Thalamic inputs to striatal interneurons in monkeys: synaptic organization and co-localization of calcium binding proteins

Pergamon

PII:

Neuroscience Vol. 89, No. 4, pp. 1189–1208, 1999 Copyright  1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/99 $19.00+0.00 S0306-4522(98)00367-4

THALAMIC INPUTS TO STRIATAL INTERNEURONS IN MONKEYS: SYNAPTIC ORGANIZATION AND CO-LOCALIZATION OF CALCIUM BINDING PROTEINS M. SIDIBE u † and Y. SMITH*‡ *Division of Neuroscience, Yerkes Regional Primate Research Center and Department of Neurology, 954 Gatewood Road N.E., Emory University, Atlanta, GA 30329, U.S.A. †Centre de Recherche en Neurobiologie, Hoˆpital de l’Enfant-Je´sus and Universite´ Laval, Que´bec, Canada Abstract––Recent studies indicate that extrinsic inputs from sensorimotor regions of the cerebral cortex and the centromedian intralaminar thalamic nucleus terminate preferentially upon specific subpopulations of striatal output neurons in monkeys. The objective of the present study was to verify whether this specificity of innervation also characterizes the synaptic interactions between thalamic inputs from the centromedian nucleus and the four major populations of striatal interneurons. This was achieved by double labelling techniques at the electron microscope level, combining the anterograde transport of biotinylated-dextran amine with the immunostaining for specific markers of striatal interneurons (somatostatin, parvalbumin, choline acetyltransferase and calretinin). Injections of biotinylated-dextran amine in the centromedian nucleus led to dense bands of anterograde labelling which, in double immunostained sections, largely overlapped with the four populations of interneurons in the postcommissural region of the putamen. In the electron microscope, biotinylated-dextran amine-containing terminals formed asymmetric axo-dendritic synapses with somatostatin-, parvalbumin-, and choline acetyltransferase-containing elements. However, synapses between anterogradely labelled terminals and calretinin-positive neurons were not found. In sections processed to localize biotinylated-dextran amine and parvalbumin or calretinin, double-labelled terminals (biotinylated-dextran amine/parvalbumin and biotinylated-dextran amine/calretinin), morphologically similar to thalamostriatal boutons, were found in the striatum indicating that calcium binding proteins may be expressed by thalamostriatal neurons. To test this possibility, we combined the retrograde transport of lectin-conjugated horseradish peroxidase from the putamen with parvalbumin and calretinin immunostaining and found that, indeed, most of the retrogradely labelled cells in the centromedian nucleus displayed parvalbumin and calretinin immunoreactivity. Moreover, co-localization studies revealed that calretinin and parvalbumin co-exist in single neurons of the centromedian nucleus. In conclusion, striatal interneurons immunoreactive for somatostatin, parvalbumin and choline acetyltransferase, but not those containing calretinin, receive strong inputs from the centromedian nucleus in monkeys. Moreover, our findings indicate that parvalbumin and calretinin co-exist in individual thalamostriatal neurons. In combination with our previous data, these results suggest that thalamic information may be conveyed to striatal projection neurons both, directly via excitatory synaptic inputs, or indirectly via striatal interneurons. The relative importance of those direct and indirect thalamic influences upon the activity of striatal output neurons remains to be established.  1999 IBRO. Published by Elsevier Science Ltd. Key words: striatum, thalamus, somatostatin, parvalbumin, calretinin, choline acetyltransferase.

The mammalian striatum (caudate–putamen) enclose two broad categories of neurons: the spiny and aspiny cells. The spiny cells, the overwhelming ‡To whom correspondence should be addressed. Abbreviations: ABC, avidin–biotin–peroxidase complex; BDA, biotinylated dextran-amine; BDHC, benzidine dihydrochloride; BSA, bovine serum albumin; ChAT, choline acetyltransferase; CM, centromedian nucleus; DAB, 3,3 -diaminobenzidine; MD, mediodorsal nucleus; Ni-DAB, nickel-enhanced diaminobenzidine; NMDA, N-methyl--aspartate; PB, phosphate buffer; PBS, phosphate-buffered saline; PF, parafascicular nucleus; s-PF, subparafascicular nucleus; TMB, tetramethylbenzidine; WGA–HRP, wheatgerm agglutinin (lectin)conjugated horseradish peroxidase.

majority of which are medium-sized, account for 90–95% of all striatal neurons and are recognized as the output neurons of the striatum.28,39,55,70 On the other hand, the aspiny cells represent 5–10% of the total neuronal population of the striatum and are generally considered as interneurons.21,22,36,37 Based on their chemical content, four major classes of aspiny interneurons have been identified in the neostriatum: (i) the cholinergic neurons, (ii) the parvalbumin-containing neurons, which also contain GABA, (iii) the somatostatin-containing neurons which also display neuropeptide Y, nitric oxide and glutamate decarboxylase immunoreactivity and (iv) the calretinin-immunoreactive neurons.38 Although

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they represent a minor neuronal population, these interneurons give rise to rich intrastriatal terminal fields that may influence broad regions of the striatum (see Refs 10, 38 and 66 for reviews). Striatal neurons are the targets of two major excitatory inputs arising from the cerebral cortex and the caudal intralaminar thalamic nuclei which, in monkeys, include the centromedian (CM) and parafascicular (PF) nuclei. It is well established that those inputs display a high degree of functional specificity in their pattern of striatal innervation so that the sensorimotor cortical areas and the CM project preferentially to the post-commissural part of the putamen whereas inputs from associative and limbic cortices as well as the PF terminate selectively in the caudate nucleus and the rostroventral striatum.53a,61,62 At the electron microscope level, cortical and thalamic terminals form asymmetric synapses preferentially with spines and dendrites of projection neurons.23,40,62,64,67 Although morphologically similar, the striatal output neurons are largely segregated into two major categories based on their content in neuropeptides and expression of dopamine receptors.25b The so-called ‘‘direct’’ striatofugal neurons, that mainly express D1 dopamine receptors and contain substance P and dynorphin, innervate preferentially the internal pallidum and the substantia nigra pars reticulata. On the other hand, the ‘‘indirect’’ striatofugal neurons that mainly express D2 dopamine receptors and contain enkephalin, project preferentially to the external pallidum.25a Recent evidence suggest that cortical and thalamic influences upon these two populations of striatal output neurons are likely to be much more specific than previously thought. For instance, microstimulation or local disinhibition of the sensorimotor cortex induces expression of immediate-early genes selectively in enkephalin-immunoreactive neurons in the rat9 and monkey54 striatum. On the other hand, inputs from CM terminate much more frequently on ‘‘direct’’ than ‘‘indirect’’ striatofugal neurons in squirrel monkeys.64 Although much less attention has been paid to the analysis of extrinsic inputs to striatal interneurons, anatomical and molecular data obtained so far suggest a certain degree of specificity in their pattern of cortical and thalamic inputs. For instance, the proximal part of cholinergic interneurons receives massive inputs from the thalamus but is almost completely devoid of cortical afferents in rats.43,50 On the other hand, parvalbumin-immunoreactive neurons receive a prominent cortical input in squirrel monkeys.44 In keeping with these ultrastructural findings, recent data showed that local disinhibition of the primary motor cortex leads to strong expression of c-fos in parvalbumin- but not in choline acetyltransferase (ChAT)-immunoreactive neurons in the rat.9 Apart from the cholinergic interneurons which, as mentioned above, are mainly driven by thalamic inputs, the synaptic innervation and poten-

tial roles of thalamic influences upon other populations of striatal interneurons still remain to be established. As part of an ongoing project on the synaptic organization of the thalamostriatal pathway in primates,62,64,65,67 the objective of this study is to compare the pattern of synaptic innervation of the four main populations of striatal interneurons by thalamic inputs from CM in squirrel monkeys (Saimiri sciureus). The results of this study have been presented in abstract form.65 EXPERIMENTAL PROCEDURES

Animals, injections of tracers and perfuse-fixation Five adult male (body weight 900–1100 g) squirrel monkeys (Saimiri sciureus; Yerkes Primate Center, Atlanta, GA) were used in the present study. Four animals were anaesthetized with a mixture of ketamine hydrochloride (Ketaset, 70 mg/kg, i.m.) and xylazine (10 mg/kg, i.m.) before being fixed in a stereotaxic frame. The depth of anaesthesia was determined by monitoring hearth rate, muscle tone as well as corneal and toe-pinch reflexes. The surgery, anaesthesia and post-operative care were performed according to the rules established by the Canadian Council on Animal Care and the National Institute of Health guide for the care and use of laboratory animals. Another animal, that did not receive intracerebral injection, was perfuse-fixed and used for co-localization studies of parvalbumin and calretinin in the CM. All efforts were made to minimize animal suffering and to reduce the number of animals used. The injections of tracers were performed as follows: (i) two monkeys received bilateral injections of biotinylated dextran-amine (BDA) in the CM, and (ii) two monkeys received an injection of BDA in the CM on one side and an injection of wheatgerm agglutinin (lectin)-conjugated horseradish peroxidase (WGA–HRP) in the post-commissural region of the putamen on the other side. The BDA (Molecular Probes, Eugene, Oregon, U.S.A.; 5% in distilled water) was loaded in glass micropipettes with a tip diameter ranging from 20–30 µm. It was then injected iontophoretically in CM with 5 µA positive current for 20 min using a 7 s ON/7s OFF cycle. The WGA–HRP (5% in 0.9% saline; Sigma) was delivered by pressure through a glass micropipette (50–70 µm tip diameter) sealed with wax to a 1.0 µl Hamilton microsyringe. Ten to 30 nl of WGA–HRP were injected in the putamen at rate of 1–2 nl/min. The stereotaxic coordinates were chosen according to the atlas of Emmers and Akert.24 During the first two days after the surgery, the animals received injections of analgesic (buprenorphine; 0.01 mg/kg, s.c.) twice daily. After the appropriate survival period (seven to 10 days for BDA and 24–36 h for WGA–HRP), the animals were deeply anaesthetized with an overdose of pentobarbital (50 mg/kg, i.v.) and perfuse-fixed with 500–700 ml of cold oxygenated Ringer solution followed by 1.5 l of fixative containing a mixture of 3% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer (PB, 0.1 M, pH 7.4). This was followed by 1 l of cold PB. After perfusion, the brains were cut in 10-mm-thick blocks in the transverse plane and placed in cold phosphate-buffered saline (PBS, 0.01 M, pH 7.4) until sectioning. The blocks were cut into 60-µm-thick transverse sections with a vibrating microtome, collected in cold PBS and treated for 20 min with sodium borohydride (1% in PBS). The sections were then processed to reveal first, the injected and transported BDA or WGA–HRP, and second, the different markers of striatal interneurons.

Thalamic inputs to striatal interneurons in monkeys Biotinylated dextran-amine histochemistry combined with somatostatin, parvalbumin, calretinin, and choline acetyltransferase immunohistochemistry Light microscopy. To reveal the injected and transported BDA,56,72 a series of sections containing the injection sites and the striatum were incubated for 12–16 h at room temperature with avidin–biotin–peroxidase complex (ABC; Vector Laboratories, Burlingame, CA, U.S.A.; 1:100 dilution)30 in PBS containing 0.3% Triton X-100 and 1% bovine serum albumin (BSA; Sigma Chemical Company, St Louis, MO, U.S.A.). They were then washed in PBS and Tris buffer (0.05 M, pH 7.6) before being placed in a solution containing 3,3 -diaminobenzidine tetrahydrochloride (DAB, 0.025%; Sigma Chemical Company), 0.01 M imidazole (Fisher Scientific, Nepean, Ontario, Canada) and 0.006% hydrogen peroxide for 10–15 min. After several washes in PBS, sections of the striatum and the thalamus were preincubated for 1 h at room temperature in a solution containing 0.3% Triton X-100 and 1% non-immune sera (rabbit for somatostatin antibodies, horse for parvalbumin antibodies, and goat for ChAT and calretinin antibodies) diluted in PBS and then incubated for 12–16 h with rat anti-somatostatin (Medicorp, Montreal, Quebec, Canada; 1:50 dilution), mouse anti-parvalbumin (Swant, Switzerland; 1:10,000 dilution)13 rabbit anti-ChAT (Chemicon, Temecula, CA, U.S.A.; 1:1000 dilution), or rabbit anti-calretinin (Swant, Switzerland; 1:10,000 dilution)63 in PBS containing the appropriate normal serum (1% dilution) and 0.3% Triton X-100. The sections were then rinsed three times in PBS and incubated for 90 min at room temperature in biotinylated secondary antibodies (rabbit anti-rat for somatostatin, horse anti-mouse for parvalbumin and goat anti-rabbit for ChAT and calretinin; 1:200 dilution) diluted in PBS for 90 min at room temperature. After three rinses in PBS, the sections were incubated in ABC (1:100 in PBS/0.3% Triton X-100/1% BSA) for 90 min at room temperature. They were then washed twice in PBS and then placed in Tris buffer containing DAB (0.025%), ammonium nickel sulfate (Ni–DAB; Fisher Scientific, 0.35%) and hydrogen peroxide (0.0006%) for 10 min (see Ref. 79 for more details). The reaction was stopped by extensive rinsings in PBS. The sections were then mounted onto gelatin-coated slides, dehydrated and a coverslip was applied with Permount. Electron microscopy. A series of sections that included the striatum were processed for the simultaneous visualization of the transported BDA, and somatostatin, parvalbumin, ChAT and calretinin immunoreactivities as described above, except that no Triton X-100 was used, and the incubation in the primary antibodies lasted for 48 h at 4C. To enhance the penetration of the antibodies and the ABC into the tissue, the sections were placed in a cryoprotectant solution (PB, 0.05 M, pH 7.4, containing 25% sucrose and 10% glycerol) for 20–30 min, and frozen at
80C for 20 min. They were then thawed and washed many times in PBS before being processed to localize BDA with DAB. Once the tracer has been revealed, the sections were processed to localize the different populations of interneurons with tetramethylbenzidine (TMB, Sigma Chemical Company) according to the protocol introduced by LlewellynSmith et al.47 Briefly, after having been washed many times in PB (0.1 M, pH 6.0), and pre-incubated for 20 min in the TMB solution, the reaction was initiated by adding hydrogen peroxide to a final concentration of 0.003% in a fresh TMB solution. The reaction was terminated after 7–10 min by extensive washings in PB (0.1 M, pH 6.0). The TMB reaction product was stabilized by incubating the sections for 5–7 min in a solution containing: 100 ml of PB (0.1 M, pH 6.0), 100 mg of DAB, 1 ml of 0.4% NH4Cl, 1 ml of 20% -glucose, 2 ml of 1% CoCl2 and 100 µl of glucose oxidase.47 The sections were then washed many times in

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PB (0.1 M, pH 6.0) before being processed for electron microscopy. Control experiments. As controls, a series of sections were processed to reveal BDA and, then, were incubated in solutions where the primary antibodies were replaced by non-immune sera. The other steps of the immunocytochemical reactions and the TMB staining were the same as described above. Processing for electron microscopy The sections were washed in PB (0.1 M, pH 6.0) before being postfixed in osmium tetroxide (1% solution in PB) for 20 min. They were then dehydrated in a graded series of alcohol and propylene oxide. Uranyl acetate was added to the 70% ethanol (30 min) to improve the contrast in the electron microscope. The sections were then embedded in resin (Durcupan ACM, Fluka; EM Sciences, Fort Washington, PA, U.S.A.) on microscope slides and put in the oven for 48 h at 60C. After a detailed examination in the light microscope, regions of interest were drawn, sometimes photographed, cut out from the slides and glued on the top of resin blocks with cyanoacrylate glue. Serial ultrathin sections were cut on a Reichert–Jung Ultracut E ultramicrotome and collected on Pioloform-coated single slot copper grids. They were stained with lead citrate58 and examined in a Phillips EM300 electron microscope. Wheatgerm agglutinin (lectin)-conjugated horseradish peroxidase histochemistry combined with parvalbumin and calretinin immunohistochemistry The WGA–HRP was revealed using the TMB method introduced by Mesulam51 and modified by Olucha et al.53 Briefly, the sections were washed three times in cold PB (0.1 M, pH 6.2) before being put into the same buffer containing 0.25% ammonium molybdate (VI) tetrahydrate (Aldrich Chemical Co.) and 0.5% TMB (Sigma). After a 5 min pre-incubation, 0.003% hydrogen peroxide was added to the solution every 5 min throughout the incubation time of 30 min. The sections were then washed many times in PB (0.1 M, pH 6.2) and the TMB reaction product was stabilized with DAB according to the method of Rye and co-workers.60 After several washes in PBS, a series of WGA–HRP-stained sections throughout the CM were processed to reveal either parvalbumin or calretinin immunoreactivities with DAB as described above. Double immunostaining for parvalbumin and calretinin A series of sections through the CM were processed for the co-localization of parvalbumin and calretinin immunoreactivities at the light microscope level. Some sections were processed to reveal parvalbumin first with DAB, and then calretinin with benzidine dihydrochloride (BDHC; Sigma Chemicals, St Louis, MO, U.S.A.), whereas in other sections, the order was reversed so that calretinin was revealed, first, with DAB and parvalbumin, second, with BDHC. The concentration of antibodies, steps of the immunocytochemical procedures and histochemical reaction in DAB were the same as described above. The sections that were incubated in BDHC were washed three to five times in PB (0.01 M, pH 6.8) before being pre-incubated in a solution of BDHC that was prepared according to Levey et al.46 After a 10-min pre-incubation, the reaction was initiated by adding hydrogen peroxide to a final concentration of 0.005% in fresh BDHC solution. The reaction was terminated after 5–7 min by extensive washing in PB (0.01 M, pH 6.8). The sections were then mounted onto gelatin-coated slides, dehydrated and a coverslip was applied with Permount.

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Table 1. Number of blocks prepared from the different experimental cases, total number of biotinylated dextran-aminelabelled terminals examined and proportion of biotinylated dextran-amine-labelled boutons in contact with processes of interneurons in the monkey striatum Experimental protocol

Experimental cases

Number of blocks (Number of sections examined)

Total number of BDA-labelled terminals examined

Total number of BDA-labelled boutons in contact with interneurons

BDA/somatostatin

case case case case

1 2 3 4

2 1 2 1

(40) (20) (90) (35)

167

62

BDA/parvalbumin

case case case case

1 2 3 4

2 1 1 1

(70) (30) (45) (25)

160

67

BDA/ChAT

case case case case

1 2 3 4

0 0 2 (75) 1 (45)

104

55

BDA/calretinin

case case case case

1 2 3 4

3 1 3 1

116

0

(110) (50) (120) (35)

As controls, each of the primary antibodies were omitted in turn, while the remaining of the procedure was the same. Analysis of the material The thalamic and striatal injection sites as well as the distribution of anterogradely labelled fibres and retrogradely labelled cells were charted from coronal sections at magnifications between 1 and 10 with a drawing tube connected to a Nikon light microscope. Adjacent sections stained with Cresyl Violet were used to facilitate the delineation of the thalamic nuclei. The synaptic relationships between thalamic terminals and the different populations of interneurons were analysed at the electron microscope level in selected areas of overlap in the dorsolateral part of the post-commissural putamen (Fig. 3). The selection of the appropriate regions was performed after examination of the sections at the light microscope level. We selected those areas that contained a large density of anterogradely labelled thalamic fibres in register with perikarya and dendritic processes of interneurons. Since the thalamic input terminates preferentially on dendrites, we particularly selected areas where the dendritic processes of striatal interneurons were labelled over a long distance as depicted in Fig. 2. Once such an area was found, it was cut out from the slide, mounted on blocks of resin, cut into serial ultrathin sections and examined in the electron microscope. The number of sections examined from each experimental group is given in Table 1. In order to ensure that the material contained both DAB- and TMBlabelled elements, the most superficial sections of the blocks were selected for electron microscope analysis. Once a BDA-labelled terminal was found to be apposed to a TMB-labelled structure, it was followed in two to four serial sections to verify the possibility of synaptic contact between these elements. To compare the relative frequency of

thalamic inputs to the four populations of interneurons, the number of anterogradely labelled terminals from CM in contact with either labelled or unlabelled striatal elements were counted in series of ultrathin sections that always contained TMB-labelled structures in the vicinity (less than 2 µm from) of DAB-labelled boutons. To chart the distribution of single and double-labelled cells in the CM after WGA–HRP injection in the putamen, we examined five sections selected a regular intervals through the rostrocaudal extent of the CM. The exact location of single and double labelled neurons was then plotted on drawing paper at a magnification of 10 using a camera lucida. Based on the atlas of Emmers and Akert,24 three representative levels were chosen for the illustration in Fig. 7. To estimate the proportion of single versus doublelabelled neurons in the co-localization studies, we scanned, at 40, five sections through the rostrocaudal extent of CM. When a BDHC-containing neuron was encountered, we verified whether it also contained DAB and categorized it as a single or double-labelled neuron. RESULTS

Biotinylated dextran-amine injection sites and anterograde labelling in the striatum In four of the six hemispheres (case nos 1–4) that received injections of BDA into the thalamus, the tracer involved the entire rostrocaudal and dorsoventral extent of the central part of the CM, with slight contamination of the subparafascicular (s-PF) and mediodorsal (MD) nuclei (Fig. 1). The parafascicular (PF) nucleus was not contaminated by

Abbreviations used in the figures Ax CR den FR HRP

axon calretinin dendrite fasciculus retroflexus horseradish peroxidase

IC PV SS Sp

internal capsule parvalbumin somatostatin spine

Thalamic inputs to striatal interneurons in monkeys

Fig. 1. Injection site of BDA in the central part of the CM in case no. 2. Scale bar=1.0 mm.

these injections (Fig. 1). In a fifth hemisphere (case no. 5), the injection was much bigger and involved the entire caudal intralaminar nuclear complex including the CM, PF, s-PF and central lateral nuclei as well as the MD. In a sixth case (case no. 6), the injection site was small and confined to the dorsal third of the CM. Because of the specificity of the injection site and the high density of anterograde labelling in the post-commissural putamen, all the material selected for electron microscopy came from case nos 1–4 (see below). As reported in previous studies, BDA injections confined to the central part of the CM (Fig. 1) led to dense bands of anterogradely labelled fibres in the post-commissural region of the putamen.61,62,64,67 On the other hand, the injection that involved both the CM and PF (case no. 5) resulted in massive anterograde labelling throughout the whole striatum including, not only the putamen, but also the caudate nucleus and the nucleus accumbens.61 The small injection in the dorsal CM (case no. 6) led to sparse labelling in the pre-commissural region of the putamen. Topographical relationships between anterogradely labelled thalamic fibres and striatal interneurons The pattern of distribution and morphological characteristics of the different populations of striatal interneurons were in keeping with data obtained in previous studies in rats and monkeys (see Refs 10, 37 and 38 for reviews). In brief, the four populations of interneurons were homogeneously distributed throughout the whole extent of the caudate nucleus and the putamen, except for the calretinin-positive neurons that were more abundant in the rostral half

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of the striatum (see also Ref. 6). The perikarya, that were of various shapes (polygonal, pyramidal, ovoid, fusiform) and sizes (longer diameter ranging from 15 to 40 µm), gave rise to three to five smooth varicose dendrites which, in many cases, formed a field that extended up to 400 µm in diameter (Fig. 2). Although two categories of calretinin-containing neurons have been described in the primate striatum,8a,53b the dorsolateral putamen mostly contained mediumsized calretinin-positive neurons. As previously reported, the so-called ‘‘giant’’ calretininimmunoreactive neurons were rare and found predominantly in the ventral regions of the monkey striatum.8a In light microscopic sections prepared for the simultaneous visualization of BDA-containing fibres and striatal interneurons, the labelled structures were differentiated by the colour of the reaction product associated with them; the thalamic fibres contained the brown amorphous DAB deposit whereas the processes of interneurons displayed a dark blue staining typical of the Ni–DAB deposit (Fig. 3). After tracer injections in CM, tight zones of overlap between interneurons and thalamostriatal fibres were found in the dorsal half of the post-commissural putamen (Fig. 3). When examined at high magnification, close appositions between BDA-containing varicosities and labelled dendrites were commonly found in those regions. To verify the possibility of synaptic contacts between these elements, sections adjacent to those that contained areas of overlap were processed to visualize BDA and markers of striatal interneurons in the electron microscope. In these sections, that had been postfixed in osmium, the DAB deposit in the thalamic fibres and the TMB deposit in interneurons could not be distinguished by their colour but, rather, displayed a different texture. Whereas the DAB deposit was amorphous and filled homogeneously varicose fibres, the TMB reaction product was crystalline and more heterogeneously distributed in the perikaryon and dendrites of labelled neurons. These differences in texture between the DAB and TMB reaction products were easier to distinguish in the electron microscope (Figs 4–6). Synaptic relationships between thalamic afferents and striatal interneurons A total of 22 blocks were prepared for the analysis in the electron microscope (Table 1). They were all selected from dense zones of overlap in the dorsal post-commissural putamen in case nos 1–4 (see Fig. 3), except for the sections processed for ChAT and BDA that came only for case nos 3 and 4. Although no difference in the pattern of labelling was found between the different cases, the ultrastructure was better preserved in case nos 1 and 3, which explains why more blocks were prepared and more sections were examined from these two cases.

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Overall distribution of staining. As mentioned above, the appearance of the two reaction products were easily distinguishable at the electron microscope level, the DAB reaction product was amorphous, electron-dense and attached to the outer surface of microtubules, mitochondria, and electron-lucent vesicles as well as with the inner surface of the plasmalemma (Figs 4–6). On the other hand, the TMB reaction product was in the form of electron-dense crystals that were randomly dispersed throughout the labelled elements (Figs 4–6). In general, the two reaction products were associated with different structures in the double labelled sections; the DAB deposit was found exclusively in pre-synaptic elements incuding terminals and unmyelinated axons, whereas the TMB reaction product was preferentially associated with dendrites and perikarya (Figs 4–6), though subpopulations of TMB-labelled axon terminals were also encountered. In the sections processed for parvalbumin or calretinin with BDA, terminals containing both reaction products were frequently found (see below). Although the TMB-labelled perikarya were of various shapes and sizes, they all had an indented nucleus, a typical feature of striatal interneurons.20,22 The ultrastructural features and pattern of synaptic distribution of the BDA-containing terminals were in keeping with those described for the CM terminals in our previous studies62,64,67 i.e. they were medium-sized (0.5–1.5 µm in diameter), contained round or oval electron-lucent vesicles which filled the entire terminal, had at least one mitochondrion and formed asymmetric synapses predominantly with dendritic shafts, but also with spines (Figs 4–6, see below). Biotinylated dextran-amine and somatostatin immunostaining. The postsynaptic targets of 167 BDAcontaining terminals were examined in somatostatinimmunostained sections. Seventy-three percent formed synapses with dendritic shafts, 27% contacted spines and none established contacts with perikarya. More than a third of the postsynaptic targets contacted by these terminals displayed somatostatin immunoreactivity (Table 1, Fig. 4A,B). Twelve TMB-containing terminals were found, and in the four cases where the synaptic specialization could be seen, it was of the symmetric type. None of them contacted somatostatin-immunoreactive elements. No double labelled terminals was found in these sections.

Fig. 2. Different populations of striatal interneurons characterized by their immunoreactivity for somatostatin (SS, A), parvalbumin (PV, B), choline acetyltransferase (ChAT, C) and calretinin (CR, D). Note the extensive dendritic labelling of the four populations of interneurons. Scale bar=25 µm.

Biotinylated dextran-amine and parvalbumin immunostaining. In the parvalbumin-immunostained tissue, examination of 160 BDA-labelled terminals collected from five blocks revealed that 76% formed asymmetric synapses with dendritic shafts whereas 24% contacted dendritic spines. About a third of those postsynaptic targets were parvalbuminimmunoreactive (Table 1, Fig. 4C,D). All were

Thalamic inputs to striatal interneurons in monkeys

Fig. 3. Colour photomicrographs showing zones of overlap of BDA-containing thalamic fibres (brown deposit, DAB) and striatal interneurons (dark-blue deposit, Ni–DAB) in the dorsal part of the post-commissural putamen. The arrows point to immunoreactive interneurons. Scale bar=0.5 mm.

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Fig. 4. CM input to somatostatin- (A, B) and parvalbumin- (C, D) immunoreactive striatal interneurons. The BDA-containing terminals are labelled with DAB whereas the somatostatin- and parvalbuminimmunoreactive structures contain crystals of TMB. The arrowheads indicate asymmetric synapses between BDA-labelled terminals and somatostatin- or parvalbumin-immunoreactive dendrites (den, A–C) and a spine (Sp, D). Scale bars: (A)=1.0 µm; (B–D)=0.5 µm.

Thalamic inputs to striatal interneurons in monkeys

Fig. 5. CM inputs to ChAT-immunoreactive striatal interneurons (A–C). No synaptic contact was found between thalamic terminals and calretinin-containing striatal interneurons, despite the fact that the two sets of labelled elements were frequently apposed (D). The BDA-containing terminals are labelled with DAB whereas the ChAT- and calretinin-immunoreactive neurons contain TMB. In A and C, the arrowheads indicate asymmetric synapses between BDA-labelled terminals and ChAT-immunoreactive dendrites (den), whereas in (D) it shows an asymmetric synapse between a BDA-labelled terminal and an unlabelled dendrite. The stars indicate calretinin-immunoreactive elements in the vicinity of the anterogradely labelled terminal. The BDA-labelled bouton in (A) also establishes an asymmetric synapse with an unlabelled spine (Sp). Note the presence of axon terminals labelled with TMB in B (indicated by TMB) and C (indicated by a star). Scale bar=1.0 µm.

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Fig. 6. Anterogradely labelled thalamostriatal terminals that display calretinin (BDA/CR in A–B) and parvalbumin (BDA/PV in D) immunoreactivity. (C) shows an unmyelinated axon (Ax) labelled for BDA and calretinin. Other terminals in the same region are labelled with BDA only (A, B, D). The arrows in A, B and D point to aymmetric synapses between BDA-containing terminals and unlabelled dendrites (den, A, B) or spines (Sp, D). Scale bars: (A, B, D)=0.5 µm; (C)=1.0 µm.

Thalamic inputs to striatal interneurons in monkeys

dendritic shafts except for a single spine that received thalamic input on its head (Fig. 4D). Forty-two percent of the BDA-labelled terminals also contained the TMB reaction product indicating that they displayed parvalbumin immunoreactivity (Fig. 6D). No ultrastructural difference was noticed between single and double-labelled terminals, which were frequently encountered in the same area (Figs 4C–D, 6D). Seventeen single labelled terminals with TMB were also visualized. In the eight cases, where we could see the synaptic specialization, it was predominantly of the asymmetric type (73%) and associated with dendrites, while a minority (27%) formed symmetric synapses with large dendrites and perikarya. Biotinylated dextran-amine and choline acetyltransferase immunostaining. Three blocks were prepared for the analysis of the relationships between ChATcontaining neurons and CM terminals. Seventy-eight percent of the 104 BDA-containing terminals examined in those sections formed asymmetric synapses with dendritic shafts whereas 22% contacted dendritic spines. More than half of the structures that received thalamic input displayed ChAT immunoreactivity (Table 1, Fig. 5A–C). Thirteen ChATimmunoreactive boutons were found (Fig. 5B, C). Six of them formed a clear symmetric synapse with dendrites and spines, while another established an asymmetric axo-spinous synaptic contact (Fig. 5B). We could not see the synaptic specialization of the six remaining TMB-labelled terminals (Fig. 5C). Double-labelled terminals were not found in these sections. Biotinylated dextran-amine and calretinin immunostaining. Eight blocks were selected for this analysis. Among the 116 BDA-containing terminals encountered, 66% contacted unlabelled dendrites, whereas 34% formed synapses with unlabelled spines. Despite the fact that they were commonly apposed to each other, no clear synapses between calretinin-positive structures and BDA-containing terminals were found (Table 1, Fig. 5D). When examined through serial sections, it was often the case that the BDA-labelled terminals established synaptic contacts with neighbouring non-immunoreactive dendrites (Fig. 5D). As was the case for the parvalbumin-immunostained tissue, 43% of the BDA-labelled terminals that formed asymmetric synapses with dendrites and displayed ultrastructural features of thalamic terminals also contained the TMB reaction product (Fig. 6A–C). Myelinated and non-myelinated doublelabelled axons were also encountered (Figs 5D, 6C). Only five single-labelled terminals with TMB were found. None of them established clear synaptic contact in the plane of section. Control experiments. In the control experiments, where the primary antibodies to localize the various markers of interneurons were omitted, the tissue was

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entirely devoid of TMB crystals in the electron microscope. Importantly, none of the DABcontaining terminals had crystals of TMB associated with them in parvalbumin- and calretininimmunostained sections. Retrograde labelling of thalamostriatal neurons combined with parvalbumin and calretinin immunoreactivity As mentioned above, a substantial proportion of anterogradely labelled thalamostriatal terminals contained crystals of TMB in parvalbumin and calretinin-immunostained sections (Fig. 6). One of the possible explanation for this double labelling is that thalamostriatal neurons in CM display parvalbumin and/or calretinin immunoreactivity. To test this possibility, we placed unilateral injections of WGA–HRP in the post-commissural putamen in two monkeys and processed serial sections of the CM for either WGA–HRP/parvalbumin or WGA–HRP/ calretinin (Figs 7, 8). The WGA–HRP was revealed with TMB, which resulted in dark granules, whereas the parvalbumin and calretinin immunoreactivity was localized with DAB (Fig. 8A–C). There was no significant difference in the pattern of staining between the two animals. In both cases, the retrogradely labelled cells were found in the central and dorsal part of the CM while the parvalbuminand calretinin-immunoreactive neurons were homogeneously distributed throughout the nucleus (Fig. 7). Three different categories of labelled neurons were identified in the CM following these injections: (i) single-labelled neurons for parvalbumin and calretinin that only contained the DAB reaction product, (ii) single WGA–HRP-labelled cells that only contained dark granules of TMB and (iii) double labelled neurons that displayed both DAB and TMB reaction products (Figs 7, 8A–C). Quantitative estimates of double-labelled cells in five sections taken through the rostrocaudal extent of the CM in each case, revealed that 91% and 88% of retrogradely labelled cells in CM displayed parvalbumin or calretinin immunoreactivity, respectively (Figs 7, 8A–C). Co-localization of parvalbumin and calretinin in single centromedian nucleus neurons The fact that the majority of retrogradely labelled thalamostriatal neurons display parvalbumin and calretinin immunoreactivities suggest that both calcium binding proteins may co-exist at the single cell level in CM. We tested this possibility by double immunostaining using DAB and BDHC as chromogens. In the sections where calretinin was revealed with DAB and parvalbumin localized with BDHC, 61% of the parvalbumin-immunoreactive neurons (n=174) in the CM displayed calretinin immunoreactivity (Fig. 8D). Roughly, a similar proportion

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Fig. 7. Schematic drawings showing the distribution of retrogradely labelled neurons through the rostrocaudal extent of CM following injections of WGA–HRP in the post-commissural putamen. (A–C) show the distribution of single- and double-labelled neurons in parvalbumin-immunostained sections whereas (D–F) show the distribution of labelled neurons after calretinin immunostaining. Each dot represents three neurons. The number in parentheses indicate the approximate rostrocaudal stereotaxic level according to the atlas of Emmers and Akert.24 Note that most of the retrogradely labelled cells are labelled for parvalbumin and calretinin.

(58%) of calretinin-positive neurons (n=153) displayed parvalbumin immunoreactivity when the reactions were reversed, i.e. when parvalbumin was revealed first with DAB and calretinin second with BDHC. In contrast, calretinin and parvalbumin immunoreactivities were segregated in different populations of neurons in the striatum (Fig. 8E), the cerebral cortex and the substantia nigra, which is in keeping with previous studies in rats and monkeys.14,16,38,57

In the control experiments, where the primary antibodies for the second immunohistochemical reaction were omitted, none of the DAB-labelled neurons in the CM contained the BDHC reaction product. DISCUSSION

The main finding of this study is that somatostatin-, parvalbuminand ChATimmunoreactive striatal interneurons receive a

Thalamic inputs to striatal interneurons in monkeys

Fig. 8. (A–C) Photomicrographs showing examples of retrogradely labelled neurons in CM after injections of WGA–HRP in the post-commissural putamen. Sections in A and B are immunostained for parvalbumin, whereas the section in C is stained for calretinin. In this material, parvalbumin and calretinin are revealed with DAB whereas WGA–HRP is localized with TMB. The majority of retrogradely labelled neurons display parvalbumin (PV/HRP) and calretinin (CR/HRP) immunoreactivities, but a few single HRP-labelled neurons were also encountered (HRP in B). The double-labelled cells are intermingled with many single-labelled neurons for parvalbumin or calretinin. (D–E) show examples of immunostained neurons in CM (D) and putamen (E) in sections processed to reveal calretinin and parvalbumin immunoreactivities. In these cases, calretinin was localized first with DAB (brown amorphous deposit), whereas parvalbumin was revealed second with BDHC (blue crystalline deposit). Calretinin and parvalbumin largely co-exist in single CM neurons (D) whereas, as previously shown, they are segregated in different populations in the striatum (E). Scale bars: (A–C)=10 µm; (D, E)=10 µm.

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substantial input from CM, whereas calretinincontaining neurons are largely avoided by CM afferents in monkeys. This suggests that thalamic inputs can modulate the activity of striatal projection neurons either directly, via asymmetric excitatory synapses,64,75 or indirectly via interneurons.10,66 By virtue of their polarities, it is likely that direct and indirect thalamic inputs have opposite effects upon the activity of striatofugal neurons. The other major finding of our study is that the two calcium binding proteins, parvalbumin and calretinin, are expressed in thalamostriatal neurons. Although the functional role of these calcium binding proteins remains to be elucidated, it is worth noting that, in contrast to monkeys, calretinin and parvalbumin are not expressed by thalamostriatal neurons in rats.12,57 These findings, therefore, urge caution in making generalizations about the distribution and possible functions of calcium binding proteins in the CNS of primates and non-primates. In the following account, we will consider these observations in the light of previous anatomical findings and speculate on their significance for information processing in the striatum. Technical considerations The validity of the findings of this study rely upon three important factors: (i) the specificity of the source of the anterogradely labelled terminals, (ii) the specificity of the TMB staining for striatal interneurons, and (iii) the selection of appropriate areas for electron microscope analysis. We considered that most of the anterogradely labelled terminals visualized in the post-commissural putamen arose from CM, which was the major target of the BDA injection sites. Although the mediodorsal and subparafascicular nuclei were contaminated by the injections, none of these nuclei project significantly to the post-commissural putamen.61,68 Furthermore, the ultrastructural features and overall pattern of synaptic organization of the anterogradely labelled terminals visualized in the present study are largely congruent with those of thalamostriatal boutons described previously.64,67 To differentiate the BDA staining in the anterogradely labelled terminals from the different markers of striatal interneurons, we used two different chromogens to reveal the peroxidase reaction products. Although DAB is commonly used in immunocytochemistry, such is not the case for TMB. One can therefore argue that TMB might not be as sensitive and specific as DAB for revealing striatal interneurons. Various evidence indicate that such is not the case: (i) the overall pattern of distribution of TMB in striatal interneurons is congruent with that disclosed in previous studies using other chromogens,6,7,19,19a,71a (ii) all the neuronal perikarya that displayed TMB staining had an invaginated nucleus, a typical ultrastructural feature of

striatal interneurons,6,7,19,19a,20 and (iii) except for one parvalbumin-immunoreactive spiny dendrite (Fig. 4D), spines never contained crystals of TMB, suggesting that TMB was confined to aspiny interneurons. Moreover, we recently used TMB to localize retrogradely labelled striatopallidal neurons and found it to be highly reliable and specific for projection neurons.64 Quantitative data, like those presented in Table 1, must always be interpreted with caution when obtained from pre-embedding immunostained material. Various factors such as the limited penetration of antibodies into the tissue, the variable degree of overlap of anterogradely labelled terminals and striatal interneurons and the quality of dendritic immunostaining surely deserve attention when interpreting those data. In the present study, we tried to partly overcome these problems by selecting areas for electron microscope analysis that were comparable in their location, density of anterograde labelling and quality of interneuron immunostaining at the light microscope level. Of course, our analysis was shifted towards those striatal regions that contained dense plexuses of labelled terminals in register with a large number of immunostained interneurons. In the electron microscope, only those anterogradely labelled boutons that were less than 2 µm away from a TMB-labelled structure were considered in the estimates of the proportion of thalamic inputs to striatal interneurons (Table 1). We thought that being so selective would be a good way to increase the chance that the lack of TMB staining in striatal elements contacted by anterogradely labelled boutons was not due to a limited and heterogeneous penetration of antibodies into the sections. On the other hand, this might have led to overestimate the proportion of thalamic terminals in contact with interneurons. For that reason, the absolute values shown in the last column of Table 1 should not be seen as representative of the proportion of CM terminals in contact with interneurons versus projection neurons in the striatum, but rather as indications that CM inputs to somatostatin-, parvalbumin- and ChAT-containing interneurons is substantial and significantly stronger than thalamic inputs to calretinin-positive neurons. Thalamic inputs to somatostatin- and parvalbuminimmunoreactive interneurons According to our findings, somatostatin- and parvalbumin-immunoreactive cells are major targets of CM terminals in squirrel monkeys. These observations are different from those recently obtained in rats showing that striatal interneurons immunoreactive for neuropeptide Y which, in the striatum, largely co-exists with somatostatin69,73 do not receive inputs from PF.35 Whether this represents a difference in the pattern of synaptic innervation of striatal interneurons by CM and PF efferents or a species difference between rats and primates remains to be

Thalamic inputs to striatal interneurons in monkeys

established. A feature common to parvalbumin- and somatostatin-containing interneurons is that both populations contain GABA and form synapses with medium-sized projection neurons.7,8,17,26,37a,71a They can, therefore, mediate feedforward processing of thalamic inputs to projection cells.26 However, since the two populations of neurons display different physiological properties, they are likely to respond differently to thalamic activation and play different roles in the local circuitry of the striatum.37 Moreover, another important feature that differentiates these two groups of GABAergic interneurons is the extent of intrastriatal axonal arborizations. Whereas somatostatin-containing neurons have long axons (up to 1000 µm) that run in straight lines and emit few collaterals, parvalbumin-positive cells have a much more dense, but restricted (400–600 µm in diameter), axonal field.37,38 This suggests that activation of somatostatin-positive neurons by CM inputs may lead to widespread, but weak, inhibition of a large number of projection neurons, whereas activation of parvalbumin-containing neurons is likely to result in restricted, but strong, inhibitory influences over a small number of projection neurons. Another well established substrate for intrastriatal inhibitory interactions are the recurrent collaterals of projection neurons.77 However, recent electrophysiological data showed that interconnections between medium spiny neurons are relatively weak and, therefore, unlikely to underlie the fast inhibition commonly observed in the striatum after stimulation of extrinsic afferents.32 This reinforces the functional importance of feedforward inhibition through GABAergic interneurons in the synaptic circuitry of the striatum.33 It would be important to elucidate the roles of feedback versus feedforward inhibition generated in projection neurons by activation of the thalamostriatal pathway. Another potential function of CM input to somatostatin-immunoreactive interneurons might be to facilitate the release of nitric oxide, which is recognized as a messenger molecule involved in the control of blood regulation, synaptic efficacy and glutamate toxicity.11 Of particular interest, nitric oxide was recently found to inhibit N-methyl-aspartate (NMDA) receptor functions in cultured striatal neurons.48 This implies that nitric oxide released from interneurons after stimulation of thalamic afferents may modulate the activity of spiny neurons via inhibition of NMDA receptors at corticostriatal and thalamostriatal synapses. Thalamic inputs to cholinergic interneurons In keeping with previous findings in rats,43,50 cholinergic interneurons were found to be frequently contacted by CM terminals in monkeys. Recent microdialysis studies suggest that thalamic afferents are, in fact, the main excitatory drive of striatal cholinergic interneurons in rats.5,15 The functional

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importance of the thalamic input to cholinergic interneurons is supported by recent electrophysiological studies showing that the CM/PF is required for the expression of the sensory responsiveness acquired by ‘‘tonically active neurons’’, which resemble physiologically and morphologically the cholinergic interneurons,2,3 through behavioural conditioning.49 This is not surprising since CM is well recognized as a brain structure involved in maintaining a state of high vigilance and attention.41,71,74 The ChAT-immunoreactive terminals form symmetric synapses with proximal dendrites and perikarya of medium sized spiny neurons.31 Therefore, a potential role of thalamic inputs to ChATimmunoreactive neurons might be to increase the extracellular level of acetylcholine which, via activation of muscarinic receptors, has been found to stabilize the activity of neighbouring projection neurons either at a depolarized state, in which neurons can fire, or at a hyperpolarized state, in which neurons cannot fire.1,29,52 Via activation of cholinergic interneurons, the CM may, thus, act as modulator of striatofugal neurons to allow basal ganglia functions to be expressed. Moreover, because of their widespread intrinsic axonal projections, it is likely that, once activated, cholinergic interneurons influence the membrane potentials of striatofugal neurons distributed over large areas of the striatum.37,76 As discussed above, electron microscope studies could not demonstrate the existence of synaptic contacts between cortical terminals and cholinergic interneurons in the rat striatum.43,50 On the other hand, Wilson and colleagues76 showed that giant aspiny neurons, similar in morphology to cholinergic interneurons, respond monosynaptically to cortical stimulation. The most likely explanation of this discrepancy between anatomical and electrophysiological data is that cortical inputs are sparse and located on distal dendrites of cholinergic neurons. However, because cholinergic neurons are highly sensitive to incoming inputs, even the small number of distal cortical terminals are likely to be functionally important.37,38,76 It might, therefore, be of great interest to investigate the functional interactions between the proximal thalamic and distal cortical inputs to control the activity of individual cholinergic interneurons. Lack of centromedian nucleus inputs to calretininimmunoreactive interneurons One of the major finding of this study is the differential density of thalamic innervation of calretinin-immunoreactive cells in comparison to other populations of striatal interneurons. Of course, the validity of these findings rely upon a comparable sampling of material at the electron microscope level for the four populations of interneurons. To make sure that such was the case, we paid a particular

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attention at selecting striatal zones of overlap that were quite similar in both the extent of dendritic labelling of interneurons and the density of anterogradely labelled CM terminals. In fact, at the electron microscope level we found many thalamostriatal terminals apposed to calretinin-labelled dendrites, but none of these appositions ended up in synaptic contacts when followed through serial sections. The BDA-labelled boutons, rather, established asymmetric synapses with neighbouring non-immunoreactive dendrites or spines, suggesting that CM afferents tend to avoid calretinin-containing elements. However, we cannot rule out the possibility that the CM inputs to calretinin-positive neurons might be located on the most distal part of the dendritic tree, and that these dendritic processes were not labelled in our material. Whether calretinin-positive neurons throughout the striatum are totally devoid of thalamic inputs or receive afferents from other intralaminar or specific thalamic nuclei68 remains to be established. Another possibility is that calretininpositive neurons receive most of their excitatory inputs from the cerebral cortex, but not much from the thalamus. If such is the case, this would be the opposite of cholinergic interneurons which, as mentioned above, receive most of their excitatory inputs from the intralaminar nuclei in rats and monkeys.15,43,50 Future physiological studies of the role of calretinin-containing neurons in the neuronal network of the striatum should help to understand the significance of these electron microscope obvservations. Calcium binding proteins in thalamostriatal neurons The results of our study demonstrate that parvalbumin and calretinin are expressed, and likely to be co-localized, in individual thalamostriatal neurons in monkeys. The existence of an extrinsic source of parvalbumin-immunoreactive terminals in the striatum was previously suggested by Bennett and Bolam7 in the light of electron microscopic data showing that most of the parvalbumin-immunoreactive terminals in the monkey striatum form asymmetric synapses. Our anterograde and retrograde tracing experiments revealed that, indeed, a large number of parvalbumin-immunoreactive terminals in the putamen arise from the CM in squirrel monkeys (see also Refs 7 and 34). Surprisingly, such is not the case in rodents where parvalbumin-containing terminals in the striatum always form symmetric synapses, suggesting that they all arise from parvalbumin/GABA interneurons.8,42 In line with these observations, immunocytochemical studies showed that neurons in PF, which are known as the main source of thalamostriatal fibres in rodents,27 do not display parvalbumin immunoreactivity in rats.12 This differential distribution of calcium binding proteins in thalamostriatal neurons between rats and primates also applies to calretinin. In contrast to previous data

showing the absence of calretinin immunoreactivity in the rat PF,57,78 we found that almost 90% of thalamostriatal neurons in CM are immunoreactive for calretinin in squirrel monkeys. Whether or not these differences in the expression of calretinin and parvalbumin have any functional consequences on the activity of thalamostriatal neurons between rats and primates remain to be established. The co-existence of calretinin and parvalbumin in individual neurons is a feature which, to our knowledge, is common during development,80 but not very frequent in the adult CNS,4,18,25,34,45 except in the superior colliculus, where double-stained neurons were commonly found in humans.45 Various observations indicate that the high frequency of colocalization described in this study is not the result of non-specific interactions between chromogens used in the first and second immunocytochemical reactions. First, we examined other brain areas in the same sections and found that, as previously reported, parvalbumin and calretinin were largely segregated in different neuronal populations in the striatum,25,37,38 the cerebral cortex,14,18 and the substantia nigra.16,57,59 Second, single-labelled neurons with DAB or BDHC, intermingled with the doublelabelled cells, were found in the CM. Third, in control sections, DAB-labelled CM neurons were devoid of BDHC deposit. Because of the lack of information on the physiological properties of CM neurons, the significance of the co-expression of calcium binding proteins in those cells is still unclear. Of course, by virtue of their calcium-buffering properties, one may speculate that calretinin and parvalbumin jointly modulate the activity of thalamostriatal neurons via different mechanisms which include: altering the duration of action potentials, facilitating neuronal bursting activities by inhibiting Ca2+-dependent K+ currents and protecting them against neurotoxicity induced by Ca2+ overload during prolonged period of activation. Future studies are clearly needed to test these hypotheses. CONCLUSIONS

The findings of this study strongly suggest that thalamic inputs from CM may modulate the activity of striatofugal neurons, not only via direct asymmetric excitatory synapses,64 but also indirectly via activation of GABAergic and cholinergic interneurons. The relative importance of the direct and indirect thalamic influences upon the activity of individual striatofugal neurons surely deserve further considerations. Another issue that remains to be established is the mechanism by which cortical and thalamic inputs interact to drive individual striatal neurons. Although the cholinergic interneurons are contacted preferentially by thalamic afferents, the parvalbumin- and somatostatin-containing neurons appear to receive a comparable innervation from the thalamus and the cerebral cortex.44,73a On the other

Thalamic inputs to striatal interneurons in monkeys

hand, calretinin-positive neurons are quite unique since they appear to be devoid or, at least much more weakly innervated than other striatal interneurons, by CM afferents. These observations, combined with the fact that individual populations of interneurons display different membrane properties that allow them to be more or less sensitive to their synaptic inputs37,38,76 suggest that the effects and interactions between thalamic and cortical afferents are likely to be different and specific for each population of interneurons. Another important issue that deserves consideration in the interpretation of the functional significance of increased activity of striatal interneurons is the extent of transmission of the resulting effects to projection neurons. The fact that some interneurons have sparse but widespread axonal fields (somatostatin-immunoreactive and cholinergic) compared to others that have a much denser but more restricted axonal arborization (parvalbuminimmunoreactive),38 suggest that the striatofugal

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neurons may be modulated differently by the three classes of thalamic-receiving neurons. Finally, another interesting issue raised by our findings is the potential role of calcium binding poteins in controlling the activity of thalamostriatal neurons in monkeys versus rats. As mentioned above, the functional significance of this species difference is not clear yet but, because of the calcium-buffering properties of parvalbumin and calretinin,4 it is likely that this might influence the firing pattern and release of transmitter of individual thalamostriatal neurons. A better knowledge of the physiological properties of thalamostriatal neurons in rats and primates is essential to fully understand to functional implications of these findings. Acknowledgements—The authors thank Jean-Franc¸ois Pare´ for technical assistance and Frank Kiernan for photography. This research was supported by NIH grants R01 NS37948-01 and RR00165 and internal funds from the Department of Neurology at Emory University.

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