Electrophysiological and Morphological Evidence for a Gabaergic Nigrostriatal Pathway

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The Journal of Neuroscience, June 1, 1999, 19(11):4682–4694

Electrophysiological and Morphological Evidence for a GABAergic Nigrostriatal Pathway

Manuel Rodrı´guez1 and Toma´ s Gonza´ lez-Herna´ ndez2 Departments of 1Physiology and 2Anatomy, Faculty of Medicine, University of La Laguna, La Laguna, Tenerife, Canary Islands, Spain

The electrophysiological and neurochemical characteristics of gic neurons, a percentage that reached 81–84% after 6-OHDA the nondopaminergic nigrostriatal (NO-DA) cells and their func- injection. Electrophysiologically, NO-DA cells showed a behavior tional response to the degeneration of dopaminergic nigrostri- similar to that found in other nigral GABAergic (nigrothalamic) atal (DA) cells were studied. Three different criteria were used to cells. In addition, the 6-OHDA degeneration of DA cells induced a identify NO-DA cells: (1) antidromic response to striatal stimu- modiﬁcation of their electrophysiological pattern similar to that lation with an electrophysiological behavior (ﬁring rate, inter- found in GABAergic nigrothalamic neurons. Taken together, the spike interval variability, and conduction velocity) different from present data indicate the existence of a small GABAergic nigro- that of DA cells; (2) retrograde labeling after striatal injection of striatal pathway and suggest their involvement in the pathophys- HRP but showing immunonegativity for DA cell markers (ty- iology of Parkinson’s disease. rosine hydroxylase, calretinin, calbindin-D28k, and cholecystokinin); and (3) resistance to neurotoxic effect of 6-hydroxydomine Key words: nigrostriatal pathway; dopaminergic cells; (6-OHDA). Our results showed that under normal conditions, GABAergic cells; GABA; glutamic acid decarboxylase; parval- 5–8% of nigrostriatal neurons are immunoreactive for GABA, glu- bumin; calretinin; calbindin-D28k; cholecystokinin; Parkinson’s tamic acid decarboxylase, and parvalbumin, markers of GABAer- disease

The substantia nigra (SN) is a major output center of the basal paminergic (Grace and Bunney, 1980, 1983a). Most studies have ganglia (DeLong, 1990; Gerfen et al., 1990; Graybiel, 1990; been focused on this neuron type (Grace and Bunney, 1980, Obeso et al., 1997), participating in their sensory-motor functions 1983a–c, 1984a,b, 1985a,b; DeLong et al., 1983; Schwarz et al., (DeLong et al., 1983; Hikosaka and Wurtz, 1983a–c; Schwarz et 1984; Schultz, 1986; Chiodo, 1988; Schultz and Romo, 1990), al., 1984; Schultz, 1986). This, together with its direct implication because they are probably conditioned by their direct involvement on the physiopathology of Parkinson’s disease (Albin et al., 1989; in various neurological disorders, including Parkinson’s disease DeLong, 1990; Graybiel, 1990; Obeso et al., 1997; Viteck et al., (Ehringer and Hornykiewicz, 1960), whereas the higher conduc- 1997), has increased the interest in studying this mesencephalic tion velocity neurons have remained practically unexplored. center. The SN is composed primarily of two projection neurons. The main aim of this study was the electrophysiological and Dopaminergic (DA) cells, located in the pars compacta (SNc) and neurochemical characterization of nondopaminergic nigroestria- pars reticulata (SNr) and projecting to the striatum, and GABA tal (NO-DA) cells. Before analyzing their electrophysiological cells, located in the pars reticulata and projecting to the thalamus, behavior, SN cells were classified as nigrostriatal or nigrothalamic superior colliculus, and pedunculopontine nucleus (Dahlstro¨m according to their antidromic response (Guyenet and Aghajanian, and Fuxe 1964; Kilpatrick et al., 1980; Childs and Gale, 1983; Chiodo, 1988; Hedreen and DeLong, 1991; Parent and Hazrati, 1978; Grace and Bunney, 1980, 1983a–c; Ruffieux and Schultz, 1995; Hazrati, 1995a,b). Some preliminary evidence suggests 1980; Sanderson et al., 1986; Chiodo, 1988; MacLeod et al., 1990; that, besides the dopaminergic nigrostriatal system, there are Castellano and Rodrı´guez, 1991; Castellano et al., 1993). In the nondopaminergic nigral cells projecting to the striatum. Thus, not neurochemical study, nigrostriatal neurons were first identified by all SN cells projecting to the striatum show catecholamine fluo- the retrograde transport of horseradish peroxidase (HRP) from rescence (van der Kooy et al., 1981) or tyrosine hydroxylase the striatum. Then they were classified as DA or GABA cells immunoreactivity (Swanson, 1982; Gerfen et al., 1987). Early using different neurochemical markers. For DA cells, besides electrophysiological studies classified nigrostriatal cells as belong- tyrosine hydroxylase (TH, the rate-limiting enzyme in the DA ing to slow conduction velocity (0.5 m/sec) pars compacta neurons synthesis), we used the peptide cholecystokinin (CCK; Ho¨kfelt et and higher conduction velocity (1.7 m/sec) pars reticulata neurons al., 1980, 1985; Chiodo, 1988; Freeman and Chiodo, 1988) and the (Deniau et al., 1978; Guyenet and Aghajanian, 1978). The slow calcium-binding proteins calretinin (CR) and calbindin (CB; conduction velocity neurons were subsequently identified as do- Goodman et al., 1979; Rogers, 1987; Yamada et al., 1990; Heiz- mann and Hunziker, 1991; Iacopino et al., 1994; Liang et al., Received Dec. 28, 1998; revised March 5, 1999; accepted March 24, 1999. 1996), three substances contained in DA neurons (Re´sibois and This work was supported by Ministerio de Sanidad y Consumo, FIS, Grant 98/1499) and Gobierno Autonomo de Canarias Grant PI1998/008, Spain. Rogers, 1992; Liang et al., 1996; McRitchie et al., 1996). For Correspondence should be addressed to Manuel Rodrı´guez, Department of Phys- GABA cells, besides GABA and glutamic acid decarboxylase iology, Faculty of Medicine, University of La Laguna, La Laguna, Tenerife, Canary Islands, Spain. (GAD, the synthesizing enzyme for GABA), we used parvalbu- Copyright © 1999 Society for Neuroscience 0270-6474/99/194682-13$05.00/0 min (PV), a calcium-binding protein that, in the SN, is expressed Rodrı´guez and Gonza´ lez-Herna´ ndez • GABAergic Nigrostriatal Pathway J. Neurosci., June 1, 1999, 19(11):4682–4694 4683

by and restricted to GABAergic neurons (Reiner and Anderson, striatal stimulation, mostly consisting of the initial segment component of 1993; Rajakumar et al., 1994). the action potential. GABAergic nigrothalamic neurons were identified by their biphasic extracellular wave form (often showing a duration of Ͻ1 In addition, taking into account experimental (Arbuthnott, msec) and a short-latency antidromic response (a conduction velocity of 1974; Ruffieux and Schultz, 1980; Sanderson et al., 1986; Mitchell Ͼ1 m/sec) evoked from the ventromedial nucleus of the thalamus. el al., 1989; DeLong, 1990; MacLeod et al., 1990; Robledo and The basal activity of neurons was recorded 10 min after their anti- Fe´ger, 1992; Herrero et al., 1993; Burbaud et al., 1995) and dromic identification. Extracellular signals were simultaneously digitized clinical (Bernheimer et al., 1973; Conde´, 1992) data suggesting on a Pentium-based computer by using a 16 bit analog-to-digital con- verter (LTI-C30; Madrid, Spain), recorded on magnetic tape for off-line that some Parkinsonian motor disturbances, acquired after the analysis, and displayed on a storage oscilloscope. Only recordings with destruction of DA cells, are induced by a functional modification single-unit activity were used. Action potentials were considered as in SN GABAergic cells projecting to the thalamus, we have also displayed by the same neuron when the spike shape, analyzed with both studied the effect of experimentally produced degeneration of hardware (SD1 spike discriminator; Tucker–Davis Technologies, DA cells on the electrophysiological behavior of NO-DA cells. Gainesville, Florida) and software (hybrid multilayer artificial neural network; Garcia et al., 1998) procedures, did not change during the recording session. The firing rate (number of spikes per second) and MATERIALS AND METHODS variation coefficient of interspike intervals (VC; SD of interspike intervals/mean of interspike intervals) were computed for a recording time of Animals. Experiments were performed on male Sprague Dawley rats at least 4 min. (300–350 gm; Panlab, Barcelona, Spain). Animals were housed at 22°C, At the end of each experiment, the recording sites were marked by two per cage, under normal laboratory conditions on a standard 12 hr iontophoretic injection of Pontamine Sky Blue (Ϫ20 ␮A, 30 min), and light/dark schedule (with lights on from 3 A.M. to 3 P.M.) and with free the stimulus sites were marked by means of an iron deposited by passing access to food and water. Experimental protocols were in accordance a current of 10 ␮A for 30 sec. Rats were perfused with 0.9% saline and with the European Community Council Directive of November 24, 1986 1% potassium ferricyanide in 10% formaldehyde. Brains were post-fixed (86/609/EEC) regarding the care and use of animals for experimental in the same solution for 6–10 hr and cryoprotected overnight in 30% procedures. Adequate measures were also taken to minimize pain and ␮ discomfort. sucrose at 4°C. Serial sections (50 m thick) were obtained with a A group of animals were lesioned using a procedure that induces a freezing microtome and stained with formal thionine. marked and selective destruction of DA neurons (Ho¨kfelt and Unger- Morphological studies. These studies were performed by combining stedt, 1973; Castro et al., 1985). Under anesthesia (80 mg/kg ketamine retrograde transport of HRP and immunocytochemistry for different plus 12 mg/kg xylazine, i.p.), rats received a double unilateral injection markers of nigral neurons. Under anesthesia (80 mg/kg ketamine and 12 mg/kg xylazine, i.p.) and aseptic conditions, rats received a single injec- of 6-hydroxydopamine hydrochloride (6-OHDA; Sigma, St. Louis, MO; ␮ 8 ␮gin4␮l of a 0.9% saline solution with 0.3 ␮g/␮l ascorbic acid/ tion of 0.2–0.4 l of 40% HRP (Boehringer Mannheim, Mannheim, injection; 1 ␮l/min) or vehicle in the medial forebrain bundle (Castro et Germany) in the striatum 42–68 hr before killing. Given that under al., 1985; Burunat et al., 1988; Barroso and Rodrı´guez, 1996), according normal conditions the soma concentrations of some neuropeptides (i.e., to the stereotaxic coordinates of Paxinos and Watson (1988): 4.0 mm CCK) and enzymes (i.e., GAD) are so low that they are difficult to detect ␭ with immunocytochemistry, a group of rats were injected in the lateral anterior to , 1.6 mm to the right of the midline, and 8 mm below the dura ␮ (first injection); and 4.6 mm anterior to ␭, 1.4 mm to the right of the ventricle (ipsilateral to the HRP injection) with 10 l of colchicine (10 midline, and 8 mm below the dura (second injection). The behavioral mg/ml, Sigma) dissolved in saline 24 hr before killing. This is a success- evaluation of lesion degree (Castro et al., 1985) was performed 2–3 ful procedure to block the axonal transport, increasing the immunore- weeks later. Animals showing more than eight turns per minute in activity in somata (Fallon et al., 1983; Seroogy et al., 1989; Campbell et response to apomorphine hydrochloride (1 mg/kg, i.p.; Sigma) were al., 1991). Rats were heavily anesthetized and transcardially perfused included in the study. with 0.9% saline and a fixative solution of 1% paraformaldehyde and Electrophysiological studies. Unlesioned, sham-lesioned, and 6-OHDA 1.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were lesioned (4–6 weeks after lesion) rats were anesthetized with chloral removed and stored in the same fixative at 4°C for 4–12 hr. Midbrains hydrate (400 mg/kg, i.p.), and the extracellular activity of SN neurons were initially obtained by using a brain blocker (Activational System, ␮ was monitored according to the procedure of Castellano and Rodrı´guez Warren, MI) and then cut at 30 m with a vibratome. Sections were (1991). Recordings (2.8–3.4 mm anterior to ␭, 1.8–2.4 mm lateral to the collected in parallel series, treated for inhibition of endogenous peroxi- midline, and 7–8.5 mm below the cortical surface) were obtained from dase with 3% H2O2 , and processed for double labeling. First, they were Ј rats kept between 36.5 and 37.0°C by using electrodes (glass tubing filled processed for histochemical localization of HRP by using 3,3 - with 2 M NaCl containing 2% Pontamine Sky Blue; BDH Chemicals, diaminobenzidine (DAB, Sigma) intensified by cobalt chloride (Sigma; Poole, England) with a 6–9 m⍀ impedance (at 1000 Hz). The signal was Adams, 1977) or nickel amonium sulfate (Serva, Heidelberg, Germany; amplified (3000ϫ) and filtered (200–5000 Hz) in a high input impedance Adams, 1981) as chromogen, which produces a black granular reaction differential amplifier (CAN96T; Telcan, Tenerife, Spain). product, and thereafter for immunocytochemical detection of distinct To identify nigrostriatal and nigrothalamic cells (Castellano et al., markers (one series per marker) using nonintensified DAB, which pro- 1993; Rodrı´guez and Barroso, 1996), stimulating electrodes were placed duces a brown diffuse reaction product. in the head of the caudate nucleus (0.0 mm anterior to bregma, 3.2 mm As primary antibodies we used a mouse anti-TH monoclonal antibody to the right of the midline, and 5 mm ventral to the brain surface) and in (1:8000, Sigma), a mouse anti-GABA monoclonal antibody (1:12,000; the ventral thalamus (2.5 mm posterior to bregma, 1.5 mm to the right of Matute and Streit, 1986), a rabbit anti-GAD polyclonal antibody (1:2000; the midline, and 6.5–7 mm ventral to the brain surface). The collision test Chemicon, Temecula, CA), a rabbit anti-CCK-8 polyclonal antibody was applied to determine the antidromic activation from these nuclei, (1:12000, Sigma), a mouse anti-CB monoclonal antibody (1:1000, Sigma), using a Grass (West Warwick, RI) S-8800 and a bipolar stimulating a rabbit anti-CR polyclonal antibody (1:5000, Chemicon), and a mouse electrode made with stainless steel rods (250 ␮m diameter; A-M Systems, anti-PV monoclonal antibody (1:6000, Sigma). After HRP histochemis- Everet, WA). Two rods were electroetched with a 60 Hz alternating try, sections were washed in 0.1 M PBS, pH 7.4, and immersed for 60 min current [1:1 (v/v) 96% sulfuric acid/85% phosphoric acid] and insulated in the preincubation solution (PIS): 3% normal goat serum (NGS; Vector with Sylgar 184 (Dow Corning, Wiesbaden, Germany), and their conduc- Laboratories, Burlingame, CA) for GAD, CCK, and CR, or 3% normal tive tips (0.5 mm) were placed 500 ␮m apart. Neurons were considered horse serum (NHS, Vector Laboratories) for TH, GABA, CB, and PV; antidromically activated (0.5 mA/0.3 msec square pulse) when they and 0.5% BSA (Serva) and 0.1% Triton X-100 (TX-100, Sigma) in PBS. showed (1) an antidromic spike per stimulus and a fixed latency from the Sections were then incubated overnight in the primary antibody dissolved stimulus artifact (Ͻ0.05 msec) during high-rate stimulation (50 Hz) and in PIS. In GAD and GABA immunocytochemistry TX-100 was omitted. (2) a collision between the spontaneously occurring action potentials and Sections were washed several times in PBS and incubated for 60 min in the stimulation-elicited spikes. DA neurons were identified by a 2–5 msec 1:200 biotinylated goat anti-rabbit antiserum (for GAD, CCK, and CR; triphasic extracellular wave form (with a positive first phase), often Vector Laboratories) or 1:200 biotinylated horse anti-mouse antiserum displaying a prominent notch (initial segment–somatodendritic break) in (for TH, GABA, CB, and PV; Vector Laboratories) and either 1:200 the initial positive rising, a spontaneous activity Ͻ10 spikes/sec, and a NGS or 1:200 NHS in PBS. Immunoreactions were visualized by incu- long-latency antidromic response (conduction velocity of ϳ0.5 m/sec) to bation for 2 hr in ExtrAvidin (1:5000, Sigma) in PBS and for 10 min in 4684 J. Neurosci., June 1, 1999, 19(11):4682–4694 Rodrı´guez and Gonza´ lez-Herna´ ndez • GABAergic Nigrostriatal Pathway

0.005% DAB (Sigma) and 0.001% H2O2 in cacodylate buffer. After Thus, the conduction velocity was different in the three cell several rinses in PBS, sections were mounted on gelatinized slides, groups (ANOVA, F ϭ 42.19; p Ͻ 0.0001), with the nigrothalamic dehydrated, cleared in xylene, and coverslipped with DPX (BDH). Con- neurons showing values lower than those of NO-DA cells ( p Ͻ trol experiments to demonstrate the specificity of the immunolabeling Ͻ were performed by removing the primary or secondary antibodies, re- 0.0001) and higher than those of DA neurons ( p 0.0001). sulting in negative staining. Statistical differences were also found for firing rate and VC The percentage of nigrostriatal neurons that were immunostained for (Fig. 1h). Both NO-DA ( p Ͻ 0.0001) and nigrothalamic ( p Ͻ the different markers was computed by counting the number of double- 0.0001) cells showed a firing rate higher than that found in DA labeled neurons in a total of 900 HRP-positive cells per series in each ϭ Ͻ unlesioned rat [700 in SN, 160 in ventral tegmental area (VTA), and 40 cells (ANOVA, F 67.42; p 0.0001), with no difference in retrorubral field (RRF)] and 70 HRP-positive cells per series in each between NO-DA and nigrothalamic neurons ( p ϭ 0.43). VC also lesioned rat (all then in SN). In each series, HRP-positive neurons were showed higher values in NO-DA ( p Ͻ 0.01) and nigrothalamic ␮ randomly elected in 10 sections separated by a distance of 210 m from ( p Ͻ 0.0001) cells than in DA cells (ANOVA, F, ϭ 15.34; p Ͻ each other and taken from rostral SN to caudal RRF. The quantitative 0.0001), with no statistical difference between NO-DA and nigro- study was performed with the aid of a Magiscan image analysis system ϭ using the Genias program (Joyce-Loebl) and dividing the SN, VTA, and thalamic cells ( p 0.51). Whereas a group of DA and nigrotha- RRF into fields of 400 ϫ 400 ␮m at a magnification of 200ϫ. Taking into lamic neurons showed a bursting activity, most NO-DA cells account that antisera only penetrate 8–10 ␮m from the surface, whereas presented a regular tonic discharge, and only a few of them HRP-positive neurons are evident at all depths of the section, we have displayed irregular discharges. only counted those single- and double-labeled neurons exposed on the surface of sections, excluding diffusely HRP-labeled cells. Statistical comparisons. The statistical analyses were performed using a Experiment 2: morphological and neurochemical one-way ANOVA followed by appropriate post hoc tests (Statistica; identification of nondopaminergic nigrostriatal cells Ͻ Statsoft, Tulsa, OK). In all cases, a level of p 0.05 was considered Although as expected, after HRP injection in the striatum, most critical for assigning statistical significance. Experiment 1: electrophysiological identification of nondopaminergic ni- HRP-positive neurons in the basal midbrain were immunoposi- grostriatal cells. The extracellular electrophysiological activity of 374 tive for TH, a significant number of them were TH-negative nigral cells was recorded in 33 unlesioned rats. Electrical stimulation of (15.3%). They were preferentially located in the SNr (Figs. 2, 3), the head of the caudate nucleus and ventral thalamus was used for in a lower proportion in VTA, and only a few of them in RRF identifying the target of recorded neurons. Multiunitary recordings were (Fig. 3). excluded, and only cells showing antidromic response to striatal (n ϭ 142) or thalamic (n ϭ 62) stimulation were finally included. As mentioned above, to identify these nigrostriatal neurons Experiment 2: morphological and neurochemical identification of non- neurochemically, alternate sections were immunostained with dif- dopaminergic nigrostriatal cells. To identify cells projecting to the stria- ferent nigral neuron markers. In this experiment, we focused on tum, 12 rats were injected in the caudate nucleus with HRP. Two days GABAergic neuron markers, using GAD, GABA, and PV immu- later, 6 of them were injected in the lateral ventricle with colchicine. After2dinthecase of the colchicine-untreated rats and3dinthecase nocytochemistry in both colchicine-treated and untreated rats. of colchicine-treated rats, they were heavily anesthetized, perfused, and Double-labeled neurons were found in both colchine-treated processed for the histochemical and immunocytochemical studies. and untreated rats (Fig. 4). The number of the GAD–HRP- Experiment 3: effect of dopaminergic nigrostriatal cells degeneration on positive cells (Fig. 4a–c) was slightly higher in the colchicine- nondopaminergic nigrostriatal neurons. Forty-four rats were used to eval- treated rats, in which they reached levels of 5.2% of the total uate the effects of DA cell degeneration on nondopaminergic nigrostriatal and nigrothalamic neurons. Two to 3 weeks after 6-OHDA (n ϭ 22) number of HRP-positive cells. In untreated rats, this proportion or vehicle (n ϭ 22) injections, rats were behaviorally tested to determine was 3.8%. The number of GABA–HRP-positive neurons (Fig. the lesion degree. Twenty to 40 d later they were anesthetized for the 4d,e) was 1.5% of the total number of HRP neurons. PV–HRP- electrophysiological (16 sham and 16 6-OHDA-injected rats) or neuro- positive neurons (Fig. 4f) displayed the largest proportion of chemical (6 sham and 6 6-OHDA-injected rats) studies, following the same protocols used in experiments 1 and 2, but in this experiment double-labeled neurons (7.7%). The percentage of GABA–HRP- colchicine was not used. and PV–HRP-labeled cells did not change after colchicine injection. RESULTS The distribution pattern of double-labeled neurons was very Experiment 1: electrophysiological identification of similar to that of single HRP-stained neurons in material pro- nondopaminergic nigrostriatal cells cessed for TH with the largest number of GABA-projecting During this experiment 123 DA cells, 19 NO-DA cells, and 62 (77.8%), GAD-projecting (74.3%), and PV-projecting (80.2%) nigrothalamic cells were recorded. DA cells showed the charac- neurons localized in the SNr (Fig. 4a,b,d,e,f), only a few of them teristic triphasic action potential with the first phase being posi- in VTA (Fig. 4c), and none in the SNc and RRF. Morphologi- tive (often displaying a notch on the initial rising) and a duration cally, they constitute a polymorphic population of elongated and of 2–6 msec (Fig. 1c). The DA cells showed a stable antidromic round cells, with their largest diameter ranging between 15 and 23 latency (oscillations Ͻ0.05 msec) and were identified as nigrostri- ␮m. Although the aim of this study was not to investigate the atal neurons by the collision test (Fig. 1f). The conduction veloc- topography of this projection, we observed that the localization of ity was 0.52 Ϯ 0.023 m/sec (mean Ϯ SE; Fig. 1h) with an double-labeled cells in the SNr varied depending on the injection antidromic latency of 7–20 msec (Fig. 1g). NO-DA cells showed a site in striatum. As shown in Figure 5, neurons projecting to the biphasic short-duration (often Ͻ1 msec) spike with a positive first medial part of striatum localize in medial regions of the SNr, and phase (Fig. 1a) and an antidromic response to striatal stimulation those projecting to the lateral part of striatum localize in the with a positive collision test (Fig. 1d). The conduction velocity lateral part of the SNr. In the rostrocaudal axis, this topographic was 3.48 Ϯ 1.80 m/sec (Fig. 1h) with an antidromic latency of relation was not as evident as in the mediolateral one, because 0.5–5 msec (Fig. 1g). Nigrothalamic neurons displayed a charac- after injection in any place in the striatum, double-labeled cells teristic biphasic action potential with a positive first phase and a were present at any rostrocaudal level of the SNr. However, after duration of Ͻ1 msec (data not shown). The thalamic stimulation rostral injections, the largest number of double-labeled cells was induced a stable antidromic response with a positive collision test in the rostral half of the SNr, and after caudal injections, they (Fig. 1e) and a conduction velocity of 1.65 Ϯ 1.03 m/sec (Fig. 1h). localize perferentially in caudal regions of the SNr. Rodrı´guez and Gonza´ lez-Herna´ ndez • GABAergic Nigrostriatal Pathway J. Neurosci., June 1, 1999, 19(11):4682–4694 4685

Figure 1. Electrophysiological identiﬁcation of NO-DA, DA, and nigrothalamic cells. An example of the spike shape (a–c) and collision test (d–f)in NO-DA, nigrothalamic, and DA cells, respectively, is shown. The antidromic response consisted of the full spike in NO-DA cells (d) and nigrothalamic cells (e) and the initial segment in DA cells (f ). Because the antidromic stimulation of DA cells often displays the initial segment but not the somatodendritic component of the spike (spike dissociation), the antidromic spike shown at the top of f is clearly different (somatodendritic component virtually absent) from the spontaneous spike showed at the bottom of f. g, Antidromic latency histogram of nigrostriatal cells. Values are expressed as a percentage of the total number of NO-DA (n ϭ 19) or DA (n ϭ 123) recorded neurons. h, Basal activity and conduction velocity of DA and NO-DA nigrostriatal (NS) and nigrothalamic (NT) cells. *p Ͻ 0.0001 versus NS DA cells; **p Ͻ 0.01 versus NS DA cells. °p Ͻ 0.0001 versus NT cells. Arrows in c and d indicate the initial part of the striatal stimulus artifact. Values represent the mean Ϯ SE. 4686 J. Neurosci., June 1, 1999, 19(11):4682–4694 Rodrı´guez and Gonza´ lez-Herna´ ndez • GABAergic Nigrostriatal Pathway

Figure 3. Mesencephalic distribution of cells projecting to the striatum according to their tyrosine hydroxylase immunoreactivity (n ϭ 10,800 HRP cells). TH (ϩ), Tyrosine hydroxylase-immunopositive cells; TH (Ϫ), tyrosine hydroxylase-immunonegative cells.

onstrated by CCK (see Fig. 8), CR (Figs. 6e,f, 8) and CB (Figs. 6g,h, 8) immunocytochemistry. By comparing the number of double labeled neurons in sham and lesioned rats (Fig. 7), we found that after lesion, the percentage of HRP cells showing immunoreactivity for TH decreased from 82.3 to 1.2%, for CCK from 61.4 to 0.9%, for CB from 21.1 to 1.6%, and for CR from 86.1 to 1.6%. Despite the massive degeneration of DA cells, a number of retrogradely labeled neurons survived the neurotoxic effect of 6-OHDA (Fig. 6a,c,d), most of them showing immunoreactivity for different GABA cell markers (Figs. 7, 8). Thus, when compared with the sham group, the percentage of HRP cells showing immunoreactivity for GABA (Fig. 7b) increased from 1.2 to 84.3%, those showing immunoreactivity for GAD (Fig. 7a)increased from 4.9 to 81.3%, and those showing immunoreactivity for PV (Fig. 7c,d) increased from 8.3 to 83.0%. The electrophysiological study showed a functional modification of NO-DA and nigrothalamic cells after the 6-OHDA degeneration of DA neurons (Fig. 9). Although no statistical differences were found in the firing rate ( p ϭ 0.64) and conduction velocity ( p ϭ 0.45), NO-DA cells showed a higher VC in the 6-OHDA lesioned than in the sham-lesioned rats ( p Ͻ 0.05). This effect was similar to that found in the nigrothalamic GABAergic neurons, with no modification in the firing rate ( p ϭ 0.45) and conduction velocity ( p ϭ 0.22) but with an increase in the VC ( p Ͻ 0.05). Figure 2. HRP-TH double labeling after HRP injection in the striatum of unlesioned rats. a, Rostrolateral region of the SN; b, boxed area in a; c, DISCUSSION caudomedial region of the SN. Arrows in b and c indicate single HRP- stained neurons in focus with double-labeled neurons (arrowheads). Scale In this study we present evidence supporting the existence of bars: a, 200 ␮m; b, c,50␮m. GABAergic nigrostriatal cells with electrophysiological and neurochemical characteristics similar to those of GABAergic nigro- Experiment 3: effect of dopaminergic nigrostriatal thalamic cells but different from DA nigrostriatal neurons. After cells degeneration on nondopaminergic 6-OHDA injection, which produced the disappearance of the DA nigrostriatal neurons neurons, these cells remained intact, showing a change in the The injection of 6-OHDA into the very center of the medial electrophysiological behavior similar to that found in nigrotha- forebrain bundle led to the degeneration of practically all nigral lamic cells. Taken together, present findings indicate the exis- DA-neurons ipsilateral to injection (Figs. 6a,b, 8), affecting their tence of a GABAergic nigrostriatal pathway and suggest its in- different neurochemical subpopulations in the same way, as dem- volvement in the pathophysiology of Parkinson’s disease. Rodrı´guez and Gonza´ lez-Herna´ ndez • GABAergic Nigrostriatal Pathway J. Neurosci., June 1, 1999, 19(11):4682–4694 4687

Figure 4. Double-labeled neurons for HRP and GAD (a–c), HRP and GABA (d, e), and HRP and PV (f ) in the SNr (a, b, d–f) and VTA (c)of unlesioned rats. The arrow in a indicates the neuron shown in b.Inc, d, and f, arrows indicate double-labeled neurons; arrowheads indicate single immunostained neurons. Scale bars: a, 200 ␮m; b, f,20␮m; c–e,30␮m.

Data supporting a nondopaminergic (Swanson, 1982; Gerfen et al., 1987) and provide further mor- nigrostriatal pathway phological evidence showing that they are not immunoreactive Our morphological data show a subpopulation of SN neurons for CR, CB, and CCK. retrogradely labeled after HRP injection in the striatum that is The electrophysiological studies also identified a group of ni- nonimmunoreactive for TH, CCK, CR, or CB, proteins expressed grostriatal neurons with a conduction velocity several times by DA cells (Ho¨kfelt et al., 1980, 1985; Re´sibois and Rogers, higher than that found in the DA cells. Although this cell type 1992; Liang et al., 1996; McRitchie et al., 1996). The fact that was mentioned in the early works of Deniau et al. (1978) and these neurons do not degenerate after the injection of 6-OHDA, Guyenet and Aghajanian (1978), they were never systematically a neurotoxin selectively taken up by a transporter only contained studied. As shown in Figure 1f, these cells displayed a higher in DA neurons (Ho¨kfelt and Ungerstedt, 1973; Cohen and Heik- basal firing rate and VC than those of DA neurons. In the kila, 1974; Walkinshaw and Waters, 1994; Ciliax et al., 1995; 6-OHDA lesioned rats, surviving nigral neurons showing an Betarbet et al., 1997; Miller et al., 1997), also supports the exis- antidromic response to striatal stimulation presented a conduc- tence of NO-DA cells. Present findings confirm previous morpho- tion velocity and basal firing rate similar to those found for logical studies reporting nigrostriatal cells without catecholamine NO-DA cells in unlesioned rats. This finding also indicates that fluorescence (van der Kooy et al., 1981) or TH immunoreactivity 6-OHDA does not affect NO-DA cells. 4688 J. Neurosci., June 1, 1999, 19(11):4682–4694 Rodrı´guez and Gonza´ lez-Herna´ ndez • GABAergic Nigrostriatal Pathway

Figure 5. Distribution of GAD-IR nigrostriatal neurons. a, Example of HRP injection in the striatum. b, Schematic drawing showing the localization of the HRP injection in some representative cases. c, Distribution of GAD-HRP neurons in the SN in two cases. Solid circles indicate the localization of double-labeled neurons after injection in the lateral half of striatum (shown in a, black area in b). Open circles indicate the localization of double-labeled neurons after injection in the medial half of striatum (shadowed area in b). Each circle corresponds to three neurons. Modiﬁed drawing from Paxinos and Watson (1988). The distance of each section to the interaural line is shown in millimeters. ac, Anterior commissure; cc, corpus callosum; f, fornix; GP, globus pallidus; ic, internal capsule; lv, lateral ventricle; MT, medial terminal nucleus of the accessory optical tract; S, septum; SNl, substantia nigra pars lateralis; St, striatum. Scale bar, 1 mm. Rodrı´guez and Gonza´ lez-Herna´ ndez • GABAergic Nigrostriatal Pathway J. Neurosci., June 1, 1999, 19(11):4682–4694 4689

Figure 6. TH (a–d), CR (e, f), and CB ( g, h) immunocytochemistry after 6-OHDA injection in the right medial forebrain bundle and HRP injection in the ipsilateral striatum. a, e, g, Lesioned side; b, f, h, unlesioned side. c, d, Boxed areas in a; arrows indicate single HRP-stained neurons. Scale bars: a, b, e–h, 150 ␮m; c, d,35␮m. 4690 J. Neurosci., June 1, 1999, 19(11):4682–4694 Rodrı´guez and Gonza´ lez-Herna´ ndez • GABAergic Nigrostriatal Pathway

Figure 8. Percentage of nigrostriatal cells showing TH, CCK, CB, CR, GABA, GAD, and PV immunoreactivity in sham (n ϭ 4200 HRP cells per series) and 6-OHDA-injected (n ϭ 420 HRP cells per series) rats.

Data supporting a GABAergic nigrostriatal pathway Double labeling for HRP and GABA, GAD, or PV showed that 5–8% of nigrostriatal cells are GABAergic. This percentage reached 81–84% of all retrogradely labeled neurons in 6-OHDA lesioned rats. The difference observed in the experiment 2, between the percentage of nigrostriatal neurons showing TH immunonegativity (15%) and those showing GAD, GABA, and PV immunopositivity (5–8%), could suggest that not all NO-DA cells are GABAergic. The fact that after lesion (experiment 3) almost all TH, CCK, CR, and CB nigrostriatal neurons disappeared, whereas Ͼ80% of HRP neurons were immunopositive for GAD, GABA, and PV, indicates that if a subpopulation of NO-DA cells is not GABAergic, this is quantitatively negligible. It is possible that, despite our effort counting only those neurons lying in the surface of sections, and because of the limited penetration of antisera (8–10 ␮m) compared with that of HRP (evident at all depths of the sections), the number of single HRP-stained neurons was overestimated. So, we must be cautious when interpret- ing our quantitative data. GABAergic nigrostriatal neurons are restricted to the SNr, and their distribution varies as a function of the injection site in striatum. In the mediolateral axis, they follow a topographic pattern similar to that previously described for the dopaminergic nigrostriatal projection (Fallon and Moore, 1978) and for strionigral descending fibers (Gerfen, 1985). In the rostrocaudal axis, the topography of GABAergic nigrostriatal neurons is also biased according to the injection site, matching that of the dopaminergic ones (Fallon and Moore, 1978) but differing from that of descending strionigral fibers that, independently of the injection site, are distributed homogeneously throughout the rostrocaudal axis of the SNr (Gerfen, 1985). Although our material does not allow us to establish a precise spatial correspondence in the GABAergic nigrostrial pathway, we can suggest that this may be involved in Figure 7. Double-labeled neurons for HRP and GAD (a), HRP and GABA (b), and HRP and PV (c, d) in SNr of 6-OHDA lesioned rats. d, the modular organization of the nigro-strio-nigral loop (Deniau Boxed area in c; arrows in b and d indicate double-labeled neurons; and Chevalier, 1992; Parent and Hazrati, 1995; Deniau and arrowhead in d indicates a single PV-immunostained neuron. Scale bars: Thierry, 1997). a, b,20␮m; c, 200 ␮m; d,30␮m. A further support for the GABAergic nature of NO-DA cells comes from electrophysiological data. The firing rate and VC of Rodrı´guez and Gonza´ lez-Herna´ ndez • GABAergic Nigrostriatal Pathway J. Neurosci., June 1, 1999, 19(11):4682–4694 4691

inergic cells transporting HRP from the striatum to VTA and RRF. Recently, GABAergic cells have been reported in the VTA (Steffensen et al., 1998). Our data show that the striatum is one of the targets for these cells, although the electrophysiological and neurochemical details of this projection require further study.

GABAergic nigrostriatal pathway and dopaminergic cell degeneration In the current model of the organization of basal ganglia, the symptoms of different diseases have been explained on the basis of modifications in the firing rate of specific cell groups (Albin et al., 1989; Alexander and Crutcher, 1990a,b; DeLong, 1990), disturbances that may be attenuated by new neurosurgical therapies that modify the balance between excitatory and inhibitory inputs (Marsden et al., 1997; Obeso et al., 1997; Pollak et al., 1997; Viteck et al., 1997). It has been suggested that a reduction of striatal dopamine leads to activation of the inhibitory GABAergic nigrothalamic input (Bernheimer et al., 1973; Mitchell et al., 1989; DeLong, 1990; MacLeod et al., 1990; Conde´, 1992; Herrero et al., 1993; Burbaud et al., 1995), and a subsequent reduction of thalamo-cortical activation (DeLong, 1990; Obeso et al., 1997). The final result could be a cortical inhibition that may be directly involved in different motor disturbances and especially in the hypokinesia often found in Parkinson’s disease. This hypothesis is supported by previous experiments showing that the degeneration of dopaminergic nigrostriatal cells produces an increase of 2-deoxyglucose uptake (Mitchell el al., 1989) and GAD mRNA levels (Herrero et al., 1993; Vila et al., 1996) in SNr cells. How- ever, the overexpression of GAD and GABA does not necessarily imply an increase of GABAergic cell activity or GABA release. Contradictory results have been reported about the functional consequences of the DA cell degeneration on the GABA turn- over (Tanaka et al., 1986; Lindgren, 1987; Samuel et al., 1988; Houssain and Weiner, 1995) and firing rate (Arbuthnott, 1974; Ruffieux and Schultz, 1980; Sanderson et al., 1986; MacLeod et al., 1990; Robledo and Fe´ger, 1992; Burbaud et al., 1995) of SNr cells. Our data show that the increase of GAD expression previously reported by others in 6-OHDA lesioned rats is not associated with changes in the firing rate, at least in nigrothalamic and Figure 9. Functional effect of DA cell degeneration on NO-DA and nigrostriatal GABAergic neurons. However, the VC increased in nigrothalamic (NT) cells. *p Ͻ 0.05 versus sham-lesioned rats. Values Ϯ both groups. Despite the fact that we did not block 6-OHDA represent the mean SE. uptake into noradrenergic fibers, the effect appears to be secondary to the degeneration of DA neurons, because this was similar NO-DA cells were higher than those of DA cells (Grace and to that consistently reported in previous studies for nigrothalamic Bunney, 1980, 1983a–c, 1984a,b, 1985a,b; DeLong et al., 1983; neurons (Ruffieux and Schultz, 1980; Sanderson et al., 1986; Schwarz et al., 1984; Schultz, 1986; Chiodo, 1988) but similar to MacLeod et al., 1990). It has been suggested that a firing pattern those of other SN GABAergic neurons (Sanderson et al., 1986; code could be a more important feature than the mean firing rate Burbaud et al., 1995). Contrary to that reported in DA cells for explaining the basal ganglia activity (Chesslet and Delfs, 1996; (Grace and Bunney, 1984a,b; Overton and Clark, 1997), NO-DA Wichmann and DeLong, 1996; Parent and Ciccheti, 1998). How- cells showed no clear evidence of burst discharge. Most of them ever, the characteristics of this code remain unknown (Egger- displayed tonic discharges with different degrees of regularity, a mont, 1990), and only indirect measurements of the information pattern similar to that reported in two subgroups of nigrothalamic processing in the basal ganglia can be taken. A suitable measure neurons: the regular tonic and the irregular tonic discharging in this case is the VC, an indicator of the interspike interval groups (Sanderson et al., 1986; Burbaud et al., 1995). On the variability that is invariant to the firing rate. From this point of other hand, the conduction velocity suggests that these GABAer- view, the modification of VC observed in nigrothalamic and gic nigrostriatal cells are probably composed of myelinated fibers nigrostriatal GABAergic cells after 6-OHDA lesion suggests that of a relatively small diameter (3.48 Ϯ 2.03 m/sec), in contrast to DA cells modulate the SNr cell activity in a complex manner and the unmyelinated axon of DA neurons (0.52 Ϯ 0.02 m/sec). do not induce a simple modification of the firing rate. Previous It is known that, besides the dopaminergic nigrostriatal projec- evidence showed that whereas the GABAergic neurons of SNr tion, there are DA cells projecting to the striatum from both VTA process and transmit information concerning different sensory or and RRF (Parent et al., 1983; Charara and Parent, 1984; Lewis motor functions (DeLong et al., 1983; Hikosaka and Wurtz, and Sesack, 1997). Along with these cells, we found nondopam- 1983a–c; Schwarz et al., 1984; Joseph and Boussaoud, 1985; 4692 J. Neurosci., June 1, 1999, 19(11):4682–4694 Rodrı´guez and Gonza´ lez-Herna´ ndez • GABAergic Nigrostriatal Pathway

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