Increased GABAergic transmission in neuropeptide Y-expressing neurons in the dopamine-depleted murine striatum
Abstract
As the main input nucleus of the basal ganglia, the striatum plays a central role in planning, control, and execution of movement and motor skill learning. More than 90% of striatal neurons, so-called medium spiny neurons (MSN), are GABAergic projection neurons, innervating primarily the substantia nigra pars reticulata or the globus pallidus internus. The remaining neurons are GABAergic and cholinergic interneurons, synchronizing and controlling striatal output by reciprocal connections with MSN. Besides prominent local cholinergic influence, striatal function is globally regulated by dopamine (DA) from the nigrostriatal pathway. Little is known about whether DA depletion, as occurs in Parkinson’s disease, affects the activity of striatal interneurons. Here we focused on neuropeptide Y (NPY)-expressing interneurons, which are among the major subgroups of GABAergic interneurons in the striatum. We investigated the effects of striatal DA depletion on GABAergic transmission in NPY interneurons by electrophysiologically recording GABAergic spontaneous (s) and miniature (m) inhibitory postsynaptic currents (IPSCs) in identified NPY interneurons in slices from 6-hydroxydopamine (6-OHDA)- and vehicle-injected transgenic NPY-humanized Renilla green fluorescent protein (hrGFP) mice with the whole cell patch-clamp technique. We report a significant increase in sIPSC and mIPSC frequency as well as the occurrence of giant synaptic and burst sIPSCs in the 6-OHDA group, suggesting changes in GABAergic circuit activity and synaptic transmission. IPSC kinetics remained unchanged, pointing to mainly presynaptic changes in GABAergic transmission. These results show that chronic DA depletion following 6-OHDA injection causes activity-dependent and -independent increase of synaptic GABAergic inhibition onto striatal NPY interneurons, confirming their involvement in the functional impairments of the DA-depleted striatum.
NEW & NOTEWORTHY Neuropeptide Y (NPY) interneurons regulate the function of striatal projection neurons and are upregulated upon dopamine depletion in the striatum. Here we investigated how dopamine depletion affects NPY circuits and show electrophysiologically that it leads to the occurrence of giant synaptic and burst GABAergic spontaneous inhibitory postsynaptic currents (IPSCs) and to an activity-independent increase in GABAergic miniature IPSC frequency in NPY neurons. We suggest that degeneration of dopaminergic terminals in the striatum causes functional changes in striatal GABAergic function.
INTRODUCTION
The striatum is the main input nucleus of the basal ganglia. It is critically involved in the control of movement and motor skill learning and consists almost entirely of GABAergic neurons. Approximately 95% of these neurons are medium spiny neurons (MSN), projecting to other basal ganglia nuclei/substantia nigra and thereby controlling thalamic function via the direct, movement promoting, and the indirect, movement suppressing, pathway. The remaining neurons are GABAergic and cholinergic interneurons (Koós and Tepper 1999; Rymar et al. 2004). Although inferior in number, GABAergic interneurons play a crucial role in the regulation and synchronization of MSN and hence striatal output. At least five immunohistochemically and electrophysiologically distinct types of GABAergic interneurons have been described in the murine striatum: parvalbumin-expressing (PV) fast-spiking interneurons, calretinin-expressing interneurons, tyrosine hydroxylase (TH)-expressing interneurons, cholecystokinin-expressing interneurons. and neuropeptide Y (NPY)/somatostatin (SOM)/neuronal nitric oxide synthase (NOS)-expressing low-threshold spiking (LTS) interneurons (Boccalaro et al. 2019; Muñoz-Manchado et al. 2018; Tepper et al. 2010; Tepper and Koós 2016). The group of NPY-positive interneurons is subdivided into two electrophysiologically and morphologically distinct subpopulations: the NPY-LTS interneurons, which have slightly larger somata, coexpress SOM and NOS, form two to four primary dendrites with few up to 800-µm-extending branches, and account for ~80% of the NPY-positive interneurons, and the NPY neuroglial form (NPY-NGF) interneurons, which do not coexpress SOM and NOS, form up to nine primary highly branching dendrites that have their peak density at ∼80 µm and account for the remaining 20% of NPY-positive neurons (Ibáñez-Sandoval et al. 2011; Tepper and Koós 2016). NPY-LTS interneurons receive suprathreshold glutamatergic input from the cerebral cortex and have a low connection probability of <15% to striatal MSN. NPY-NGF interneurons receive their action potential (AP)-triggering excitation not from the cerebral cortex but from the thalamus and have a high connection probability to MSN of >85% (Ibáñez-Sandoval et al. 2011).
NPY interneurons, like almost all cells in the striatum, are innervated by dopaminergic terminals and are therefore controlled by dopamine (DA) originating from the substantia nigra pars compacta (SNc) (Cao et al. 2007; Gerfen and Surmeier 2011; Kerkerian et al. 1986; Lindefors et al. 1990; Pelletier and Simard 1991). DA plays a key role in balancing the activity of the direct and indirect pathways and hence in the initiation and execution of proper movements. Chronic DA depletion in the basal ganglia, as it occurs in Parkinson’s disease (PD), leads to the dysregulation of striatal circuits, affecting in particular the balance between the direct and indirect pathways, leading to severe motor impairments. The underlying causes are complex and probably involve all subtypes of striatal neurons.
Several studies have shown that NPY and NPY-expressing cells are involved in or affected by the changes occurring in PD. An increase in NPY-positive cell numbers and protein levels has been reported in the putamen of PD patients (Cannizzaro et al. 2003) as well as in the striatum of DA-depleted rats with 6-hydroxydopamine (6-OHDA) (Abrous et al. 1994; Ma et al. 2014). Furthermore, intracerebral administration of NPY decreased the extent of 6-OHDA-induced neurodegeneration (Decressac et al. 2012; Pain et al. 2019), whereas extracellular application of DA depolarized and triggered AP firing in NPY-LTS interneurons (Centonze et al. 2002), most likely mediated by D5 receptors (Rivera et al. 2002; Tepper and Koós 2016). NPY interneurons receive GABAergic input from MSN and from other interneurons, especially PV interneurons, and possibly from extrinsic sources. Therefore, the effects of DA depletion on their activity could be indirect and involve changes in GABAergic function.
Here we have addressed this possibility by investigating the effects of striatal DA depletion on GABAergic transmission onto NPY interneurons in the striatum in order to evaluate potential changes within GABAergic circuits. To this end, we performed and analyzed whole cell patch-clamp recordings of AP-dependent and AP-independent GABAergic synaptic input to NPY-positive striatal neurons in DA-depleted and control mice.
MATERIALS AND METHODS
Animals.
Electrophysiological recordings were performed in unilaterally injected 10- to 14-wk-old male NPY-hrGFP mice (IMSR_JAX:006417). These mice were raised on a C57BL/6 background and express humanized Renilla green fluorescent protein (hrGFP) under the control of the mouse Npy promoter, to label NPY-expressing neurons. NPY-hrGFP-positive mice were identified with UV light radiation a few days after birth. Only male mice were used in this study to avoid sex-related differences. Mice were bred and housed under standard conditions with a 12:12-h light-dark cycle at the Laboratory Animal Service Center of the University of Zurich. In total, 13 male NPY-hrGFP mice were used in this study, of which 7 animals were injected with 6-OHDA and 6 animals were injected with the vehicle ascorbic acid.
All experiments were performed according to the ARRIVE guidelines on animal use and care and were approved by the Cantonal Veterinary Office of Zurich.
Stereotaxic 6-hydroxydopamine injections.
To deplete dopaminergic (DA) terminals in the striatum, small doses of the neurotoxin 6-hydroxydopamine (6-OHDA) were injected unilaterally into the striatum of 8-to 9-wk-old male NPY-hrGFP mice. 6-OHDA induces the formation of reactive oxygen species (ROS) by autoxidation and affects the mitochondrial respiratory chain of the neuron (Glinka et al. 1996; Kumar et al. 1995; Sachs and Jonsson 1975), thereby leading to the degeneration of DA terminals.
Mice were anesthetized by the inhalation of 1.5% isoflurane (Attane; Piramal Healthcare, India) in oxygen, head-fixed in a stereotaxic frame, and unilaterally injected in the right dorsal striatum with the coordinates x = +1.75 mm, y = +0.9 mm, z = −4–2.8 mm relative to bregma. Stereotaxic injections were performed as described in Deprez et al. (2015). Animals of the 6-OHDA-group were injected with 6 μg (≈ 400 nL) of 6-OHDA (Tocris Bioscience catalog no. 2547), and animals of the control/vehicle group were injected with 400 nL of 0.2% ascorbic acid in PBS (vehicle substrate). To prevent the oxidation of the 6-OHDA solution, it was prepared directly before use and was kept at 4°C in the dark until the injection. Directly after the surgery and up to the 4th day after injection, 6-OHDA-injected animals showed spontaneous turning behavior toward the side of injection.
Acute brain slice preparation.
After brief anesthesia with isoflurane, mice were euthanized 15–20 days after the injection with either 6-OHDA or 0.2% ascorbic acid by decapitation, and the brains were quickly removed and put into ice-cold oxygenated (95% O2-5% CO2) slicing solution (in mM: 65 NaCl, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 1.25 NaH2PO4, 25 NaHCO3, 105 sucrose, and 25 glucose; pH = 7.4). The contralateral hemisphere was marked, 350-µm-thick coronal slices were cut (Leica VT1200S vibrating microtome; Leica Biosystems, Germany), and the slices were transferred briefly into a potassium gluconate-containing solution (in mM: 130 C6H11KO7, 15 KCl, 0.2 EGTA, 20 HEPES, and 25 glucose; pH = 7.4), before being transferred into a 35°C oxygenated artificial cerebrospinal fluid (aCSF; in mM: 125 NaCl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 25 NaHCO3 and 25 glucose; pH = 7.4, 315 mosM) for 25–30 min. After the incubation at 35°C the slices were transferred to oxygenated aCSF solution at room temperature, where they were kept until use.
Electrophysiology and data analysis.
The recording chamber of the electrophysiological setup was constantly perfused with oxygenated aCSF at a flow rate of 1.5 mL/min. Single coronal slices were transferred into the chamber for recording and fixed with a harp slice grid. An upright Zeiss Examiner.A1 microscope (×63/1.0 water-immersion objective; Zeiss, Germany), a HXP 120 V light source (Zeiss, Germany), and an Andor Zyla sCMOS camera with Andor Solis software (Oxford Instruments, Abingdon, UK) were used to visualize and identify NPY-hrGFP-positive cells. Borosilicate glass pipettes with a tip diameter of ∼1 µm (2–4 MΩ) were used for whole cell patch-clamp recordings and filled with CsCl-containing internal solution (in mM: 120 CsCl, 4 MgCl2, 10 HEPES, 10 EGTA, 2 Mg-ATP, and 0.5 Na-GTP; pH = 7.3 with CsOH). The recordings were carried out with a MultiClamp 700B amplifier; a Digidata 1440A was used for data acquisition (both Molecular Devices, California). GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded from hrGFP-positive neurons with aCSF containing 20 μM NBQX (Alomone Labs catalog no. N-186) and 0.5 μM strychnine (Tocris Bioscience catalog no. 2785). For the recording of miniature inhibitory postsynaptic currents (mIPSCs), 1 μM TTX (Latoxan Laboratory catalog no. L8503) was added to the external solution used for sIPSC recordings. For recordings of the tonic current inhibition, the slices were perfused with 10 μM gabazine (SR 95531; Tocris Bioscience catalog no. 1262). Each external solution was oxygenated and perfused for 5 min. For all recordings, the holding potential was set to −70 mV.
Recordings were excluded from analysis if the holding/leak current was larger than −100 pA or the access resistance was greater than 30 MΩ. A Butterworth low-pass filter (2 kHz) was used to filter the recorded traces, and the last 90 s of every 5-min perfusion period was analyzed with Mini Analysis Software 6.0.7 (Synaptosoft, New Jersey). The minimum cutoff for IPSC detection was set to 5 pA. Events were excluded if the decay time was faster than the rise time or the decay time was faster than 1.5 ms. After the analysis and sorting of the data, the interevent interval (IEI), peak amplitude, and decay time constants were plotted in cumulative distribution plots and average frequency, amplitude, and decay, as well as the total number of events within the 90 s of analysis and the maximum peak amplitude of every recording were calculated. Data plotted in the histograms are means ± SE, and data presented in the bar graphs are means ± SD.
Immunohistochemical labeling.
To validate GFP-positive neurons as being NPY-expressing cells, we performed immunohistochemical experiments. Mice were anesthetized by an intraperitoneal injection of pentobarbital sodium (Nembutal; 50 mg/kg), transcardially perfused with aCSF solution (pH 7.4), and killed by decapitation as described in Notter et al. (2014). Quickly, the brains were removed and postfixed for 90 min in a solution containing 0.15 M Na-phosphate buffer and 4% paraformaldehyde (pH 7.4) at 4°C. Thereafter, the fixed brains were cryoprotected overnight in a 30% sucrose solution, frozen, and cut into 40-µm-thick coronal sections. For immunohistochemical labeling, sections were washed three times for 10 min with 50 mM Tris and incubated with the primary antibody against NPY (1:1,000; Peninsula Laboratories catalog no. T-4069, RRID: AB_2314974) in a solution containing 50 mM Tris, 150 mM NaCl, 0.2% Triton X-100, and 2% normal goat serum (pH 7.4) at 4°C overnight. On the next day, the sections were washed with 50 mM Tris (3 times for 10 min) and incubated for 30 min at room temperature in a 50 mM Tris, 150 mM NaCl, 0.05% Triton X-100, 2% normal goat serum (pH 7.4) solution containing the secondary antibody coupled to Cy3 (1:500; Jackson ImmunoResearch Laboratories). Next, the sections were washed (3 times for 10 min) with 50 mM Tris, mounted onto gelatin-coated slides, and coverslipped with a fluorescent mounting medium (Dako, North America Inc., United States, catalog no. S3023). Images were acquired by confocal laser scanning microscopy (Carl Zeiss LSM 700) with a ×20 and a ×40 objective (numerical aperture of 1.4). Representative images were prepared with ImageJ (ImageJ imaging software, National Institutes of Health, Bethesda, MD).
Tyrosine hydroxylase immunoperoxidase labeling.
After electrophysiological recordings, the extent of DA depletion was assessed in brain slices of 6-OHDA-injected mice by an immunoperoxidase labeling for tyrosine hydroxylase (TH). Slices were postfixed for 10 min in 4% paraformaldehyde in 0.1 M Na-phosphate buffer (pH 7.4) after electrophysiological recordings and cryoprotected with a 30% sucrose solution overnight. The fixed slices were rinsed (3 times for 10 min) with Tris-Triton and incubated in a rabbit polyclonal anti-TH antibody (EMD Millipore catalog no. AB152, RRID: AB_390204) diluted in Tris buffer containing 0.2% Triton X-100 and 2% normal goat serum at 4°C and under continuous agitation (100 rpm) overnight. On the second day, the sections were incubated for 30 min with biotinylated goat anti-rabbit antibody (Vector Laboratories catalog no. BA-1000, RRID: AB_2313606) at room temperature and washed (3 times for 10 min) with Tris-Triton. Thereafter, the sections were incubated for 30 min in avidin-peroxidase complex (ABC) solution (Vectastain Elite Kit; Vector Laboratories, United States, catalog no. PK-6100) at room temperature and subsequently washed with Tris-Triton (3 times for 10 min). Finally, the slices were light-protected and preincubated for 5 min in a diaminobenzidine solution (0.5 g/L in Tris-Triton; pH 7.7) before the peroxidase reaction was triggered by adding a 1% H2O2 solution until the final concentration of 0.01% H2O2 was reached. The reaction was stopped by transfer of the sections into ice-cold PBS. After a final washing step (3 times for 10 min in Tris-Triton), the sections were mounted on gelatinized glass slides and left to dry overnight. The dried sections were dehydrated by processing them through an ascending ethanol series (5 min each). Finally, the mounted slices were washed three times in xylene before being coverslipped with Eukitt quick-hardening mounting medium (Sigma-Aldrich, United States). DA depletion was considered successful when the TH immunoreactivity was strongly reduced or absent on the side of 6-OHDA injection.
RESULTS
To confirm that GFP-positive neurons are in fact NPY-expressing neurons, immunofluorescence labeling was performed on fixed brain sections from NPY-hrGFP mice with an anti-NPY antibody. In all cells investigated a perfect match (colabeling of NPY-GFP and NPY, n = 13) was observed, as shown for a representative field in Fig. 1.
Fig. 1.Immunofluorescence labeling of neuropeptide Y (NPY)-positive neurons in an NPY-humanized Renilla green fluorescent protein (hrGFP) mouse. A and B: representative image of NPY-hrGFP-positive neurons (green; A) in a mouse striatum immunoreactive for NPY (red; B). C: an overlap of the NPY-hrGFP and the anti-NPY antibody is shown in yellow. Scale bars: represent 20 µm (merged image) and 5 µm (inset).
Since the interpretation of our results depends on the effect of 6-OHDA, the extent of DA depletion in the striatum of 6-OHDA-injected animals was assessed by TH immunoreactivity post hoc in every slice after completion of the electrophysiological recordings. Three representative examples are shown in Fig. 2. Figure 2, A–C, show the immunoperoxidase staining as seen in the microscope; Fig. 2, A′–C′, show the threshold values (binary scale) of the respective image above. TH immunoreactivity in these images was considered to be either absent (Fig. 2, A and C) or strongly decreased (Fig. 2B). This degree of depletion was consistently achieved with a dose of 6 μg of 6-OHDA in the mice used for the recordings. All cells were recorded in the central zone of the striatum, where the depletion is maximal, but away from the immediate core of the injection.
Fig. 2.Tyrosine hydroxylase (TH) immunoperoxidase labeling. A–C: 3 examples of post hoc immunoperoxidase staining against TH in coronal brain sections of 6-hydroxydopamine-injected mice after electrophysiological recordings to evaluate dopamine (DA) depletion. A′–C′: threshold figures of the corresponding immunoperoxidase images in A–C illustrating the strong DA depletion seen in each of these cases.
Pharmacologically isolated GABAergic sIPSCs and mIPSCs were recorded in acute slices in the whole cell patch-clamp configuration in striatal NPY-positive interneurons in mice 15–20 days after the unilateral injection of 0.2% ascorbic acid (vehicle) or 6-OHDA. This time point was selected to assess the effects of chronic DA depletion on circuit activity and GABA synapse function. For sIPSC recordings 17 cells from six ascorbic acid-injected animals and 18 cells from six 6-OHDA-injected animals and for mIPSC recordings 13 cells from five ascorbic acid-injected animals and 15 cells from six 6-OHDA-injected animals were used for analysis.
To ensure that the recording quality was comparable in vehicle- and 6-OHDA-injected mice, only cells with an access resistance <30 MΩ were used for analysis, resulting in similar values for the average access resistance in the two groups (sIPSCs: 16.1 ± 4.4 MΩ vs. 17.9 ± 7.7 MΩ for vehicle and 6-OHDA, respectively, unpaired t test, t33 = 0.835, P = 0.41; mIPSCs: 16.8 ± 3.0 MΩ vs. 16.0 ± 4.7 MΩ for vehicle and 6-OHDA, respectively, unpaired t test, t26 = 0.486, P = 0.631). Likewise, similar average rise times were seen in the two conditions (sIPSCs: 2.7 ± 0.4 ms vs. 2.7 ± 0.3 ms for vehicle and 6-OHDA, respectively, unpaired t test, t33 = 0.231, P = 0.819; mIPSCs: 2.6 ± 0.2 ms vs. 2.6 ± 0.2 ms for vehicle and 6-OHDA, respectively, unpaired t test, t26 = 0.065, P = 0.948).
Data from the analysis of sIPSC are shown in Fig. 3. Figure 3A shows a 30-s trace of a typical sIPSC recording from an NPY interneuron (detected by high transgenic GFP expression) in a vehicle-injected (black trace) and a 6-OHDA-injected (red trace) mouse. To show similar activation and inactivation kinetics in the vehicle-injected and 6-OHDA-injected groups, the comparison of a normalized single event recorded from NPY-positive cells of a vehicle- and a 6-OHDA-injected animal is shown in Fig. 3A′. Recordings obtained in slices from 6-OHDA-injected mice had shorter interevent intervals (IEIs; Kolmogorov–Smirnov test, P < 0.0001; Fig. 3, B and B′) and significantly higher frequencies (1.4 ± 0.2 Hz vs. 1.9 ± 0.2 Hz for vehicle and 6-OHDA, respectively, unpaired t test, t33 = 2.417, P = 0.021; Fig. 3B″) than slices from vehicle-injected mice. Hence, more sIPSCs occurred within the 90-s time window of analysis (123 ± 13 vs. 170 ± 14 events in 90 s for vehicle and 6-OHDA, respectively, unpaired t test, t26 = 2.517, P = 0.017; Fig. 3B‴). Two cells recorded from two mice in the 6-OHDA group received bursts of sIPSCs (Fig. 3E). Single giant events greater than −200 pA could be seen in 4 of 18 recorded cells in three of six 6-OHDA-injected animals. Bursts and giant events were never observed in recordings from vehicle-injected animals (Fig. 3, A and C‴). However, despite these giant events, no significant differences could be seen in the cumulative distribution plot (Kolmogorov–Smirnov test, P = 0.126; Fig. 3C′) of the current amplitudes as well as in the analysis of the average amplitude (21.5 ± 1.4 pA vs. 23.0 ± 2.6 pA for vehicle and 6-OHDA, respectively, unpaired t test, t33 = 0.534, P = 0.597; Fig. 3C″) and the maximal amplitude (119 ± 11 pA vs. 179 ± 33 pA for vehicle and 6-OHDA, respectively, unpaired t test, t33 = 1.71, P = 0.097; Fig. 3 C‴). Finally, there were no differences between the decay time kinetics of vehicle-injected and 6-OHDA-injected animals [Kolmogorov–Smirnov test, P = 0.234 (Fig. 3D′) and 15.6 ± 0.4 ms vs. 15.9 ± 0.6 ms average decay time constant for vehicle and 6-OHDA, respectively, unpaired t test, t33 = 0.392, P = 0.697 (Fig. 3D″)]. Decay kinetics could be best fitted by monoexponential functions.
Fig. 3.Spontaneous GABAergic postsynaptic currents (sIPSCs) in neuropeptide Y (NPY)-positive striatal interneurons. A and A′: example traces and a magnified single synaptic event of sIPSC recordings from NPY-positive cells of a vehicle-injected and a 6-hydroxydopamine (6-OHDA)-injected NPY-humanized Renilla green fluorescent protein (hrGFP) mouse. B–B‴: event frequency given as the interevent interval (IEI) depicted in a bar graph (B) and a cumulative distribution plot (B′), the average frequency per recorded cell (B″), and the total number of events occurring within the 90 s of analysis (B‴). C–C‴: analysis of the sIPSC amplitude shown in a bar graph (C) and a cumulative distribution plot (C′) and the average (C″) as well as the maximum (C‴) peak amplitude per cell. Note the occurrence of giant events in some cells of the 6-OHDA group despite the overall similarity in the average peak amplitude. D–D″: decay kinetics of the sIPSCs shown in a bar graph (D) and a cumulative distribution plot (D′) and the average decay time constant per recorded cell (D″). E: example of bursts of sIPSCs recorded in an NPY-positive cell from a 6-OHDA-treated mouse. Data were analyzed from 6 ascorbic acid-injected (17 cells) and 6 6-OHDA-injected (18 cells) animals with the Kolmogorov–Smirnov test (B′, C′, D′) and an unpaired t test (B″, B‴, C″, C‴, D″). *P < 0.05, ****P < 0.0001.
To investigate GABAergic synapse function in striatal NPY-positive interneurons of DA-depleted animals, GABAergic mIPSCs were recorded from vehicle-injected and 6-OHDA-injected animals in a set of slices different from that used for recording spontaneous events. In Fig. 4A, original mIPSC traces recorded from a vehicle-injected and a 6-OHDA-injected animal are shown. NPY-positive interneurons from 6-OHDA-injected animals showed significantly more mIPSCs compared with mIPSCs recorded from NPY interneurons of vehicle-injected animals, resulting in a higher frequency [Kolmogorov–Smirnov test, P < 0.0001 (Fig. 4B′) and 1.2 ± 0.1 Hz vs. 1.7 ± 0.2 Hz for vehicle and 6-OHDA, respectively, unpaired t test, t26 = 2.532, P = 0.018 (Fig. 4, B–B″)] and in an increased number of mIPSCs within the 90-s time window of analysis (110 ± 11 vs. 154 ± 14 events in 90 s for vehicle and 6-OHDA, respectively, unpaired t test, t26 = 2.409, P = 0.023; Fig. 4B‴). There were no differences in mIPSC amplitude [Kolmogorov–Smirnov test, P = 0.052 (Fig. 4C′); average amplitude: 17.1 ± 1.4 pA and 16.9 ± 1.1 for vehicle and 6-OHDA, unpaired t test, t26 = 0.079, P = 0.937 (Fig. 4C″); and maximal amplitude: 73.1 ± 9.3 pA vs. 61.3 ± 7.2 pA for vehicle and 6-OHDA, respectively, unpaired t test, t26 = 1.014, P = 0.32 (Fig. 4C‴)] and decay time constants [Kolmogorov–Smirnov test, P = 0.249 (Fig. 4D′); average decay time: 15.9 ± 0.55 ms vs. 15.4 ± 0.39 ms for vehicle and 6-OHDA, respectively, unpaired t test, t26 = 0.81, P = 0.426 (Fig. 4D″)]. Although the compared frequency, amplitude, and decay time constants of sIPSCs and mIPSCs revealed that most of the former are unitary events, no bursts and no giant events were seen upon addition of TTX to the recording solution, confirming the AP-dependent nature of these events.
Fig. 4.Miniature GABAergic postsynaptic currents (mIPSCs) of neuropeptide Y (NPY)-positive striatal neurons. A and A′: example traces and a magnified single synaptic event of mIPSC recordings from NPY-positive cells of a vehicle-injected and a 6-hydroxydopamine (6-OHDA)-injected NPY-humanized Renilla green fluorescent protein (hrGFP) mouse. B–B‴: calculated event frequency and interevent intervals (IEI) shown as a bar graph (B) and a cumulative distribution plot (B′) and the average frequency per recorded cell (B″) and the total number of events occurring within the 90 s of analysis (B‴). C–C‴: analysis of the mIPSC amplitude depicted as a bar graph (C) and a cumulative distribution plot (C′) and the average (C″) and maximum (C‴) peak amplitude per cell. D–D″: similar decay kinetics of the mIPSCs shown in a bar graph (D) and a cumulative distribution plot (D′) and the average decay time constant per recorded cell (D″). Data were analyzed from 5 ascorbic acid-injected (13 cells) and 6 6-OHDA-injected (15 cells) animals with the Kolmogorov–Smirnov test (B′, C′, D′) and an unpaired t test (B″, B‴, C″, C‴, D″). *P < 0.05, ****P < 0.0001.
After mIPSC recordings and to investigate currents through extrasynaptic GABAA receptors (GABAARs), the GABAAR-specific antagonist gabazine was added to the external aCSF solution to record GABAergic tonic inhibition. Tonic currents were recorded from 11 cells each from five vehicle-injected and five 6-OHDA-injected animals. Extrasynaptic GABAAR function was determined by analyzing the shift in holding current caused by selectively blocking GABAARs. Gabazine suppressed all mIPSCs, and no shift of the baseline current could be seen in either the vehicle-injected or the 6-OHDA-injected group. There was no significant difference in holding current between the two groups induced by gabazine (Δ from baseline: 0.28 ± 0.1 pA vs. 0.56 ± 0.1 pA for vehicle and 6-OHDA, respectively, unpaired t test, t20 = 1.684, P = 0.108; Fig. 5), suggesting that NPY interneurons receive little, if any, tonic GABAergic inhibition in either condition.
Fig. 5.Lack of tonic inhibition in neuropeptide Y (NPY)-positive striatal neurons. A: example traces of tonic inhibition recorded from NPY-positive neurons in the presence of the specific GABAA receptor inhibitor gabazine from a vehicle- and a 6-hydroxydopamine (6-OHDA)-injected mouse. B: analysis of GABAergic tonic inhibition recorded from 11 cells each of 5 vehicle-injected and 5 6-OHDA-injected animals. Tonic currents are given as Δ holding current from the baseline.
DISCUSSION
This study reveals that chronic 6-OHDA-induced DA depletion in the striatum increases GABAergic input onto NPY-positive neurons in the mouse striatum. Our data show a significant increase in sIPSC and mIPSC frequency after DA depletion and the occurrence of giant synaptic events, as well as burst sIPSCs in a subset of cells of 6-OHDA-treated mice, but no changes in tonic GABAergic currents. Although the averaged sIPSC amplitudes were not significantly different and the fraction of events with amplitudes between 20 and 60 pA was similar in the vehicle and 6-OHDA groups, a shift occurred from high-amplitude events of 80–120 pA in the vehicle group to amplitudes of 140 up to 560 pA in the 6-OHDA group. This shift in high-amplitude events was not observed in mIPSC recordings upon suppression of AP firing by TTX, indicating that giant synaptic events are not unitary. However, the significant increase in IPSC frequency remained present in mIPSC recordings, underscoring that it is not due to changes in network activity only. The increased AP-dependent IPSC amplitudes and the increase in sIPSC and mIPSC frequency in 6-OHDA-treated animals could be explained by the presence of more GABA release sites per synapse and/or by an increase of postsynaptic GABAA receptors. This conclusion implies that DA depletion induces changes in GABAergic circuits involving NPY interneurons. Dehorter et al. (2009), who observed giant sIPSCs in MSN of the DA-depleted striatum, hypothesized that these events were due to alterations in the basic activity pattern of LTS interneurons, which changed from single spikes to burst firing pattern, as well as to an increase in the efficacy of GABAergic synaptic transmission. Innervation of NPY-positive cells by other LTS interneurons (Markram et al. 2004) could explain the burst IPSCs observed in our sIPSC recordings. However, to fully interpret our findings, it would be important to establish the source(s) of GABAergic inputs to NPY interneurons as well as their degree of interconnectivity.
As indicated in introduction, there are two main subpopulations of NPY interneurons in the striatum, which could not be distinguished for technical reasons in this study. Although LTS-NPY interneurons represent the majority, it is statistically likely that our sample also includes some NPY-NGF interneurons. By analyzing our recordings, we could not find criteria for a possible post hoc subclassification into two distinct groups. Therefore, we surmise that both subpopulations receive GABAergic inputs to a similar degree, although the source of this innervation (MSN, interneurons, extrinsic inputs) could be different. If we assume the changes underlying the increased frequency of mIPSCs and sIPSCs to be presynaptic (increased number of release sites), then these changes likely affect both subpopulations of NPY interneurons.
On the system level, our results suggest that altered MSN-interneuron as well as interneuron-interneuron interconnections upon DA depletion either contribute to the imbalance of direct and indirect pathways in the DA depleted striatum or represent functional compensations. Several studies have shown an upregulation of NPY-expressing neurons in tissue from PD patients and upon DA depletion (Abrous et al. 1994; Cannizzaro et al. 2003; Ma et al. 2014; Schwarting and Huston 1996), as well as a neuroprotective function of NPY in animal models of PD (Decressac et al. 2012; Duarte-Neves et al. 2016; Pain et al. 2019). It was hypothesized that a decrease in D1 tonic control is involved in the upregulation of NPY mRNA expression in the DA-depleted striatum (Cannizzaro et al. 2003), but also corticostriatal and nigrostriatal pathways have been considered to regulate NPY-expressing striatal neurons (Cannizzaro et al. 2003; Kerkerian et al. 1988; Salin et al. 1994). For example, blocking NMDA receptors in DA-depleted brains led to a reduction of 6-OHDA-induced upregulation of NPY-positive neurons (Salin et al. 1994). Hence, one might speculate that excitatory transmission from cortical neurons onto NPY interneurons is increased upon the loss of DA. The increased IPSCs in NPY-positive cells observed here could therefore represent a compensatory mechanism to limit their overexcitation. A possible increase in glutamatergic corticostriatal transmission in the DA-depleted striatum was already suggested by Cannizzaro et al. (2003).
The main finding of this study is that 6-OHDA-induced degeneration of DA terminals in the striatum causes functional changes in GABAergic synapses in the striatum. The underlying mechanisms are unknown but might be related to altered signaling cascades controlled by D1 and/or D2 receptors and regulating GABAA receptor expression, trafficking, or posttranslational regulation, as well as regulating synapse formation and plasticity. The alterations shown here for NPY interneurons, which are known to have a low connection probability with MSN, suggest that DA depletion induces widespread changes in striatal GABAergic function, which might underlie major pathological dysfunctions in PD.
GRANTS
This research was supported by Swiss National Science Foundation Grant 310030-166130 to J.-M.F.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
L.R. and J.-M.F. conceived and designed research; L.R. performed experiments; L.R. analyzed data; L.R. and J.-M.F. interpreted results of experiments; L.R. prepared figures; L.R. drafted manuscript; L.R. and J.-M.F. edited and revised manuscript; L.R. and J.-M.F. approved final version of manuscript.
ACKNOWLEDGMENTS
We thank the animal caretakers of the Laboratory Animal Service Center of the University of Zurich for the maintenance of mouse colonies and identification of hrGFP-expressing mice and Cornelia Schwerdel for expert technical assistance.
REFERENCES
- . The increase in striatal neuropeptide Y immunoreactivity induced by neonatal dopamine-depleting lesions in rats is reversed by intrastriatal dopamine-rich transplants. Brain Res 656: 169–173, 1994. doi:10.1016/0006-8993(94)91379-X.
Crossref | PubMed | ISI | Google Scholar - . Cell type-specific distribution of GABAA receptor subtypes in the mouse dorsal striatum. J Comp Neurol 527: 2030–2046, 2019. doi:10.1002/cne.24665.
Crossref | PubMed | ISI | Google Scholar - . Increased neuropeptide Y mRNA expression in striatum in Parkinson’s disease. Brain Res Mol Brain Res 110: 169–176, 2003. doi:10.1016/S0169-328X(02)00555-7.
Crossref | PubMed | Google Scholar - . Mechanism of dopamine mediated inhibition of neuropeptide Y release from pheochromocytoma cells (PC12 cells). Biochem Pharmacol 73: 1446–1454, 2007. doi:10.1016/j.bcp.2007.01.003.
Crossref | PubMed | ISI | Google Scholar - . Activation of dopamine D1-like receptors excites LTS interneurons of the striatum. Eur J Neurosci 15: 2049–2052, 2002. doi:10.1046/j.1460-9568.2002.02052.x.
Crossref | PubMed | ISI | Google Scholar - . Neuroprotection by neuropeptide Y in cell and animal models of Parkinson’s disease. Neurobiol Aging 33: 2125–2137, 2012. doi:10.1016/j.neurobiolaging.2011.06.018.
Crossref | PubMed | ISI | Google Scholar - . Dopamine-deprived striatal GABAergic interneurons burst and generate repetitive gigantic IPSCs in medium spiny neurons. J Neurosci 29: 7776–7787, 2009. doi:10.1523/JNEUROSCI.1527-09.2009.
Crossref | PubMed | ISI | Google Scholar - . Postsynaptic gephyrin clustering controls the development of adult-born granule cells in the olfactory bulb. J Comp Neurol 523: 1998–2016, 2015. doi:10.1002/cne.23776.
Crossref | PubMed | ISI | Google Scholar - . Neuropeptide Y (NPY) as a therapeutic target for neurodegenerative diseases. Neurobiol Dis 95: 210–224, 2016. doi:10.1016/j.nbd.2016.07.022.
Crossref | PubMed | ISI | Google Scholar - . Modulation of striatal projection systems by dopamine. Annu Rev Neurosci 34: 441–466, 2011. doi:10.1146/annurev-neuro-061010-113641.
Crossref | PubMed | ISI | Google Scholar - . Nature of inhibition of mitochondrial respiratory complex I by 6-hydroxydopamine. J Neurochem 66: 2004–2010, 1996. doi:10.1046/j.1471-4159.1996.66052004.x.
Crossref | PubMed | ISI | Google Scholar - . A novel functionally distinct subtype of striatal neuropeptide Y interneuron. J Neurosci 31: 16757–16769, 2011. doi:10.1523/JNEUROSCI.2628-11.2011.
Crossref | PubMed | ISI | Google Scholar - . Striatal neuropeptide Y neurones are under the influence of the nigrostriatal dopaminergic pathway: immunohistochemical evidence. Neurosci Lett 66: 106–112, 1986. doi:10.1016/0304-3940(86)90174-6.
Crossref | PubMed | ISI | Google Scholar - . Pharmacological characterization of dopaminergic influence on expression of neuropeptide Y immunoreactivity by rat striatal neurons. Neuroscience 26: 809–817, 1988. doi:10.1016/0306-4522(88)90101-7.
Crossref | PubMed | ISI | Google Scholar - . Inhibitory control of neostriatal projection neurons by GABAergic interneurons. Nat Neurosci 2: 467–472, 1999. doi:10.1038/8138.
Crossref | PubMed | ISI | Google Scholar - . Free radical-generated neurotoxicity of 6-hydroxydopamine. J Neurochem 64: 1703–1707, 1995. doi:10.1046/j.1471-4159.1995.64041703.x.
Crossref | PubMed | ISI | Google Scholar - . Neuropeptide gene expression in brain is differentially regulated by midbrain dopamine neurons. Exp Brain Res 80: 489–500, 1990. doi:10.1007/BF00227990.
Crossref | PubMed | ISI | Google Scholar - . The effects of unilateral 6-OHDA lesion in medial forebrain bundle on the motor, cognitive dysfunctions and vulnerability of different striatal interneuron types in rats. Behav Brain Res 266: 37–45, 2014. doi:10.1016/j.bbr.2014.02.039.
Crossref | PubMed | ISI | Google Scholar - . Interneurons of the neocortical inhibitory system. Nat Rev Neurosci 5: 793–807, 2004. doi:10.1038/nrn1519.
Crossref | PubMed | ISI | Google Scholar - . Diversity of interneurons in the dorsal striatum revealed by single-cell RNA sequencing and PatchSeq. Cell Rep 24: 2179–2190.e7, 2018. doi:10.1016/j.celrep.2018.07.053.
Crossref | PubMed | ISI | Google Scholar - . A protocol for concurrent high-quality immunohistochemical and biochemical analyses in adult mouse central nervous system. Eur J Neurosci 39: 165–175, 2014. doi:10.1111/ejn.12447.
Crossref | PubMed | ISI | Google Scholar - . Inflammatory process in Parkinson disease: neuroprotection by neuropeptide Y. Fundam Clin Pharmacol 33: 544–548, 2019. doi:10.1111/fcp.12464.
Crossref | PubMed | ISI | Google Scholar - . Dopaminergic regulation of pre-proNPY mRNA levels in the rat arcuate nucleus. Neurosci Lett 127: 96–98, 1991. doi:10.1016/0304-3940(91)90903-7.
Crossref | PubMed | ISI | Google Scholar - . Molecular phenotype of rat striatal neurons expressing the dopamine D5 receptor subtype. Eur J Neurosci 16: 2049–2058, 2002. doi:10.1046/j.1460-9568.2002.02280.x.
Crossref | PubMed | ISI | Google Scholar - . Neurogenesis and stereological morphometry of calretinin-immunoreactive GABAergic interneurons of the neostriatum. J Comp Neurol 469: 325–339, 2004. doi:10.1002/cne.11008.
Crossref | PubMed | ISI | Google Scholar - . Mechanisms of action of 6-hydroxydopamine. Biochem Pharmacol 24: 1–8, 1975. doi:10.1016/0006-2952(75)90304-4.
Crossref | PubMed | ISI | Google Scholar - . Reversal of the adaptive response of neuropeptide Y neurons in the rat striatum to nigrostriatal dopamine deafferentation by the N-methyl-d-aspartate antagonist dizocilpine maleate. Neuroscience 61: 93–105, 1994. doi:10.1016/0306-4522(94)90063-9.
Crossref | PubMed | ISI | Google Scholar - . Unilateral 6-hydroxydopamine lesions of meso-striatal dopamine neurons and their physiological sequelae. Prog Neurobiol 49: 215–266, 1996. doi:10.1016/S0301-0082(96)00015-9.
Crossref | PubMed | ISI | Google Scholar - GABAergic interneurons of the striatum. In: Handbook of Basal Ganglia Structure and Function, edited by Steiner H, Tseng K. London: Elsevier, 2016, p. 157–178.
Crossref | Google Scholar - . Heterogeneity and diversity of striatal GABAergic interneurons. Front Neuroanat 4: 150, 2010. doi:10.3389/fnana.2010.00150.
Crossref | PubMed | ISI | Google Scholar
