Rathner et al. 2001; Smith et al. 1998; Stornetta et al. 2005; Strack et al. 1989). It follows that any bilateral section of dorsolateral funiculi will block the sympathetic control of vasomotion, therefore eliciting vasodilatation of the tail and paws. It is not surprising therefore that such bilateral lesion reduced the effect on TFL of morphine injected either systemically (Abbott et al. 1996; Barton et al. 1977; Ryan et al. 1985; Rydenhag and Andersson 1981) or into the RVM/rMR (Llewelyn et al. 1986).
The earliness, suddenness, and fullness of the effects of morphine on these cells were striking. Indeed, both the blockade of the activity of on-cells and activation of off-cells were seen in their full extent within the minute following the intravenous injection of morphine. Vasoconstriction of the tail and paws then began rapidly, and the hyperthermia started within less than 5 min. A full vasoconstriction was achieved within 15 min for the tip of the tail and within 15–30 min for the other parts of the tail and the paws. The peak of hyperthermia was reached within 1 h. Such effects are akin with the kinetic of morphine concentration within the brain and its effects on the TFL following intravenous injections (Bolander et al. 1983; Plomp et a1. 1981; South et al. 2009). They support the suggestion that on- and off-cell activities are linked to sympathetic inhibition and activation, respectively (El Bitar et al. 2014b).
In a thermoneutral state where Tcore evolves cyclically within a narrow range of a few tenths of degrees, the recordings were characterized by slow regular periodic changes of the variables at a recurrence of ∼3–7 cycles/h, organized in a chronological order (El Bitar et al. 2014b). Such organization allowed cross-correlation analyses to be performed between each couple of variables and then absolute maximal correlations and the corresponding time lags to be calculated. It was found that overall the changes in firing of on-cells preceded the opposite changes of off-cells by about 30 s. The variations were particularly abrupt in the case of sympathetic blockade, initiated by on-cell activation when the core body temperature reached a few tenths of degrees above the mean Tcore. Events then occurred in the following order: 1) drops of off-cell firing, MAP, and HR; 2) vasodilatation of the tail and paws within 2–3 min; and 3) decrease of Tcore within 5 min. The opposite phenomenon occurred in the case of sympathetic activation, initiated by the blockade of on-cell firing when the core body temperature reached a few tenths of degrees below the mean Tcore. The following order of events then occurred: 1) rise of off-cell firing, MAP, and HR; 2) vasoconstriction of the tail and paws within 2–3 min; and 3) increase of Tcore within 5 min. Interestingly, the former variations were steeper than the latter.
When the ambient temperature separates this zone of dynamic stability, the balance breaks in favor of hypo- or hyperthermia. We reported that slight warming of the room produces concomitant activation of on-cells and inhibition of off-cells, followed by peripheral vasodilatation and decrease of Tcore, whereas slight cooling activates off-cells and inhibits on-cells together with peripheral vasoconstriction and increase of Tcore. This suggests that on- and off-cells are warm and cold responsive, respectively (El Bitar et al. 2014b). That morphine mimics an environmental cooling supports the hypothesis of an upward setting of the hypothalamic set point (Adler and Geller 1993; Cox et al. 1976; Spencer et al. 1990).
Both the morphine-induced peripheral vasoconstriction and hyperthermia were reversed by naloxone, as was the morphine-induced blockade of the activity of on-cells and activation of off-cells. In fact, rebound activity was seen during a short postnaloxone period, except for the central temperature. Once again, our observations match some reports of “acute tolerance” to morphine analgesia assessed with the tail-flick test. When naloxone was given after a systemic single injection of morphine, TFL was reduced to values significantly below premorphine baseline, whereas on-cell activity increased to levels greater and off-cell activity decreased to near zero (Bederson et al. 1990; Kaplan and Fields 1991). We thoroughly reproduced these neurophysiological findings together with the subsequent vasodilatations. The briefness of these effects prevented any repercussion on the central temperature.
A Series of Caveats and Reservations Regarding Nociceptive Tests
Peripheral vasomotor tone.
One cannot deny that morphine is analgesic. One cannot deny either that morphine blocks the noxious heat evoked behavioral responses assessed in the rat with the tail-flick, hot-plate, or paw-withdrawal tests. But a question emerges: does the former morphine effect specifically result from the latter one? Indeed, morphine elicits a sufficient powerful vasoconstriction to explain, at least largely, the increase of the reaction time to heat (Han and Ren 1991; Hole and Tjølsen 1993; Hole et al. 1990; Le Bars et al. 2001, 2009; Tjølsen and Hole 1997). Irwin and co-workers (1951) observed that the slope of the dose-response curves for the antinociceptive effect of morphine assessed by the tail-flick test was less steep in chronically spinalized rats than in intact rats and interpreted these data as follows: “one must suspect from our data that morphine augments supraspinal inhibitory mechanisms concerned with the tail reflex in addition to affecting the reflex arc directly” (see also Advokat and Gulati 1991; Bonnycastle et al. 1953; Proudfit and Levy 1978; Randich et al. 1992; Sinclair et al. 1988; Wood et al. 1981). However, the vasomotor tone of the tail was neglected in these and many other studies, whereas Tjølsen and Hole (1997) attributed the entire reduction in tail-flick reaction time after section of the spinal cord (Berge, 1982; Grossmann et al. 1973; Jensen et al. 1984; Woolf et al. 1980) to the increase in skin temperature.
We have already stressed that the potential variations of the skin temperature are restricted to a range limited by the ambient and the core temperature, corresponding to vasoconstriction and vasodilatation, respectively. In the former case, any further vasoconstriction would be of limited magnitude and unlikely to elicit significant increases in TFL; in the latter case, any further vasoconstriction would be of large magnitude, increasing the (initially short) TFL. When two factors of variation are involved in the experiment, this caveat in pain research is often eclipsed by the technical artifice of adjusting the radiant heat emission through a rheostat in such a way that it elicits a predetermined value of the TFL in the control period of both the control and the experimental group. In reality, this very standard practice consists of physically applying a correction (adjustment of the intensity of the radiant heat source) to any possible biological variations of the TFL during the control period (Le Bars et al. 2001). Note incidentally that the frequent calculation of the percentage of maximum possible effect (%MPE) in pharmacological investigations introduces a further arbitrary ratio, which is dependent on the choice of the time limit by the investigator (see Fig. 8C): simply reducing the time limit can “improve” the analgesic index of a substance as already quoted (Carmody 1995; Miller 1948).
An anecdotal observation, apparently puzzling, strongly supports our approach. As predicted by the calculation of the TFL shown in Fig. 8 based on the morphine-induced temperature variations, the effect of morphine is less pronounced when the proximal part of the tail is stimulated (Martínez-Gómez et al. 1994; Prentice et al. 1996; Yoburn et al. 1984). The source of these pharmacological observations is probably to be found in the larger number of arterial-venous anastomoses in the distal part of the tail (Wu et al. 1995).
Another general prediction from the model is the decreased effect of morphine on the reaction time elicited with stronger heat stimuli (although it is used clinically for severe pain). This actually has been repeatedly observed with the conventional tail-flick, paw-withdrawal, or hot-plate tests following systemic (Abram et al. 1997; Ankier 1974; Bonnycastle 1962; Granat and Saelens 1973; O'Callaghan and Holzman 1975; Plone et al. 1997; Yeomans et al. 1996; Zimet et al. 1986) or ICV administration (Qi et al. 1990; Suh et al. 1992).
Environmental temperature.
Laboratory ambient temperatures are most often controlled by air conditioning (often ∼22°C) and are well below thermoneutrality for small laboratory animals, favoring hypothermia. However, this is not always the case, and in addition, animals are very often placed in individual cylindrical plastics containers, which have an orifice to allow the tail to stick out. This confinement could result in increasing core and tail temperatures (Keim and Sigg 1976; Martin and Morrison 1978; Martin et al. 1977; Vidal et al. 1984). These effects are counteracted by morphine in a dose-dependent fashion (Tjølsen and Hole 1992). On the other hand, however, the physical restraint could represent a strong stressor, potentially a source of vasoconstriction associated with increased TFL and core temperature (Barnum et al. 2007; Bhattacharya et al. 1978; Busnardo et al. 2010, 2013; Gamaro et al. 1998; Jørgensen et al. 1984; McGivern et al. 2009; Pilcher and Browne 1983; Sanches et al. 2003; Scopinho et al. 2013; Terlouw et al. 1996; Wright and Katovich 1996), which are potentiated by morphine (Appelbaum and Holtzman 1984, 1985; Calcagnetti et al. 1990; Kelly and Franklin 1984; Woolfolk and Holtzman 1995). Note that some investigators have used wire mesh or perforated tubes as restrainers to favor the dissipation of body heat (e.g., Pilcher and Browne 1983; Terlouw et al. 1996; Wright and Katovich 1996). Regarding the test itself, some investigators most concerned with minimizing stress prefer to manipulate the animal gently with a cloth to orient its tail toward the source of heat and/or manipulate the animal daily before undertaking the actual test; this shortens the tail-flick reaction time (Milne and Gamble 1989, 1990).
In fact, stress-induced hyperthermia not only results from decreased heat dissipation through vasoconstriction in the tail and paws (Blessing 2003; Garcia et al. 2001; Vianna and Carrive, 2005) but also from increased nonshivering thermogenesis (Marks et al. 2009; Nagasaka et al. 1979, 1980; Ootsuka et al. 2008; Shibata and Nagasaka 1982). Overall, these effects are highly dependent on the ambient temperature (Aydin et al. 2011; Benoist et al. 2008). Interestingly, administered within the RVM/rMR, muscimol reduced the tachycardia and abolished the tail vasoconstriction elicited by a conditioned fear (Vianna et al. 2008).
Others have studied the tail-flick response in the anesthetized rat and used an automated homeothermic blanket system driven by the actual core temperature measured in the colon, to be stabilized at a predetermined value. Note that the introduction in the experimental setup of an external process of regulation can engender conflicts between the physiological regulations of the core temperature and the artificial feedback loops elicited by the forced regulation (unpublished observations). It is obviously not easy to compare results obtained under such different experimental conditions, which all impact to some extent on 1) basal control temperatures of both the tail and the paws and 2) the effects of opioids, notably morphine, thereon.
Conclusion
The increases or decreases of a reaction time to heat applied on the skin are generally interpreted in terms of hypo- or hyperalgesia, respectively. However, the conventional tests do not achieve the criterion of construct validity because they do not effectively measure the targeted construct, i.e., a quantitative nociceptive response, presumed to reflect the animal's perception of pain. Indeed, in both rats and mice the reaction time measured in the tests in which conventional progressive heating was used is the sum of 1) the time inevitable to reach the threshold temperature of the withdrawal and 2) the behavioral latency proper. In fact, it is predominantly a physical process that accounts for the length of time that builds up the reaction time (Benoist et al. 2008; Pincedé et al. 2012). This preponderance increases either by an intentional lessening of the power of the heat source or by a decline of the basal temperature of the skin, e.g., in this study following morphine administration. For a given power of the source of heat, there is no way of knowing whether any pharmacologically induced variation of the reaction time was produced by changes of the behavioral threshold of the reaction or the basal temperature of the skin, or both. The question of the validity of using a reaction time of a behavioral response to an increasing heat stimulus as a pain index is directly challenged here, not only for the tail but also for the paws. Interestingly, our results also provide explanations for several paradoxical effects of morphine on the tail-flick latency, such as the “quantal” effect, the increased efficacy with dimmed power of the radiant heat source, and the decreased efficacy when the proximal part of the tail is stimulated.
There is no doubt therefore that the vasomotor variations blur the analgesic properties of morphine, but it is conceivable that morphine could affect to some extent the behavioral responses if the tail temperature remains artificially fixed between trials. We checked this possibility in psychophysical terms by considering the behavioral threshold and the behavioral latency (Benoist et al. 2008; Pincedé et al. 2012). The behavioral threshold increased slightly, but the most significant observation was a small but very reproducible increase of the behavioral latency, and consequently of the reaction time. However, the order of magnitude of such increases was less than the temporal resolution of the measurements of the reaction time in the conventional tail-flick test (generally 0.1 s). How do we not conclude that the effects of morphine in such tests have hardly any relationship with its analgesic properties?
The implicit confidence in these tests is mainly based on the strong belief that they are reliable because they provide the expected pharmacological results. In other words, the tests meet the criterion of face validity, i.e., the extent to which the measure looks like what it is supposed to assess. However, face validity is the weakest evidence to demonstrate construct validity because it is essentially an apparent validity. Knowing the ubiquity of the μ-opioid receptor involved in many physiological functions, it follows that an expected effect of morphine on a supposed nociceptive test can result from an (ignored or neglected) effect on another function such as thermoregulation. Note that pharmacological criteria such as dose dependency, stereospecificity, or structure-activity relationship are fully verified because the observation is based on a real pharmacological effect, strongly supporting the belief. All μ-opioid receptor agonists do increase the tail-flick, paw-withdrawal, or hot-plate latencies in rodents. The effect is real, indeed; the artifact concerns the interpretation. In brief, the confidence in these tests as being pain related is essentially based on a hidden “post hoc, ergo propter hoc” sophism.
GRANTS
N. El Bitar was supported by a grant from the
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
N.E.B., B.P., and D.L.B. conception and design of research; N.E.B., B.P., E.G.K., and I.P. performed experiments; N.E.B., B.P., E.G.K., I.P., and D.L.B. analyzed data; N.E.B., B.P., I.P., and D.L.B. interpreted results of experiments; N.E.B. and D.L.B. drafted manuscript; N.E.B. and D.L.B. edited and revised manuscript; N.E.B., E.G.K., I.P., and D.L.B. approved final version of manuscript; D.L.B. prepared figures.
Glossary
| A | Minimal values in ordinate of a dose-responses sigmoid curve |
| a | Coefficient of the heating ramp elicited by the CO2 laser (= α0.5; °C/s) |
| α | Slope of the squared temperature variation elicited by a radiant heat source (= a2;°C2/s) |
| AT | Apparent threshold of the behavioral response to the laser stimulus (°C) |
| B | Maximal values in ordinate of a dose-responses sigmoid curve |
| D | Distance between the stimulation site and the dorsal horn entry zone (mm) |
| ΔAT | Increase of temperature elicited by the CO2 laser stimulus to trigger the reaction (= AT − T0; °C) |
| ΔHR | Variation of heart rate (beats/min) |
| ΔMAP | Variation of mean arterial blood pressure (mmHg) |
| ΔT | Temperature variation with reference to the initial temperature (= Tskin − T0; °C) |
| ΔTcore | Variation of core body temperature (°C) |
| ΔTskin | Variation of skin temperature (°C) |
| ΔTpaw-right | Variation of skin temperature of the right hind paw (°C) |
| ΔTpaw-left | Variation of skin temperature of the left hind paw (°C) |
| ΔTtail-dist | Variation of skin temperature of the distal part of the tail (°C) |
| ΔTtail-mid | Variation of skin temperature of the middle part of the tail (°C) |
| ΔTtail-prox | Variation of skin temperature of the proximal part of the tail (°C) |
| ETCO2 | End-tidal CO2 |
| HR | Mean heart rate (beats/min) |
| Lβ | Behavioral latency to the laser stimulus (ms) |
| MAP | Mean arterial blood pressure (mmHg) |
| MPE | Maximum possible effect |
| paw-left | Mid-plantar area on the left hind paw |
| paw-right | Mid-plantar area on the right hind paw |
| ROI | Region of interest |
| Tamb | Ambient temperature (°C) |
| Tβ | Behavioral threshold of the response to the laser stimulus (°C) |
| Tcore | Core body temperature (°C) |
| Tpaws | Mean temperature of the left and right hind paws (°C) |
| Tpaw-left | Temperature of the left hind paw (°C) |
| Tpaw-right | Temperature of the right hind paw (°C) |
| tR | Reaction time to the laser stimulus (ms) |
| T0 | Temperature of the skin recorded before the laser stimulation (°C) |
| Tskin | Skin temperature (°C) |
| Ttail-dist | Temperature of the distal part of the tail (°C) |
| Ttail-mid | Temperature of the mid part of the tail (°C) |
| Ttail-prox | Temperature of the proximal part of the tail (°C) |
| Twarm | Warming temperature (°C) |
| tail-dist | Distal area of the tail, located at 3 cm from the tip |
| tail-mid | Intermediate area of the tail, located at mid-tail |
| tail-prox | Proximal area of the tail, located at 3 cm from the root of the tail |
| TFL | Tail-flick latency (= tR × 103 s in the case of CO2 laser stimulation) |
| x0 | Abscissa of the inflection point of a dose-response sigmoid curve |
ACKNOWLEDGMENTS
We thank Professors François Cesselin and Léon Plaghki for advice in the preparation of the manuscript and Jean-Michel Benoist for participating in the behavioral experiments.
REFERENCES
- . The effect of lesions of the dorsolateral funiculus on formalin pain and morphine analgesia: a dose-response analysis. Pain 65: 17–23, 1996.
Crossref | PubMed | ISI | Google Scholar - . Brainstem lesions dissociate neural mechanisms of morphine analgesia in different kinds of pain. Brain Res 251: 149–155, 1982.
Crossref | PubMed | ISI | Google Scholar - . Morphine analgesia in tail-flick and formalin pain tests is mediated by different neural systems. Exp Neurol 75: 644–651, 1982.
Crossref | PubMed | ISI | Google Scholar - . Assessment of the potency and intrinsic activity of systemic versus intrathecal opioids in rats. Anesthesiology 87: 127–134, 1997.
Crossref | PubMed | ISI | Google Scholar - . Physiological functions of opioids: temperature regulation. In: Handbook of Experimental Pharmacology. Opioids II, edited by Herz A, Akil H, and Simon EJ. Berlin: Springer, 1993, vol. 104, p. 205–238.
Google Scholar - . The opioid system and temperature regulation. Annu Rev Pharmacol Toxicol 28: 429–449, 1988.
Crossref | PubMed | ISI | Google Scholar - . Spinal transection reduces both spinal antinociception and CNS concentration of systemically administered morphine in rats. Brain Res 555: 251–825, 1991.
Crossref | PubMed | ISI | Google Scholar - . Descending projections from the rostral ventromedial medulla (RVM) to trigeminal and spinal dorsal horns are morphologically and neurochemically distinct. J Chem Neuroanat 43: 103–111, 2012.
Crossref | PubMed | ISI | Google Scholar - . Serotoninergic and nonserotoninergic neurons in the medullary raphe system have axon collateral projections to autonomic and somatic cell groups in the medulla and spinal cord. J Comp Neurol 350: 357–366, 1994.
Crossref | PubMed | ISI | Google Scholar - . New hot plate tests to quantify antinociceptive and narcotic antagonist activities. Eur J Pharmacol 27:1–4, 1974.
Crossref | PubMed | ISI | Google Scholar - . Characterization of stress-induced potentiation of opioid effects in the rat. J Pharmacol Exp Ther 231: 555–565, 1984.
PubMed | ISI | Google Scholar - . Stress-induced changes in the analgesic and thermic effects of morphine administered centrally. Brain Res 358: 303–308, 1985.
Crossref | PubMed | ISI | Google Scholar - . Stress-induced changes in the analgesic and thermic effects of opioid peptides in the rat. Brain Res 377: 330–336, 1986.
Crossref | PubMed | ISI | Google Scholar - . [Narcotic antagonists.] Fortschr Arzneimittelforsch 8: 261–320, 1965.
PubMed | Google Scholar - . Influence of narcotic agents on temperature regulation. In: Advances in Biochemical Psychopharmacology. Neurochemical Mechamisms of Opiates and Endorphins, edited by Loh DH, Ross, HH. New York: Raven, 1979, vol. 20, p. 429–451.
Google Scholar - . Response of serotonin-containing neurons in nucleus raphe magnus to morphine, noxious stimuli, and periaqueductal gray stimulation in freely moving cats. Exp Neurol 88: 609–628, 1985.
Crossref | PubMed | ISI | Google Scholar - . Effect of physical restraint on the limits of thermoregulation in telemetered rats. Exp Physiol 96: 1218–1227, 2011.
Crossref | PubMed | ISI | Google Scholar - . The contribution of nucleus reticularis paragigantocellularis and nucleus raphe magnus to the analgesia produced by systemically administered morphine, investigated with the microinjection technique. Pain 12: 229–246, 1982.
Crossref | PubMed | ISI | Google Scholar - . Electron microscopic evidence of a monosynaptic pathway between cells in the caudal raphe nuclei and sympathetic preganglionic neurons in the rat spinal cord. Exp Brain Res 79: 589–602, 1990.
Crossref | PubMed | ISI | Google Scholar - . Putative pain modulating neurons in the rostral ventral medulla: reflex-related activity predicts effects of morphine. Brain Res 366: 203–210, 1986.
Crossref | PubMed | ISI | Google Scholar - . Adaptation in the corticosterone and hyperthermic responses to stress following repeated stressor exposure. J Neuroendocrinol 19: 632–642, 2007.
Crossref | PubMed | ISI | Google Scholar - . Dissociation of supraspinal and spinal actions of morphine: a quantitative evaluation. Brain Res 188: 487–498, 1980.
Crossref | PubMed | ISI | Google Scholar - . The endogenous neuromodulation system. In: Neuromodulation, edited by Krames E, Peckham PH, and Rezai A. London: Academic, 2009, p. 303–312.
Crossref | Google Scholar - . Three bulbospinal pathways from the rostral medulla of the cat: an autoradiographic study of pain modulating systems. J Comp Neurol 178: 209–224, 1978.
Crossref | PubMed | ISI | Google Scholar - . The origin of descending pathways in the dorsolateral funiculus of the spinal cord of the cat and rat: further studies on the anatomy of pain modulation. J Comp Neurol 187: 513–531, 1979.
Crossref | PubMed | ISI | Google Scholar - . Influence of pertussis toxin on thermic responses to morphine and neurotensin in rats. Eur J Pharmacol 222: 241–245, 1992.
Crossref | PubMed | ISI | Google Scholar - . Muscimol binding in rat brain: association with synaptic GABA receptors. Brain Res 148: 153–162, 1978.
Crossref | PubMed | ISI | Google Scholar - . Hyperalgesia during naloxone-precipitated withdrawal from morphine is associated with increased on-cell activity in the rostral ventromedial medulla. Somatosens Mot Res 7: 185–203, 1990.
Crossref | PubMed | ISI | Google Scholar - . Peripheral and central determinants of a nociceptive reaction: an approach to psychophysics in the rat. PLoS One 3: e3125, 2008.
Crossref | PubMed | ISI | Google Scholar - . Effects of 5-HT receptor agonists and antagonists on a reflex response to radiant heat in normal and spinally transected rats. Pain 13: 253–266, 1982.
Crossref | PubMed | ISI | Google Scholar - . Response latencies in the tail-flick test depend on tail skin temperature. Neurosci Lett 86: 284–288, 1988.
Crossref | PubMed | ISI | Google Scholar - . Study of adrenergic, imidazoline, and endothelin receptors in clonidine-, morphine-, and oxycodone-induced changes in rat body temperature. Pharmacology 87: 169–179, 2011.
Crossref | PubMed | ISI | Google Scholar - . Analgesic and hyperthermic effects of intravenously administered morphine in the rat are related to its serum levels. J Pharmacol Exp Ther 258: 511–516, 1991.
PubMed | ISI | Google Scholar - . Immobilisation stress-induced antinociception in rats: possible role of serotonin and prostaglandins. Eur J Pharmacol 50: 83–85, 1978.
Crossref | PubMed | ISI | Google Scholar - . Lower brainstem pathways regulating sympathetically mediated changes in cutaneous blood flow. Cell Mol Neurobiol 23: 527–538, 2003.
Crossref | PubMed | ISI | Google Scholar - . Raphe magnus/pallidus neurons regulate tail but not mesenteric arterial blood flow in rats. Neuroscience 105: 923–929, 2001.
Crossref | PubMed | ISI | Google Scholar - . Differential development of antinociceptive tolerance to morphine and fentanyl is not linked to efficacy in the ventrolateral periaqueductal gray of the rat. J Pain 13: 799–807, 2012.
Crossref | PubMed | ISI | Google Scholar - . Morphine concentrations in serum, brain and cerebrospinal fluid in the rat after intravenous administration of a single dose. J Pharm Pharmacol 35: 656–659, 1983.
Crossref | PubMed | ISI | Google Scholar - . The use of animals in the study of analgetic drugs. In: The Assessment of Pain in Man and Animals, edited by Keele CA, Smith R. Edinburgh: Livingston, 1962, p. 231–243.
Google Scholar - . The action of some analgesic drugs in intact and chronic spinal rats. Acta Pharmacol Toxicol (Copenh) 9: 332–336, 1953.
Crossref | PubMed | Google Scholar - . Microinjection of morphine into the rostral ventromedial medulla produces greater antinociception in male compared to female rats. Brain Res 796: 315–318, 1998.
Crossref | PubMed | ISI | Google Scholar - . Analgesic effects of intraventricular morphine and enkephalins in nondependent and morphine-dependent rats. J Pharmacol Exp Ther 222: 190–197, 1982.
PubMed | ISI | Google Scholar - . Paraventricular nucleus of the hypothalamus glutamate neurotransmission modulates autonomic, neuroendocrine and behavioral responses to acute restraint stress in rats. Eur Neuropsychopharmacol 23: 1611-1122, 2013.
Crossref | PubMed | ISI | Google Scholar - . Paraventricular nucleus modulates autonomic and neuroendocrine responses to acute restraint stress in rats. Auton Neurosci 158: 51–57, 2010.
Crossref | PubMed | ISI | Google Scholar - . Pharmacological profile of the potentiation of opioid analgesia by restraint stress. Pharmacol Biochem Behav 37: 193–199, 1990.
Crossref | PubMed | ISI | Google Scholar - . Evidence for the independence of brainstem mechanisms mediating analgesia induced by morphine and two forms of stress. Brain Res 269: 231–236, 1983.
Crossref | PubMed | ISI | Google Scholar - . Brown adipose tissue thermogenesis contributes to fentanyl-evoked hyperthermia. Am J Physiol Regul Integr Comp Physiol 288: R723–R732, 2005.
Link | ISI | Google Scholar - . Avoiding fallacies in nociceptive measurements. Pain 63: 136, 1995.
Crossref | PubMed | ISI | Google Scholar - . A reassessment of stress-induced “analgesia” in the rat using an unbiased method. Pain 152: 676–686, 2011.
Crossref | PubMed | ISI | Google Scholar - . Rat tail flick reflex: magnitude measurement of stimulus-response function, suppression by morphine and habituation. J Neurophysiol 70: 630–639, 1993.
Link | ISI | Google Scholar - . Morphine concentrations in the rat after intraperitoneal or subcutaneous injection. Subst Alcohol Actions Misuse 1: 65–70, 1980.
PubMed | Google Scholar - . Cutaneous vasodilation elicited by disinhibition of the caudal portion of the rostral ventromedial medulla of the free-behaving rat. Neuroscience 165: 984–995, 2010.
Crossref | PubMed | ISI | Google Scholar - . Action du chlorhydrate de morphine, de la caféine et de la quinine-uréthane sur le métabolisme énergétique. J Physiol Pathol Gen 26: 389–391, 1928.
Google Scholar - . Effects of medial raphe and raphe magnus lesions on the analgesic activity of morphine and methadone. Psychopharmacology (Berl) 56: 133–137, 1978.
Crossref | PubMed | ISI | Google Scholar - . Analgesic testing in animal models. In: Pharmacological Methods in the Control of Inflammation, edited by Chang JY, Lewis AJ. New York: Alan Liss, 1989, p. 195–212.
Google Scholar - . Antisense confirmation of mu- and kappa-opioid receptor mediation of morphine's effects on body temperature in rats. Drug Alcohol Depend 43: 119–124, 1996.
Crossref | PubMed | ISI | Google Scholar - . Changes in body temperature after administration of acetylcholine, histamine, morphine, prostaglandins and related agents. Neurosci Biobehav Rev 4: 175–240, 1980.
Crossref | PubMed | ISI | Google Scholar - . Changes in body temperature after administration of acetylcholine, histamine, morphine, prostaglandins and related agents: II. Neurosci Biobehav Rev 9: 479–552, 1985.
Crossref | PubMed | ISI | Google Scholar - . CC12, a P450/epoxygenase inhibitor, acts in the rat rostral, ventromedial medulla to attenuate morphine antinociception. Brain Res 1499:1–11, 2013.
Crossref | PubMed | ISI | Google Scholar - . Ethical standards for investigations of experimental pain in animals. The Committee for Research and Ethical Issues of the International Association for the Study of Pain. Pain 9: 141–143, 1980.
Crossref | PubMed | ISI | Google Scholar - . Morphine hyperthermia in the rat: an action on the central thermostats. Eur J Pharmacol 36: 33–39, 1976.
Crossref | PubMed | ISI | Google Scholar - . Effects of morphine and related drugs on core temperature of two strains of rat. Eur J Pharmacol 54: 27–36, 1979.
Crossref | PubMed | ISI | Google Scholar - . A method for determining loss of pain sensations. J Pharmacol Exp Ther 72: 74–79, 1941.
Google Scholar - . Physiology of heat loss from an extremity: the tail of the rat. Clin Exp Pharmacol Physiol 6: 69–80, 1979.
Crossref | PubMed | ISI | Google Scholar - . Involvement of the periaqueductal grey matter and spinal 5-hydroxytryptaminergic pathways in morphine analgesia: effects of lesions and 5-hydroxytryptamine depletion. Br J Pharmacol 63: 159–165, 1978.
Crossref | PubMed | ISI | Google Scholar - . Spinal adenosine modulates descending antinociceptive pathways stimulated by morphine. J Pharmacol Exp Ther 239: 88–93, 1986.
PubMed | ISI | Google Scholar - . Differentiated responses of renal and adrenal sympathetic nerve activity to intravenous morphine administration in anesthetized rats. J Pharmacol Exp Ther 253: 655–660, 1990.
PubMed | ISI | Google Scholar - . The tail-flick test. In: Methods in Narcotic Research, edited by Ehrenpreis SNeidle A. New York: Marcel Dekker, 1975, p. 101–109.
Google Scholar - . Halothane selectively inhibits nonshivering thermogenesis. Possible implication s for thermoregulation during anesthesia of infants. Anesthesiology 82: 491–501, 1995.
Crossref | PubMed | ISI | Google Scholar - . Pentobarbital selectively blocks supraspinal morphine analgesia. Evidence for GABAA receptor involvement. Pain 43: 371–376, 1990.
Crossref | PubMed | ISI | Google Scholar - . Periaqueductal grey lesions reduce morphine analgesia in the rat. Neurosci Lett 4: 99–103, 1977.
Crossref | PubMed | ISI | Google Scholar - . GABAergic modulation of nociceptive threshold: effects of THIP and bicuculline microinjected in the ventral medulla of the rat. Brain Res 450: 316–324, 1988.
Crossref | PubMed | ISI | Google Scholar - . Thermoregulatory vasomotor tone of the rat tail and paws in thermoneutral conditions and its impact on a behavioral model of acute pain. J Neurophysiol 112: 2185–2198, 2014a.
Link | ISI | Google Scholar - . “On-” and “off-” cells in the rostral ventromedial medulla of rats held in thermo-neutral conditions: are they involved in thermoregulation? J Neurophysiol 112: 2199–2217, 2014b.
Link | ISI | Google Scholar - . The rostral ventromedial medulla control of cutaneous vasomotion of paws and tail in the rat: implication for pain studies. J Neurophysiol 115: 773–789, 2016.
Link | ISI | Google Scholar - . Properties of gamma-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fractions. Brain Res 100: 81–97, 1975.
Crossref | PubMed | ISI | Google Scholar - . The fall of blood pressure caused by intravenous morphine in the rat and the cat. Br J Pharmacol Chemother 7: 542–552, 1952.
Crossref | PubMed | ISI | Google Scholar - . Opioid peptides (DAGO-enkephalin, dynorphin A(1–13), BAM 22P) microinjected into the rat brainstem: comparison of their antinociceptive effect and their effect on neuronal firing in the rostral ventromedial medulla. Brain Res 501: 116–128, 1989.
Crossref | PubMed | ISI | Google Scholar - . Cardiovascular effects of intravenous morphine in the anaesthetized rat. Eur J Pharmacol 14: 1–8, 1971.
Crossref | PubMed | ISI | Google Scholar - . Brain stem neuronal circuitry underlying the antinociceptive action of opiates. Prog Brain Res 77: 245–257, 1988.
Crossref | PubMed | ISI | Google Scholar - . Central nervous system mechanisms of pain modulation. In: Textbook of Pain, edited by Wall PD, Melzack R. London: Churchill Livingstone, 1999, p. 309–329.
Google Scholar - . Central nervous system mechanisms of pain modulation. In: Wall and Melzack's Textbook of Pain, edited by McMahon SB, Koltzenburg M. London: Churchill Livingstone, 2006, p. 125–142.
Crossref | Google Scholar - . The activity of neurons in the rostral medulla of the rat during withdrawal from noxious heat. J Neurosci 3: 2545–2552, 1983a.
Crossref | PubMed | ISI | Google Scholar - . Evidence that disinhibition of brain stem neurones contributes to morphine analgesia. Nature 306: 684–686, 1983b.
Crossref | PubMed | ISI | Google Scholar - . Dexamethasone blocks morphine-induced hypothermia in restrained rats. Life Sci 25: 1583–1589, 1979.
Crossref | PubMed | ISI | Google Scholar - . Effects of glial uptake and desensitization on the activity of gamma-aminobutyric acid (GABA) and its analogs at the cat dorsal root ganglion. J Pharmacol Exp Ther 226: 876–884, 1983.
PubMed | ISI | Google Scholar - . Cerebral delta opioid receptors mediate analgesia but not the intestinal motility effects of intracerebroventricularly administered opioids. J Pharmacol Exp Ther 229: 641–648, 1984.
PubMed | ISI | Google Scholar - . The effects of acute and repeated restraint stress on the nociceptive response in rats. Physiol Behav 63: 693–697, 1998.
Crossref | PubMed | ISI | Google Scholar - . Activation of serotonergic neurons in the raphe magnus is not necessary for morphine analgesia. J Neurosci 18: 1860–1868, 1998.
Crossref | PubMed | ISI | Google Scholar - . Serotonergic Raphe magnus cells that respond to noxious tail heat are not ON or OFF cells. J Neurophysiol 84: 1719–1725, 2000.
Link | ISI | Google Scholar - . Tail artery blood flow measured by chronically implanted Doppler ultrasonic probes in unrestrained conscious rats. J Neurosci Methods 104: 209–213, 2001.
Crossref | PubMed | ISI | Google Scholar - . Subclasses of opioids based on body temperature change in rats: acute subcutaneous administration. J Pharmacol Exp Ther 225: 391–398, 1983.
PubMed | ISI | Google Scholar - . Body temperature effects of opioids in rats: intracerebroventricular administration. Pharmacol Biochem Behav 24: 1761–1765, 1986.
Crossref | PubMed | ISI | Google Scholar - . Cutaneous arteriovenous anastomoses present in the tail but absent from the ear of the rat. J Anat 124: 355–358, 1977.
PubMed | ISI | Google Scholar - . GABAergic modulation of descending inhibitory systems from the rostral ventromedial medulla (RVM). Dose-response analysis of nociception and neurological deficits. Pain 90: 25–36, 2001.
Crossref | PubMed | ISI | Google Scholar - . The role of descending fibers from the rostral ventromedial medulla in opioid analgesia in rats. Eur J Pharmacol 449: 75–84, 2002.
Crossref | PubMed | ISI | Google Scholar - . Effect of stimulus intensity on the potency of some analgetic agents. Arch Int Pharmacodyn Ther 205: 52–60, 1973.
PubMed | Google Scholar - . Vasodilation and body warming in the rat. J Physiol 167: 311–317, 1963.
Crossref | PubMed | ISI | Google Scholar - . The dependence of the anti-nociceptive effect of morphine and other analgesic agents on spinal motor activity after central monoamine depletion. Eur J Pharmacol 24: 67–77, 1973.
Crossref | PubMed | ISI | Google Scholar - . The prediction of analgesic activity in man by animal testing. In: Pain, 15th International Symposium, Detroit, edited by Knighton RS and Dumke PR. Boston: Little Brown, 1964, p 163–182.
Google Scholar - . The temperature response in rats during acute and chronic morphine administration, a study of morphine tolerance. Arch Int Pharmacodyn Ther 129: 416–428, 1960.
PubMed | Google Scholar - . Roles of periaqueductal gray and nucleus raphe magnus on analgesia induced by lappaconitine, N-deacetyllappaconitine and morphine. Zhongguo Yao Li Xue Bao 11: 107–12, 1990.
PubMed | ISI | Google Scholar - . The importance of monitoring tail-skin temperature in measuring tail-flick latency. Pain 46: 117, 1991.
Crossref | PubMed | ISI | Google Scholar - . Effect of mu-, kappa-, and delta-selective opioid agonists on thermoregulation in the rat. Pharmacol Biochem Behav 43: 1209–1216, 1992.
Crossref | PubMed | ISI | Google Scholar - . Effect of ambient temperature on the ability of mu-, kappa- and delta-selective opioid agonists to modulate thermoregulatory mechanisms in the rat. J Pharmacol Exp Ther 268: 847–855, 1994.
PubMed | ISI | Google Scholar - . A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32: 77–88, 1988.
Crossref | PubMed | ISI | Google Scholar - . The brainstem and nociceptive modulation. In: The Science of Pain, edited by Bushnell C, Basbaum AI. Oxford: Elsevier Academic, 2009, p. 593–626.
Google Scholar - . GABA-mediated inhibition in rostral ventromedial medulla: role in nociceptive modulation in the lightly anesthetized rat. Pain 47: 105–113, 1991.
Crossref | PubMed | ISI | Google Scholar - . Disinhibition of off-cells and antinociception produced by an opioid action within the rostral ventromedial medulla. Neuroscience 63: 279–288, 1994.
Crossref | PubMed | ISI | Google Scholar - . Interference with GABA transmission in the rostral ventromedial medulla: disinhibition of off-cells as a central mechanism in nociceptive modulation. Neuroscience 63: 533–546, 1994.
Crossref | PubMed | ISI | Google Scholar - . Opioids disrupt pro-nociceptive modulation mediated by raphe magnus. J Neurosci 32: 13668–13678, 2012.
Crossref | PubMed | ISI | Google Scholar - . The pyretic action on rats of small doses of morphine. J Pharmacol Exp Ther 76: 309–315, 1942.
Google Scholar - . Modification of saccadic eye movements by GABA-related substances. I. Effect of muscimol and bicuculline in monkey superior colliculus. J Neurophysiol 53: 266–291, 1985.
Link | ISI | Google Scholar - . The tail-flick test needs to be improved. Pain 43: 391–393, 1990.
Crossref | PubMed | ISI | Google Scholar - . The tail-flick and formalin tests in rodents: changes in skin temperature as a confounding factor. Pain 53: 247–254, 1993.
Crossref | PubMed | ISI | Google Scholar - . Distribution of glycine/GABA neurons in the ventromedial medulla with descending spinal projections and evidence for an ascending glycine/GABA projection. PLoS One 7: e35293, 2012.
Crossref | PubMed | ISI | Google Scholar - . Dissociation of supraspinal and spinal morphine analgesia by reserpine. Eur J Pharmacol 97: 129–131, 1984.
Crossref | PubMed | ISI | Google Scholar - . Spinal pharmacology of antinociception produced by microinjection of mu or delta opioid receptor agonists in the ventromedial medulla of the rat. Neuroscience 118: 789–796, 2003.
Crossref | PubMed | ISI | Google Scholar - . The effects of morphine methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation. J Pharmacol Exp Ther 101: 132–143, 1951.
PubMed | ISI | Google Scholar - . Antinociceptive responses after microinjection of morphine or lanthanum in discrete rat brain sites. J Pharmacol Exp Ther 206: 46–55, 1978.
PubMed | ISI | Google Scholar - . The role of spinal pathways in dopamine mediated alteration in the tail-flick reflex in rats. Neuropharmacology 23: 149–153, 1984.
Crossref | PubMed | ISI | Google Scholar - . Comparison of antinociceptive action of morphine in the periaqueductal gray, medial and paramedial medulla in rat. Brain Res 363: 99–113, 1986.
Crossref | PubMed | ISI | Google Scholar - . Comparison of the antinociceptive effect of morphine and glutamate at coincidental sites in the periaqueductal gray and medial medulla in rats. Brain Res 476:1–9, 1989.
Crossref | PubMed | ISI | Google Scholar - . Sympathetic activity recorded from the rat caudal ventral artery in vivo. J Physiol 476: 437–442, 1994.
Crossref | PubMed | ISI | Google Scholar - . On the dominant rhythm in the discharges of single postganglionic sympathetic neurones innervating the rat tail artery. J Physiol 497: 241–259, 1996.
Crossref | PubMed | ISI | Google Scholar - . Focally recorded single sympathetic postganglionic neuronal activity supplying rat lateral tail vein. J Physiol 508: 575–585, 1998.
Crossref | PubMed | ISI | Google Scholar - . Characterization of morphine's excitatory effects. Behav Neurosci 102: 975–985, 1988.
Crossref | PubMed | ISI | Google Scholar - . Effects of dose on effector mechanisms in morphine-induced hyperthermia and poikilothermia. Psychopharmacology (Berl) 98: 269–274, 1989.
Crossref | PubMed | ISI | Google Scholar - . Immobilization-induced analgesia: possible involvement of a non-opioid circulating substance. Pharmacol Biochem Behav 20: 289–292, 1984.
Crossref | PubMed | ISI | Google Scholar - . Hyperalgesia during acute opioid abstinence: evidence for a nociceptive facilitating function of the rostral ventromedial medulla. J Neurosci 11: 1433–1439, 1991.
Crossref | PubMed | ISI | Google Scholar - . Opiate microinjections into midbrain do not affect the aversiveness of caudal trigeminal stimulation but produce somatotopically organized peripheral hypoalgesia. Brain Res 383: 271–278, 1986.
Crossref | PubMed | ISI | Google Scholar - . Physiological and biochemical concomitants of restraint stress in rats. Pharmacol Biochem Behav 4: 289–297, 1976.
Crossref | PubMed | ISI | Google Scholar - . Evidence that stress augments morphine analgesia by increasing brain tryptophan. Neurosci Lett 44: 305–310, 1984.
Crossref | PubMed | ISI | Google Scholar - . Tolerance to morphine bradycardia in the rat. J Pharmacol Exp Ther 226: 187–191, 1983.
PubMed | ISI | Google Scholar - . The effect of intracerebroventricular infusion of morphine, methionine-enkephalin and d-Ala2-enkephalinamide on body temperature of rabbits. Arch Int Physiol Biochim 90: 1–7, 1982.
PubMed | Google Scholar - . Rostral ventromedial medulla and the control of cutaneous vasoconstrictor activity following icv prostaglandin E1. Neuroscience 124: 709–717, 2004.
Crossref | PubMed | ISI | Google Scholar - . A new class of GABA agonist. Nature 268: 53–55, 1977.
Crossref | PubMed | ISI | Google Scholar - . Evidence for an intrinsic mechanism of antinociceptive tolerance within the ventrolateral periaqueductal gray of rats. Neuroscience 135: 227–234, 2005.
Crossref | PubMed | ISI | Google Scholar - . Animal models of nociception. Pharmacol Rev 53: 597–652, 2001.
PubMed | ISI | Google Scholar - . Current animal test and models of pain. In: Pharmacology of Pain, edited by Beaulieu P, Lussier D, Porreca F, and Dickenson AH. Seattle, WA: International Association for the Study of Pain, 2009, p. 475–504.
Google Scholar - . Antinociceptive activity of d-ala2-d-leu5-enkephalin (BW 180C) in the rat after modification to central 5-hydroxytryptamine function. Neuropharmacology 18: 711–717, 1979.
Crossref | PubMed | ISI | Google Scholar - . Bulbospinal neurons of the rat rostromedial medulla are highly collateralized. J Comp Neurol 506: 960–978, 2008.
Crossref | PubMed | ISI | Google Scholar - . A study of the quantal (all-or-none) change in reflex latency produced by opiate analgesics. Brain Res 201: 129–141, 1980.
Crossref | PubMed | ISI | Google Scholar - . Analgesia produced by microinjection of baclofen and morphine at brain stem sites. Eur J Pharmacol 57: 43–55, 1979.
Crossref | PubMed | ISI | Google Scholar - . Evaluation of the periaqueductal central gray (PAG) as a morphine-specific locus of action and examination of morphine-induced and stimulation-produced analgesia at coincident PAG loci. Brain Res 124: 283–303, 1977.
Crossref | PubMed | ISI | Google Scholar - . An adrenergic link in the hypothalamic pathways which mediates morphine- and beta-endorphin-induced hyperthermia in the rat. Neuropharmacology 21: 613–617, 1982.
Crossref | PubMed | ISI | Google Scholar - . Hypothalamic neuronal responses to iontophoretic application of morphine in rats. Neuropharmacology 23: 591–594, 1984.
Crossref | PubMed | ISI | Google Scholar - . A comparison of the sites at which pentazocine and morphine act to produce analgesia. Pain 16: 313–331, 1983.
Crossref | PubMed | ISI | Google Scholar - . Brainstem mechanisms of antinociception. Effects of electrical stimulation and injection of morphine into the nucleus raphe magnus. Neuropharmacology 25: 727–735, 1986.
Crossref | PubMed | ISI | Google Scholar - . Raphe pallidus and raphe obscurus projections to the intermediolateral cell column in the rat. Brain Res 222: 129–133, 1981.
Crossref | PubMed | ISI | Google Scholar - . Measurement of ‘core’ temperature in the rat. Nature 210: 854–855, 1966.
Crossref | PubMed | ISI | Google Scholar - . The spinal potentiating effect and the supraspinal inhibitory effect of midazolam on opioid-induced analgesia in rats. Eur J Pharmacol 275: 153–162, 1995.
Crossref | PubMed | ISI | Google Scholar - . Nonshivering thermogenesis without interscapular brown adipose tissue involvement during conditioned fear in the rat. Am J Physiol Regul Integr Comp Physiol 296: R1239–R1247, 2009.
Link | ISI | Google Scholar - . A possible neural basis for stress-induced hyperalgesia. Pain 142: 236–244, 2009.
Crossref | PubMed | ISI | Google Scholar - . Involvement of ventromedial medulla “multimodal, multireceptive” neurons in opiate spinal descending control system: a single-unit study of the effect of morphine in the awake, freely moving rat. J Neurosci 12: 1511–1522, 1992.
Crossref | PubMed | ISI | Google Scholar - . Hyperthermia evoked by the intracerebral injection of morphine sulphate in the rat: the effect of restraint. Brain Res 145: 127–140, 1978.
Crossref | PubMed | ISI | Google Scholar - . Effect on core temperature of restraint after peripherally and centrally injected morphine in the Sprague-Dawley rat. Pharmacol Biochem Behav 10: 313–315, 1979.
Crossref | PubMed | ISI | Google Scholar - . Restraint alters the effects of morphine and heroin on core temperature in the rat. Pharmacol Biochem Behav 7: 463–469, 1977.
Crossref | PubMed | ISI | Google Scholar - . Pharmacological inactivation in the analysis of the central control of movement. J Neurosci Methods 86: 145–159, 1999.
Crossref | PubMed | ISI | Google Scholar - . Assessing pain threshold in the rat: changes with estrus and time of day. Physiol Behav 55: 651–657, 1994.
Crossref | PubMed | ISI | Google Scholar - . Physiological identification of pontomedullary serotonergic neurons in the rat. J Neurophysiol 77: 1087–1098, 1997.
Link | ISI | Google Scholar - . Contributions of the medullary raphe and ventromedial reticular region to pain modulation and other homeostatic functions. Annu Rev Neurosci 24: 737–777, 2001.
Crossref | PubMed | ISI | Google Scholar - . Deconstructing endogenous pain modulations. J Neurophysiol 94: 1659–1663, 2005a.
Link | ISI | Google Scholar - . Ventromedial medulla: pain modulation and beyond. J Comp Neurol 493: 2–8, 2005b.
Crossref | PubMed | ISI | Google Scholar - . Descending pain modulation as a component of homeostasis. In: Handbook of Clinical Neurology. Pain, edited by Cervero F, Jensen TS. Edinburgh: Elsevier, 2006, vo. 81, p. 211–218.
Google Scholar - . From descending pain modulation to obesity via the medullary raphe. Pain 152: S20–S24, 2011.
Crossref | PubMed | ISI | Google Scholar - . Medullary circuits for nociceptive modulation. Curr Opin Neurobiol 22: 640–645, 2012.
Crossref | PubMed | ISI | Google Scholar - . Restraint alters the thermic response to morphine by postural interference. Pharmacol Biochem Behav 18: 495–499, 1983.
Crossref | PubMed | ISI | Google Scholar - . Sex differences in stress-induced hyperthermia in rats: restraint versus confinement. Physiol Behav 98: 416–420, 2009.
Crossref | PubMed | ISI | Google Scholar - . Antinociception produced by microinjection of l-glutamate into the ventromedial medulla of the rat: mediation by spinal GABAA receptors. Brain Res 620: 86–96, 1993.
Crossref | PubMed | ISI | Google Scholar - . A critique of analgesic testing methods. Ann NY Acad Sci 51: 34–50, 1948.
Crossref | PubMed | ISI | Google Scholar - . Habituation to sham testing procedures modifies tail-flick latencies: effects on nociception rather than vasomotor tone. Pain 39: 103–107, 1989.
Crossref | PubMed | ISI | Google Scholar - . Behavioural modification of bulbospinal serotonergic inhibition and morphine analgesia. Brain Res 521: 167–174, 1990.
Crossref | PubMed | ISI | Google Scholar - . The contribution of the rostral ventromedial medulla to the antinociceptive effects of systemic morphine in restrained and unrestrained rats. Neuroscience 87: 123–133, 1998.
Crossref | PubMed | ISI | Google Scholar - . Antinociceptive synergism between supraspinal and spinal sites after subcutaneous morphine evidenced by CNS morphine content. Brain Res 552: 136–140, 1991.
Crossref | PubMed | ISI | Google Scholar - . Antinociceptive tolerance revealed by cumulative intracranial microinjections of morphine into the periaqueductal gray in the rat. Pharmacol Biochem Behav 85: 214–219, 2006.
Crossref | PubMed | ISI | Google Scholar - . Opioid selective antinociception following microinjection into the periaqueductal gray of the rat. J Pain 15: 1102–1109, 2014.
Crossref | PubMed | ISI | Google Scholar - . Differential control of sympathetic outflow. Am J Physiol Regul Integr Comp Physiol 281: R683–R698, 2001.
Link | ISI | Google Scholar - . Axonal branching patterns and funicular trajectories of raphe spinal sympathoinhibitory neurons. J Neurophysiol 53: 759–772, 1985.
Link | ISI | Google Scholar - . Metabolic and cardiovascular changes during physical restrains in rats. Jpn J Physiol 30: 799–803, 1980.
Crossref | PubMed | Google Scholar - . Heat balance during physical retrains in rats. Jpn J Physiol 29: 383–392, 1979.
Crossref | PubMed | Google Scholar - . Potential role of medullary raphe-spinal neurons in cutaneous vasoconstriction: an in vivo electrophysiological study. J Neurophysiol 87: 901–911, 2002.
Link | ISI | Google Scholar - . Modulation of sympathetic and somatomotor function by the ventromedial medulla. J Neurophysiol 92: 510–522, 2004.
Link | ISI | Google Scholar - . Medullary raphe neurons facilitate brown adipose tissue activation. J Neurosci 26: 1190–1198, 2006.
Crossref | PubMed | ISI | Google Scholar - . GABA analogues: conformational analysis of effects on [3H]GABA binding to postsynaptic receptors in human cerebellum. J Neurochem 32: 249–252, 1979.
Crossref | PubMed | ISI | Google Scholar - . Influence of GABA-acting drugs on morphine-induced hyperthermia in rats. Methods Find Exp Clin Pharmacol 32: 401–406, 2010.
Crossref | PubMed | Google Scholar - . Effect of morphine on rectal temperature after acute and chronic treatment in the rat. Prog Neuropsychopharmacol 5: 363–371, 1981.
Crossref | PubMed | Google Scholar - . Quantification of the analgesic activity of narcotic antagonists by a modified hot-plate procedure. J Pharmacol Exp Ther 192: 497–505, 1975.
PubMed | ISI | Google Scholar - . Thermogenesis inhibition in brown adipocytes is a specific property of volatile anesthetics. Anesthesiology 98: 437–448, 2003.
Crossref | PubMed | ISI | Google Scholar - . Mode of neural control mediating rat tail vasodilation during heating. J Appl Physiol 59: 1533–1538, 1985.
Link | ISI | Google Scholar - . Interactive drives from two brain stem premotor nuclei are essential to support rat tail sympathetic activity. Am J Physiol Regul Integr Comp Physiol 289: R1107–R1115, 2005.
Link | ISI | Google Scholar - . Inhibition of rostral medullary raphe neurons prevents cold- induced activity in sympathetic nerves to rat tail and rabbit ear arteries. Neurosci Lett 357: 58–62, 2004.
Crossref | PubMed | ISI | Google Scholar - . Selective blockade of 5-HT2A receptors attenuates the increased temperature response in brown adipose tissue to restraint stress in rats. Stress 11: 125–133, 2008.
Crossref | PubMed | ISI | Google Scholar - . Environmental temperature effects on the thermoregulatory response to systemic and hypothalamic administration of morphine. Life Sci 7, 857–863, 1968.
Crossref | ISI | Google Scholar - . Selective effects of pirenperone on analgesia produced by morphine or electrical stimulation at sites in the nucleus raphe magnus and periaqueductal gray. Psychopharmacology (Berl) 88: 172–176, 1986.
Crossref | PubMed | ISI | Google Scholar - . The Rat Brain in Stereotaxic Coordinates. Sydney: Academic, 1982.
Google Scholar - . Pain-facilitating medullary neurons contribute to opioid-induced respiratory depression. J Neurophysiol 108: 2393–2404, 2012.
Link | ISI | Google Scholar - . Effects of naloxone and Mr 1452 on stress-induced changes in nociception of different stimuli in rats. Life Sci 33, Suppl 1: 697–700, 1983.
Crossref | PubMed | ISI | Google Scholar - . Psychophysics of a nociceptive test in the mouse. PLoS One 7: e36699, 2012.
Crossref | PubMed | ISI | Google Scholar - . EEG and laser stimulation as tools for pain research. Curr Opin Investig Drugs 6: 58–64, 2005.
PubMed | Google Scholar - . Disposition of morphine in rat brain: relationship to biological activity. J Pharmacol Exp Ther 217: 181–188, 1981.
PubMed | ISI | Google Scholar - . Individual differences in the hotplate test and effects of habituation on sensitivity to morphine. Pain 66: 265–270, 1996.
Crossref | PubMed | ISI | Google Scholar - . Antinociception from a stereospecific action of morphine microinjected into the brainstem: a local or distant site of action? Br J Pharmacol 82: 877–882, 1984.
Crossref | PubMed | ISI | Google Scholar - . Studies on rectal temperature of rats in relation to seasonal air temperature and morphine administration. Indian J Physiol Pharmacol 24: 205–215, 1980.
PubMed | Google Scholar - . Impact of shock on pain reactivity: III. The magnitude of hypoalgesia observed depends on test location. Behav Neurosci 110: 528–541, 1996.
Crossref | PubMed | ISI | Google Scholar - . Reversible inactivation of raphe magnus neurons: effects on nociceptive threshold and morphine-induced analgesia. Brain Res 201: 459–464, 1980a.
Crossref | PubMed | ISI | Google Scholar - . Effects of raphe magnus and raphe pallidus lesions on morphine-induced analgesia and spinal cord monoamines. Pharmacol Biochem Behav 13: 705–714, 1980b.
Crossref | PubMed | ISI | Google Scholar - . Time-course of alterations in morphine-induced analgesia and nociceptive threshold following medullary raphe lesions. Neuroscience 6: 945–951, 1981.
Crossref | PubMed | ISI | Google Scholar - . Morphine analgesia: blockade by raphe magnus lesions. Brain Res 98: 612–618, 1975.
Crossref | PubMed | ISI | Google Scholar - . Delimitation of neuronal substrates necessary for the analgesic action of baclofen and morphine. Eur J Pharmacol 47: 159–166, 1978.
Crossref | PubMed | ISI | Google Scholar - . Modulation of the potency and efficacy of mu-mediated antinociception by delta agonists in the mouse. J Pharmacol Exp Ther 254: 683–689, 1990.
PubMed | ISI | Google Scholar - . Sex and strain differences in morphine-induced temperature effects in WKYs and SHRs. Brain Res Bull 14: 323–326, 1985.
Crossref | PubMed | ISI | Google Scholar - . The tail of the rat, in temperature regulation and acclimatization. Can J Physiol Pharmacol 43: 257–267, 1965.
Crossref | PubMed | ISI | Google Scholar - . Intravenous morphine-induced activation of vagal afferents: peripheral, spinal, and CNS substrates mediating inhibition of spinal nociception and cardiovascular responses. J Neurophysiol 68:1027–1045, 1992.
Link | ISI | Google Scholar - . Antinociception and cardiovascular responses produced by intravenous morphine: the role of vagal afferents. Brain Res 543: 256–270, 1991.
Crossref | PubMed | ISI | Google Scholar - . Central pathway for spontaneous and prostaglandin E2-evoked cutaneous vasoconstriction. Am J Physiol Regul Integr Comp Physiol 295: R343–R354, 2008.
Link | ISI | Google Scholar - . Differential control of sympathetic drive to the rat tail artery and kidney by medullary premotor cell groups. Brain Res 834: 196–199, 1999.
Crossref | PubMed | ISI | Google Scholar - . Cold-activated raphe-spinal neurons in rats. J Physiol 535: 841–854, 2001.
Crossref | PubMed | ISI | Google Scholar - . NMDA receptors modulate morphine-induced hyperthermia. Brain Res 984: 76–83, 2003.
Crossref | PubMed | ISI | Google Scholar - . Agmatine blocks morphine-evoked hyperthermia in rats. Brain Res 1147: 89–94, 2007.
Crossref | PubMed | ISI | Google Scholar - . Effects of opioids, cannabinoids, and vanilloids on body temperature. Front Biosci 3: 822–845, 2011.
Google Scholar - . Morphine analgesia following its infusion into different liquor spaces in rat brain. Arch Int Pharmacodyn Ther 246: 108–117, 1980.
PubMed | Google Scholar - . Rat tail flick acupuncture analgesia model. Chin Med J 92: 576–582, 1979.
PubMed | ISI | Google Scholar - . Effects of invariant sympathetic activity on cutaneous circulatory responses to heat stress. J Appl Physiol 71: 521–529, 1991.
Link | ISI | Google Scholar - . Quinpirole-induced alterations of tail temperature appear as hyperalgesia in the radiant heat tail-flick test. Pharmacol Biochem Behav 59: 77–82, 1998.
Crossref | PubMed | ISI | Google Scholar - . Muscimol injections in the nucleus raphé dorsalis block the antinociceptive effect of morphine in rats: apparent lack of 5-hydroxytryptamine involvement in muscimol's effect. Br J Pharmacol 81: 25–29, 1984.
Crossref | PubMed | ISI | Google Scholar - . Differential effects of systemic versus intracranial injection of opiates on central, orofacial and lower body nociception: somatotypy in bulbar analgesia systems. Pain 9: 307–318, 1980.
Crossref | PubMed | ISI | Google Scholar - . Hyperthermic effects of morphine: set point manipulation by a direct spinal action. Br J Pharmacol 61: 91–6, 1977.
Crossref | PubMed | ISI | Google Scholar - . Spinal pain suppression mechanisms may differ for phasic and tonic Pain. Brain Res 334: 172–175, 1985.
Crossref | PubMed | ISI | Google Scholar - . Effect of DLF lesions at different spinal levels on morphine induced analgesia. Brain Res 212: 239–242, 1981.
Crossref | PubMed | ISI | Google Scholar - . Functional interactions between delta- and mu-opioid receptors in rat thermoregulation. Eur J Pharmacol 458: 101–106, 2003.
Crossref | PubMed | ISI | Google Scholar - . Central nNOS is involved in restraint stress-induced fever: evidence for a cGMP pathway. Physiol Behav 80: 139–145, 2003.
Crossref | PubMed | ISI | Google Scholar - . A comparison of the antinociceptive and temperature responses to morphine and fentanyl derivatives in rats. Arch Pharm Res 36: 501–508, 2013.
Crossref | PubMed | ISI | Google Scholar - . The importance of tail temperature monitoring during tail-flick test in evaluating the antinociceptive action of volatile anesthetics. Acta Anaesthesiol Scand 46: 451–454, 2002.
Crossref | PubMed | ISI | Google Scholar - . Lesions to ascending noradrenergic and serotonergic pathways modify antinociception produced by intracerebroventricular administration of morphine. Neuropharmacology 28: 141–147, 1989.
Crossref | PubMed | ISI | Google Scholar - . Dorsal and ventral hippocampus modulate autonomic responses but not behavioral consequences associated to acute restraint stress in rats. PLoS One 8: e77750, 2013.
Crossref | PubMed | ISI | Google Scholar - . Morphine hyperthermia in the rat: its attenuation by physostigmine. Br J Pharmacol 44: 544–548, 1972.
Crossref | PubMed | ISI | Google Scholar - . Effect of periaqueductal morphine injection on thermal response in rats. Jpn J Physiol 36:485–496, 1986.
Crossref | PubMed | Google Scholar - . Contribution of nonshivering thermogenesis to stress-induced hyperthermia in rats. Jpn J Physiol 32: 991–995, 1982.
Crossref | PubMed | Google Scholar - . Spinal vs supraspinal actions of morphine on the rat tail-flick reflex. Pain 33: 357–362, 1988.
Crossref | PubMed | ISI | Google Scholar - . Effects of Pro-Leu-Gly-NH2 (MIF) on the antinociceptive and thermoregulatory actions of morphine and oxotremorine. J Pharm Pharmacol 34: 113–115, 1982.
Crossref | PubMed | ISI | Google Scholar - . Comparison of the effects of single doses of morphine and thebaine on body temperature, activity, and brain and heart levels of catecholamines and serotonin. Psychopharmacologia 3: 291–301, 1962.
Crossref | PubMed | ISI | Google Scholar - . The mu opiate receptor is responsible for descending pain inhibition originating in the periaqueductal gray region of the rat brain. Eur J Pharmacol 156: 47–54, 1988.
Crossref | PubMed | ISI | Google Scholar - . CNS cell groups projecting to sympathetic outflow of tail artery: neural circuits involved in heat loss in the rat. Brain Res 786: 153–164, 1998.
Crossref | PubMed | ISI | Google Scholar - . Antinociception versus serum concentration relationships following acute administration of intravenous morphine in male and female Sprague-Dawley rats: differences between the tail flick and hot plate nociceptive tests. Clin Exp Pharmacol Physiol 36: 20–28, 2009.
Crossref | PubMed | ISI | Google Scholar - . Body temperature response profiles for selective mu, delta and kappa opioid agonists in restrained and unrestrained rats. J Pharmacol Exp Ther 246: 92–101, 1988.
PubMed | ISI | Google Scholar - . Alteration of thermoregulatory set point with opioid agonists. J Pharmacol Exp Ther 252: 696–705, 1990.
PubMed | ISI | Google Scholar - . Morphine effects on the cardiovascular system of awake, freely behaving rats. Arch Int Pharmacodyn Ther 223: 54–63, 1976.
PubMed | Google Scholar - . Interaction between effects of stress and morphine on body temperature in rats. Life Sci 28: 1041–1045, 1981.
Crossref | PubMed | ISI | Google Scholar - . Coexpression of vesicular glutamate transporter-3 and gamma-aminobutyric acidergic markers in rat rostral medullary raphe and intermediolateral cell column. J Comp Neurol 492: 477–494, 2005.
Crossref | PubMed | ISI | Google Scholar - . A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections. Brain Res 491: 156–162, 1989.
Crossref | PubMed | ISI | Google Scholar - . Different radiant heat intensities differentiate intracerebroventricular morphine- from beta-endorphin-induced inhibition of the tail-flick response in the mouse. Eur J Pharmacol 213: 337–341, 1992.
Crossref | PubMed | ISI | Google Scholar - . Intrathecal administration of thiorphan, bestatin, desipramine and fluoxetine differentially potentiate the antinociceptive effects induced by beta-endorphin and morphine, administered intracerebroventricularly. Neuropharmacology 29: 207–214, 1990.
Crossref | PubMed | ISI | Google Scholar - . The reduction of antinociceptive effect of morphine administered intraventricularly is correlated with the decrease of serotonin release from the spinal cord in streptozotocin-induced diabetic rats. Gen Pharmacol 27: 445–450, 1996.
Crossref | PubMed | Google Scholar - . Capsaicin pretreatment interferes with the thermoregulatory effect of morphine. Pharmacol Biochem Behav 18: 373–378, 1983.
Crossref | PubMed | ISI | Google Scholar - . Predictive value of analgesic assays in mice and rats. In: Advances in Biochemical Psychopharmacology. Narcotic Antagonists, edited by Braude MC, Harris LS, May EL, Smith JP, Villareal JE. New York: Raven, 1974, vol. 8, p. 191–211.
Google Scholar - . State-dependent effects of beta-endorphin on core temperature in stressed and non-stressed rats. Jpn J Pharmacol 39: 395–397, 1985.
Crossref | PubMed | Google Scholar - . Effect of intracerebroventricular administration of vasopressin on stress-induced hyperthermia in rats. Physiol Behav 60: 417–424, 1996.
Crossref | PubMed | ISI | Google Scholar - The biology of rodent tails: a study of form and function. (Technical report Jan 63-Feb 64). Fort Wainwright, AK: Arctic Aeromedical Laboratory, 1966, TR-65-8, p. 1–138.
Google Scholar - . Is acute morphine hyperthermia of unrestrained rats due to selective activation of brown adipose tissue thermogenesis? J Pharmacol Exp Ther 231: 422–429, 1984.
PubMed | ISI | Google Scholar - . The role of the pituitary-adrenal axis in the hyperthermia induced by acute peripheral or central (preoptic anterior hypothalamus) administration of morphine to unrestrained rats. Can J Physiol Pharmacol 63: 1590–1598, 1985.
Crossref | PubMed | ISI | Google Scholar - . Intravenous morphine infusion (IMF) to drug-naive, conscious rats evokes bradycardic, hypotensive effects, but pressor actions are elicited after IMF to rats previously given morphine. Can J Physiol Pharmacol 67: 213–222, 1989.
Crossref | PubMed | ISI | Google Scholar - . Apparent hyperalgesia after lesions of the descending serotonergic pathways is due to increased tail skin temperature. Pain 33: 225–231, 1988.
Crossref | PubMed | ISI | Google Scholar - . The effect of morphine on core and skin temperature in rats. Neuroreport 3: 512–514, 1992.
Crossref | PubMed | ISI | Google Scholar - . Animal models of analgesia. In: Handbook of Experimental Pharmacology. The Pharmacology of Pain, edited by Dickenson A, Besson J-M. Berlin: Springer, 1997, p. 1–20.
Crossref | Google Scholar - . An improved method for tail-flick testing with adjustment for tail-skin temperature. J Neurosci Methods 26: 259–265, 1989a.
Crossref | PubMed | ISI | Google Scholar - . The apparent hyperalgesic effect of a serotonin antagonist in the tail flick test is mainly due to increased tail skin temperature. Pharmacol Biochem Behav 32: 601–605, 1989b.
Crossref | PubMed | ISI | Google Scholar - . Comparison of morphine and kainic acid microinjections into identical PAG sites on the activity of RVM neurons. J Neurophysiol 88: 1707–1715, 2002.
Link | ISI | Google Scholar - . Effects on temperature of morphine injected into the preoptic/anterior hypothalamus, medulla oblongata, and peripherally in unrestrained and restrained rats. Proc Soc Exp Biol Med 156: 523–526, 1977.
Crossref | PubMed | ISI | Google Scholar - . Differential actions of intrathecal naloxone on blocking the tail-flick inhibition induced by intraventricular beta-endorphin and morphine in rats. J Pharmacol Exp Ther 232: 74–79, 1985.
PubMed | ISI | Google Scholar - . Evidence that spinal endorphin mediates intraventricular beta-endorphin-induced tail flick inhibition and catalepsy. Brain Res 302: 231–237, 1984.
Crossref | PubMed | ISI | Google Scholar - . Behavioral activities of opioid peptides and morphine sulfate in golden hamsters and rats. Psychopharmacology (Berl) 64: 215–218, 1979.
Crossref | PubMed | ISI | Google Scholar - . Brainstem sites differentially sensitive to beta-endorphin and morphine for analgesia and release of met-enkephalin in anesthetized rats. J Pharmacol Exp Ther 253: 930–937, 1990.
PubMed | ISI | Google Scholar - . Contrasting cardiovascular properties of the μ-opioid agonists morphine and methadone in the rat. Eur J Pharmacol 762: 372–381, 2015.
Crossref | PubMed | ISI | Google Scholar - . Nuclei within the rostral ventromedial medulla mediating morphine antinociception from the periaqueductal gray. Brain Res 652: 9–16, 1994.
Crossref | PubMed | ISI | Google Scholar - . Differential effects of morphine on core temperature in stressed and non-stressed rats. Eur J Pharmacol 112: 331–337, 1985.
Crossref | PubMed | ISI | Google Scholar - . Tail-flick related activity in medullospinal neurons. Brain Res 321: 135–141, 1984.
Crossref | PubMed | ISI | Google Scholar - . Multiple brain sites involved in morphine antinociception. J Pharm Pharmacol 29: 765–767, 1977.
Crossref | PubMed | ISI | Google Scholar - . Cardiovascular and behavioral responses to conditioned fear after medullary raphe neuronal blockade. Neuroscience 153: 1344–1353, 2008.
Crossref | PubMed | ISI | Google Scholar - . Changes in cutaneous and body temperature during and after conditioned fear to context in the rat. Eur J Neurosci 21: 2505–2512, 2005.
Crossref | PubMed | ISI | Google Scholar - . Regulation of body temperature and nociception induced by non-noxious stress in rat. Brain Res 297: 1–10, 1984.
Crossref | PubMed | ISI | Google Scholar - . Role of adrenal medulla in morphine-induced hyperthermia through central action. Br J Pharmacol 76: 565–568, 1982.
Crossref | PubMed | ISI | Google Scholar - . The development of tolerance to and of physical dependence on morphine following intraventricular injection in the rat. Jpn J Pharmacol 21: 383–391, 1971.
Crossref | PubMed | Google Scholar - . Body temperature effects of opioids administered into the periaqueductal grey area of rat brain. Regul Pept 7: 259–267, 1983.
Crossref | PubMed | Google Scholar - . Peripheral versus central cardiorespiratory effects of morphine. Neuropharmacology 21: 1019–1026, 1982.
Crossref | PubMed | ISI | Google Scholar - . Effects of cold acclimation and central opioid processes on thermoregulation in rats. Pharmacol Biochem Behav 54: 317–325, 1996.
Crossref | PubMed | ISI | Google Scholar - . Spinal analgesia: comparison of the mu agonist morphine and the kappa agonist ethylketazocine. Life Sci 28: 2119–2125, 1981.
Crossref | PubMed | ISI | Google Scholar - . Antinociceptive effect of peripheral segmental electrical stimulation in the rat. Pain 8: 237–252, 1980.
Crossref | PubMed | ISI | Google Scholar - . The evaluation of the analgesic action of pethidine hydrochloride (Demerol). J Pharmacol Exp Ther 80: 300–307, 1944.
Google Scholar - . Rat strain differences in the potentiation of morphine-induced analgesia by stress. Pharmacol Biochem Behav 51: 699–703, 1995.
Crossref | PubMed | ISI | Google Scholar - . Effect of restraint on drug-induced changes in skin and core temperature in biotelemetered rats. Pharmacol Biochem Behav 55: 219–225, 1996.
Crossref | PubMed | ISI | Google Scholar - . A non-uniform three-dimensional perfusion model of rat tail heat transfer. Phys Med Biol 40: 789–806, 1995.
Crossref | PubMed | ISI | Google Scholar - . Body temperature and analgesic effects of selective mu and kappa opioid receptor agonists microdialyzed into rat brain. J Pharmacol Exp Ther 281: 499–507, 1997.
PubMed | ISI | Google Scholar - . Sites of action of opiates in production of analgesia. Prog Brain Res 77: 371–394, 1988.
Crossref | PubMed | ISI | Google Scholar - . Studies on the antagonism by raphe lesions of the antinociceptive action of systemic morphine. Eur J Pharmacol 41: 399–408, 1977.
Crossref | PubMed | ISI | Google Scholar - . Systematic examination in the rat of brain sites sensitive to the direct application of morphine: observation of differential effects within the periaqueductal gray. Brain Res 114: 83–103, 1976.
Crossref | PubMed | ISI | Google Scholar - . Nociceptive responses to high and low rates of noxious cutaneous heating are mediated by different nociceptors in the rat: behavioral evidence. Pain 68: 133–140, 1996.
Crossref | PubMed | ISI | Google Scholar - . Multiplicative interaction between narcotic agonisms expressed at spinal and supraspinal sites of antinociceptive action as revealed by concurrent intrathecal and intracerebroventricular injections of morphine. J Pharmacol Exp Ther 215: 633–642, 1980a.
PubMed | ISI | Google Scholar - . Sites of antinociceptive action of systemically injected morphine: involvement of supraspinal loci as revealed by intracerebroventricular injection of naloxone. J Pharmacol Exp Ther 215: 626–632, 1980b.
PubMed | ISI | Google Scholar - . Concurrent mapping of brain sites for sensitivity to the direct application of morphine and focal electrical stimulation in the production of antinociception in the rat. Pain 4: 23–40, 1977.
Crossref | PubMed | ISI | Google Scholar - . Effect on the nociceptive threshold and EEG activity in the rat of morphine injected into the medial thalamus and the periaqueductal gray. Neuropharmacology 17: 525–532, 1978.
Crossref | PubMed | ISI | Google Scholar - . Constraints on the tailflick assay: morphine analgesia and tolerance are dependent upon locus of tail stimulation. Life Sci 34: 1755–1762, 1984.
Crossref | PubMed | ISI | Google Scholar - . The graded and quantal nature of opioid analgesia in the rat tailflick assay. Brain Res 331: 327–336, 1985.
Crossref | PubMed | ISI | Google Scholar - . Evidence for on-off control of heat dissipation from the tail of the rat. Can J Physiol Pharmacol 60: 392–398, 1982.
Crossref | PubMed | ISI | Google Scholar - . Comparison of the effects of ventral medullary lesions on systemic and microinjection morphine analgesia. Brain Res 290: 119–129, 1984.
Crossref | PubMed | ISI | Google Scholar - . Periaqueductal gray matter involvement in the muscimol-induced decrease of morphine antinociception. Naunyn Schmiedebergs Arch Pharmacol 318: 368–369, 1982.
Crossref | PubMed | ISI | Google Scholar - . Influence of stress on morphine-induced hyperthermia: relevance to drug conditioning and tolerance development. Behav Neurosci 99: 122–144, 1985.
Crossref | PubMed | ISI | Google Scholar - . Hyperthermia and antinociceptive activity of thyrotropin-releasing hormone and morphine following central administration in rats. Acta Physiol Pharmacol Bulg 14: 18–23, 1988.
PubMed | Google Scholar - . Effects of hot plate temperature on the antinociceptive activity of mixed opioid agonist-antagonist compounds. Drug Dev Res 7: 277–280, 1986.
Crossref | ISI | Google Scholar - . Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16: 109–110, 1983.
Crossref | PubMed | ISI | Google Scholar - . Effects of some GABA-mimetic drugs on the antinociceptive activity of morphine and beta-endorphin in rats. Naunyn Schmiedebergs Arch Pharmacol 316: 231–234, 1981.
Crossref | PubMed | ISI | Google Scholar
