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Local circuit neurons in the rat ventrobasal thalamus—A gaba immunocytochemical study
z%‘errroscienceVoi. 21, No. 1, pp. 229-236, 1987 Print& in Great Britain
0306-4522/87 $3.00 + 0.00 Pergamon JcwnaIs Ltd 0 1987 IBRO
LOCAL CIRCUIT NEURONS IN THE RAT VENTROBASAL THALAMUS-A GABA IMMUNOCYTOCHEMICAL STUDY ROGER M. HARRIS* and ANITA E. HENDRICKSON~ Departments of Biological Structure and tOphthalmology, University of Washington, School of Medicine, Seattle, WA 98195, U.S.A.
Abstract-The ventrobasal thalamus of seven rats was processed for immunocytochemistry using antisera to glutamate decarboxylase or y-aminobutyrate (GABA). Glutamate decarboxylase-stained sections showed a network of stained fibers and terminals but no stained cell bodies. GABA-stained sections had fewer stained fibers and terminals but did show a few stained cell bodies. Cell bodies were especially apparent when carbaxole was used for a chromogen for the ~roxida~anti~roxida~ visualization. The GABA-stained ‘cells were found to be distributed throughout the ventrobasal complex, to have smaller soma cross-sectional areas than most other cells (81 k 34 pm vs 105 i 36 pm for all cells) and to make up 0.4 +0.3% of the neuronal population of the ventrobasal complex. Injections of horseradish peroxidase into the somatosensory’ cortex (SI) retrogradely filled many neurons in the ventrobasal thalamus, but none of these labeled neurons were double labeled with GABA. These results indicate that the GABA-labeled cells probably represent a small population of local circuit neurons in the rat ventrobasal thalamus.
The neuronal
circuitry of the somatosensory thalamus follows a typical plan in most mammals. There are thalamocortical relay cells, often of several types, “,2gJ3local circuit neurons17,22,26and a neuropil containing complex synaptic glomeruli.20 This pattern has been found in cats,‘7*22~26 rabbits,16 Galago “~17and squirrel monkey. t6 The same structural organization has been found in other thalamic nuclei, including the lateral geniculate nucleus of all animals studied so far.’ The proportion of local circuit neurons in the somatosensory thalamus (the ventrobasal complex, VB) of the cat has been estimated at between 2022,26 and 30%.17 In the VB of the rat, however, there has been very little evidence for local circuit neurons. Golgi studies10*1v*24 have detailed the thalamo~ortical relay cells but have found no smaller Golgi type II cells. Large injections of horseradish peroxidase made into the somatosensory cortex have resulted in the labeling of almost all cells in the ventrobasal thalamus.g~23~31 In a careful study of this type, using thin plastic sections, Lee’ determined that less than 1% of thalamic neurons were unlabeled. A more direct search for local circuit neurons has been made possible by immun~ytffihemical methods. These studies depend on the assumption that local circuit neurons in the thalamus are inhibitory, and use y-aminobutyrate (GABA) as
*To whom correspondence should be addressed. Abbreviations: DAB, 3,3’-diaminobenzidine; GABA, y-aminobutyrate; GAD, glutamate decarboxylase; HRP, horseradish peroxidase; LCN, local circuit neuron; PBS, phosphate-buffe~d saline; VB, ventrobasal complex.
a neurotransmitter.11*17,*oMost immun~yt~hemical studies have been done by localizing glutamate decarboxylase (GAD), the rate-limiting enzyme for the synthesis of GABA. Such studies in the cat ventrobasal complex’7~22~26 and in the lateral geniculate nucleus of several mammalssJ’ have revealed sizeable numbers of GAD-stained neurons. No detailed studies of GAD-labeled cells in rat VB have been reported, but several authors have noted that such cells are very rare in this nucleus.1’~‘6~22 Recently, a more direct immunocytochemical approach has been developed using an antiserum to GABA itself.12 This antiserum appears to be more sensitive for demonstrating GABA-containing cell bodies than GAD antisera.6J2 A survey of the rat thalamus using the GABA anti~rum” showed very few stained neurons in the ventrobasal complex. In the present work, we have used the GABA antiserum in conjunction with horseradish peroxidase injections in the somatosensory cortex to locate putative GABA-containing local circuit neurons in the rat ventrobasal complex.
EXPERIMENTALPR~EDUR~ Eight Spragu*Dawley rats of about 25Og weight were used. Five rats were used for localization of neurons using antisera to GAD and GABA. These rats were perfused with one of the following fixatives in 0.05 M phosphate buffer, pH 7.4: 0.2% picric acid, 4% paraformaldehyde and 0.05% glutaraldehyde 25. , 4% paraformaldehyde, 6.05% sodium periodate and 0.34% lysine$ or 1%’ paraformaldehvde and 3% glutaraldehyde. After an 8 h waii for thorough hxation, the brains were postfixed with lo%, then 20% sucrose in phosphate buffer, removed and cryoprotected with 30% surose-phosphate buffer. Froxen sections 30 pm thick were cut through the thalamus.
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ROGERM. HARRISand
230
Tissue sections were processed for immunocytochemistry using the standard peroxidase-antiperoxidase methods.5.‘2.27 Sections through different parts of the ventrobasal complex were incubated in either antiserum to GAD (obtained from J. Y. Wu, diluted 1:300) or to GABA (Immunonuclear, diluted 1:500 to I :3000). All serum dilutions were made with phosphate-buffe~d saline (PBS) containing 1%normal goat serum and 0.2% Triton X-100 detergent. Sections were incubated in primary antiserum at 4°C for 36 h. Following a 2-h rinse in PBS containing 0.1% T&on X-100, the sections were incubated in goat anti-rabbit IgG diluted I:30 for I h at 37”C, washed for 1 h, incubated in anti-rabbit peroxidase-antiperoxidase diluted I : 100 for I h at 37°C and rinsed first in PBS and then in Tris buffer. Sections were then reacted with 0.03% diamino~nzidine and 0.~6% H,O, in Tris buffer for 8min and rinsed with PBS. The reacted sections were mounted on slides, coverslipped and examined without counterstaining. Horseradish peroxidase injections
In three rats, GABA immunocytochemistry was combined with injections of horseradish peroxidase (HRP), using the protocol of Bowker ef al.’ In two rats, 40% HRP (Sigma type VI) in 2% dimethyl sulfoxide was injected stereotaxically into the somatosensory (SI) region of the cortex in one or two injections on each side. Each injection contained 0.5 ~1 of HRP and was pressure-injected using a 1 ~1 Hamilton syringe over 5 min. In one rat, 25% HRP in 2% dimethyl disulfoxide was iontophoretically injected into the ventrobasal complex of each side, using a ZpA, 50% on-off constant current source for 5 min. The rats were allowed to survive for 48 h and then perfused with 4% paraformaldehyde plus 0.1% glutaraldehyde in phosphate buffer, followed by 10% and then 20% sucrose in phosphate buffer. The brains were infiltrated in 30% sucrose buffer and then frozen-sectioned at 30pm. Every fourth section was processed for HRP visualization using the cobalt-intensified tetramethyl benzidine method,’ mounted on a subbed slide and counterstained with Cresyl Violet. Other sections in the same area were lirst reacted using the cobalt-intensified ~amino~nzidine method’ and then processed for immunocytochemistry using the antiserum to GABA. The immunocytochemical procedure was the same as that given above except that instead of using 3,3’-diaminobenzidine (DAB) in the final visuali~tion step, 0.02% 3-amino-9~thylcarbazole mixed with 5% dimethyl formamide in 0.05 M acetate buffer was used.3 This resulted in a red-colored reaction product easily distinguishable from the blue-black DAB reaction. These sections were mounted in glycerin, coverslipped and kept at 4°C. GABA ceil an&&v
Quantitative analysis of GABA-stained cells was carried out using the three double labeled animals. The GABAlabeled cells in VB were counted in four sections from each of two animals and their locations plotted using a 4 x objective and a camera lucida attachment. An approximate
ANITA
E. HENDIUCK~~R
overall distribution was obtained by plotting all eight sections on a single drawing of VB, adjusting the positions of the cells relative to the boundaries of VB to account for differences in shape of VB at different levels. Cell size distributions were determined by drawing each labeled cell in the above sections using a 100 x oil objective. These drawings were then measured to determine the somal cross-sectional area using a micr~omputer-driven tablet system.2* Similar measurements were made on two Nisslstained sections from two different rats. In these sections all cells visible within VB in several fields were drawn. Histograms of the cell size dist~bution for GABA-stained cells and for all Nissl-stained cells were prepared. To determine the proportion of GABA-containing cells, a count of all cells in eight Nissl-stained fields (average size: 0.29 mm*) was made using a 25 x objective. Two of these fields were areas retrogradely labeled by HRP where essentially every cell was HRP-labeled. The size of each field was measured using the tablet system, yielding a density of cells for each area. This was compared with the density of GABA-labeled cells, determined from the above counts and the area of VB in each section. Comparison of the average densities of GABA-labeled cells and all Nissl-stains cells gave the proportion of GABA-labeled cells in VB.
RESULTS
In GAD- or GABA-stained sections, the ventrobasal complex (VB) was easily distinguished by its reticulated appearance, due to numerous discrete bundles of unstained myelinated fibers which course through it (Fig. I). VB is bounded laterally by the external medullary lamina and ventrally by the medial lemniscus. The medial boundary was taken to be at the onset of the myelinated bundles. In optimal sections a thin interior lamina could be seen dividing ventral posterior medial from ventral posterior lateral nuclei. The VB region was diffusely stained a light brown by GAD or GABA, in distinction to the heavily stained thalamic reticular nucleus, which was seen laterally and ventrally to VB. Sections stained for GAD showed a dense network of stained fibers and puncta throughout VB (Fig. 2). These puncta, which probably represent axon terminals, were often found surrounding unstained cell bodies. There were no obviously GAD-stained cell bodies in VB, in contrast to cells of the tha~amic reticular nucleus in the same section which were heavily stained (Fig. 2). Thus, there was no positive evidence for GABA-containing neurons in VB using the GAD antiserum.
Fig. 1. Low-power view of the lateral thalamus double stained for GABA and HRP. The ventrobasal complex (VB) is the semicircular reticulated region, bound ventrally by the medial lemniscus (ML) and laterally by the heavily stained thalamic reticular nucleus (TRN). A small band of retrogradely labeled HRP cells is visible in the center of VB. Bar = 1 mm. Fig. 2. A section through the edge of VB stained with antiserum to GAD. Note the intense fiber and terminal labeling in VB in which cell bodies are outlined by stained terminals. The thalamic reticular nucleus (TRN), unlike VB, contains heavily labeled cell bodies. Bar = 100 pm. Fig. 3. A section through part of VB stained for GABA. There is some staining of fibers and terminals and one well-stained cell body (arrow). Bar = IOOpm. Fig. 4. A section through the lateral geniculate nucleus stained for GABA. Note the numerous well-stained cell bodies, unlike the sparse staining seen in VB. Bar = 100 {tm.
LCNs in rat ventrobasal thalamus
Figs
231
Figs 5-10.
232
233
LCNs in rat ventrobasal thalamus
GABA CELLS
ALL CELLS VPM \
Fig. 1I. Composite distribution of GABA-labeled cells from eight separate sections, all plotted on the same drawing. Fi, fimbria; Hi, hippocampus; IC, internal capsule; LD, lateral dorsal; ML, medial lemniscus; OT, optic tract; PO, posterior nucleus; ST, stria terminalis; TRN, thalamic reticular nucleus; VPL, ventral posterior lateral; VPM, ventral posterior medial; ZI, zona incerta.
Fig. 12. (A) Histogram of cross-sectional areas of GABAlabeled cells from eight sections through VB in two animals (64 cells). Mean (arrow) is 81 + 34pm*. (B) Histogram of cross-sectional areas of all cells in eight Nissl-stained fields from two animals (170 cells). Mean (arrow) is 105 f 36 pm*.
Fig. 5. A section through VB stained for GABA using carbazole as the chromogen. stained cell bodies are present. Bar = 1OOpm.
Two GABA-
Fig. 6. A GABA-stained
cell in VB showing staining of the proximal parts of two dendrites. Bar = 1OOym. Fig. 7. A section through VB double stained for GABA and HRP. HRP has been injected into the SI cortex, resulting in a band of retrogradely labeled cells in VB. A single GABA-stained cell, without HRP, is shown by the arrow. Bar = 100 pm. Fig. 8. A section through VB stained for GABA and HRP, following an HRP injection into the SI cortex. A number of HRP-labeled cells are present, as well as one prominent GABA-labeled cell (arrow). Bar = 100 pm. Fig. 9. A high-powered view of the GABA-labeled cell of Fig. 8. A second, larger cell was retrogradely labeled with HRP, as shown by the prominent HRP granules. Note the lack of HRP granules in the GABA-labeled cell. Bar = 10 pm. Fig. 10. High-power view of a double labeled neuron in the thalamic reticular nucleus (*). HRP has been injected in VB, resulting in the retrograde labeling of thalamic reticular cells. Note the reddish tinge, indicating the cell is GABAergic, and the prominent HRP granules. Bar = 10pm.
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The results using the GABA antiserum were somewhat different. The staining of fibers and terminals was less intense than with the GAD antiserum, particularly in those animals fixed with low levels of glutaraldehyde. There were occasional scattered cell bodies which were labeled (Fig. 3). This was particularly noticeable in the double labeled animals, where the GABA label was a red color due to use of carbazole as the chromogen (Fig. 5). The labeled cells tended to be small, round to oval shaped, and sparse in number. In a few cells, parts of the proximal dendrites were also labeled (Fig. 6). From the counts of eight sections through VB, the average number of labeled cells was 4.1 f 2.7 per section. This paucity of GABA cells in VB was not just an artefact of staining, because numerous GABA-stained cells were seen on the same sections in the dorsal lateral geniculate (Fig. 4), hippocampus and cerebral cortex. The distribution of GABA-labeled cells was determined by plotting their positions in eight sections. The results from all eight sections were consolidated and plotted on a single plot in Fig. 11. Labeled cells were scattered throughout both the lateral and medial subdivisions of VB. There were fewer labeled cells in the medial part of VB and more labeled cells along the lateral boundary. Some of the latter cells could have actually been cells of the thalamic reticular nucleus because the boundary between VB and the reticular nucleus was sometimes unclear. To determine whether some of these GABAlabeled cells could be thalamocortical relay cells projecting to the cortex, HRP injections were made in two rats into the SI somatosensory cortex. These resulted in bands of HRP-labeled cells in VB extending in a rostralcaudal direction. Within these bands, almost all cell bodies were retrogradely labeled with HRP (Fig. 7). After staining for GABA, using carbazole as the chromogen, occasional red GABAlabeled cell bodies were found in the vicinity of the HRP-labeled cells (Figs 7-9). No double labeled cells containing both HRP and GABA were seen. In order to be certain that our technique would detect a double labeled cell, a third rat was injected with HRP into VB itself. This resulted in the retrograde transport of HRP by cells in the thalamic reticular nucleus, which project their axons into VB and are known to contain GABA.‘* Double labeled neurons with red cytoplasm containing blue-black HRP granules were easily identified (Fig. IO). A comparison between these cells (Fig. 10) and the GABA-labeled and HRP-labeled cells (Fig. 9) in VB confirmed that none of the cells in the cortex-injected rats was double labeled. This provides evidence that the GABAlabeled cells do not project to the cerebral cortex and can thus be considered local circuit neurons. The distribution of soma cross-sectional areas for GABA-stained cells is shown in Fig. 12A and a similar distribution for all cells in Nissl-stained sections in Fig. 12B. The average cross-sectional area for GABA cells was 8 1 + 34 pm2 (population stan-
ANITA E. HENDRICKSON
dard deviation); for all cells, it was 105 & 36pm2. A r-test shows these averages to be significantly different P < 0.001). The ratio of the cross-sectional areas for GABA cells to all cells is 0.77 f 0.59. Thus the GABA-labeled cells tend to be somewhat smaller than the thalamocortical relay cells, although the distributions show there is considerable overlap. GABA staining was determined by measuring the density of GABA-labeled cells and the density of all cells in the Nissl-stained sections. The density of GABA-labeled cells over eight counted sections was 2.1 + 1.4cells/mm2, while the density of cells found in Nissl-stained sections was 495 + 183. The proportion of GABA cells in VB is then 0.4 + 0.3%.
DISCUSSION Our results confirm the claim by Otterson and Storm-Mathisen’2 that the GABA antiserum is more sensitive for staining cell bodies than GAD. It was difficult to find stained cell bodies in VB in our GAD-stained sections, perhaps because of the heavy staining of fibers and terminals in the neuropil or because colchicine pretreatment, which increases cell body staining by GAD,2’ was not used. Stained cells were easily seen using the GABA antiserum, particularly when carbazole was used as the chromogen. It appears that the GABA antiserum is more effective for demonstrating cell bodies and the GAD antiserum is more selective for axonal terminals. The major result of this work is to confirm that there is a small population of GABA-containing neurons in the ventrobasal thalamus of the rat. These neurons do not project to the somatosensory cortex as indicated by the lack of double labeled neurons in rats with HRP injected into the SI cortex. It is possible that the GABA cells could project specifically to the SII cortex and not SI, but this seems unlikely since the cells that project to SII have been located primarily in the posterior pole of VB.30 Thus, we may tentatively identify these GABA cells as a sparse population of local circuit neurons (LCNs). Several authors have suggested, on the basis of previous experiments using Golgi or retrograde HRP labeling techniques, that the rat VB may be devoid of LCNs.‘0,20.23.24,3’More recent immunocytochemical studies have suggested there may be a few.“.‘3s22 Our finding that 0.4 + 0.3% of VB cells are LCNs is consistent with the estimate made by Lee’ that there are less than 1% LCNs in this nucleus. These results are similar to those found in the opossum VB, in which only a few GAD-labeled cells are seen.“x” There is, however, a marked species difference between the rat and opossum and higher mammals such as the rabbit, cat or Galago, in which VB contains 2&30% LCNS.‘~.“.~~.~’ This may represent an evolutionary trend toward increasing numbers of LCNs in VB in higher mammals, as discussed
23.5
LCNs in rat ventrobasal thalamus by Penny et aLI It is interesting to note that the dorsal lateral geniculate nucleus .in our rats does contain numerous GAEA-stained neurons as seen in other studies,*~” so that there is a difference in circuitry between the two thalamic nuclei even in the same species. The cell dist~butions in our study indicate that the GABA-labeled cells are, on average, smaller than the thalamocortical relay cells. This is in agreement with studies of GAD-labeled neurons in cat and ~~~~~~ I1.22.26 and in rabbit and opossum.‘6 Large and small neurons were also found in Nissl-stained sections of the mouse VR3* The ratio of the average cross-sectional areas for GABA cells to all cells in our work was 0.77. This agrees with the value found in opossum, 0.7’9.16Animals which contain a significant number of LCNs were found to have a significantly lower ratio of cross-sectional areas (0.59 for rabbit, 0.55 for cat, 0.52 for Galago’6). Local circuit neurons in the rat and opossum are thus larger, relative to the thalamocortical relay cells, than LCNs in higher mammals. This may imply that there is less differentiation between the two cell types in these animals than in higher animals, and gives further support to the idea that the rat and opossum are early stages in the evolutionary development of the thalamus. The functional significance of these few LCNs in the neural circuitry of the rat VB is not at all clear. It is questionable whether there are enough of them
to play a major role, as LCNs do in other mammals. Possibly these cells are involved in the processing of one specific submodality of somatosensory sensation. The small number of LCNs does indicate that the rat VB has, for the most part, a very simple circuitry. At the ultrastructural level, there appear to be few, if any, of the synaptic CompIications seen in higher mammals, such as large synaptic glomeruli or dendrodendritic synapses. 9,20Furthermore, intracellular HRP injections in rat VB4 indicate that all thalamocortical relay cells are similar in structure. This homogeneous neuronal population and lack of synaptic complexity makes the rat VB an excellent model for the study of thalamic processing. CONCLUSiONS
The rat ventrobasal thalmus does appear to contain a small population of local circuit neurons, as indicated by labeling of cells by an antiserum to GABA. These cells, which make up 0.4 + 0.3% of the population of VB, are smaller on average than the relay cells, and are dist~but~ throu~out VB. The functional significance of these local circuit neurons is not yet clear. Aclcnowledgemerats-This work was supported by National Institutes of Health Grants NS-19073 to R.M.H. and EY-01208 and EY-04536 to A.E.H. We thank J. Y. Wu for a gift of GAD antisera. We would like to thank Haila Vickland and Bente Noble for technical assistance and Doris Ringer for typing the manuscript.
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1. Adams J. C. (1977) Technical considerations 2, 141-145. 2. Berkley K. J. (1983) Afferent projections ~o~to.qe~sory ~n?egrut~o~ in the Thufum~ Amsterdam. 3. Bowker R. M., Westlund K. N., Sullivan immunocytochemical staining method for Cytochem. 4.
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Harris R. M. (1984) Anatomical structure of physiologically identified neurons of the rat ventrobasal thalamus. Sot. Neurosci. Abstr. 10, 484. Hendrickson A. E., Ogren M. P., Vaughn J. E., Barber R. P. and Wu J.-Y. (1983) Light and electron microscopic immunocytochemical localization of glutamic acid decarboxylase in monkey geniculate complex: evidence for GABAergic neurons and synapses. J. Neurosci. 3, 1245-1262. Hendrickson A., Ryan M., Noble B. and Wu J. Y. (1985) Colocalization of 3H-muscimol and antisera to GABA and glutamic acid decarboxylase within the same neurons in monkey retina. Brain Res. 348, 391-396. Jones E. G. (1985) The Talks. Plenum Press, New York. Lieberman A. R. and Webster K. E. (1974) Aspects of the synaptic organization of intrinsic neurons in the dorsal lateral geniculate nucleus. J. Neurocytol. 3, 677-710. Lee C. L. (1981) The structural organization of the rat ventrobasal complex. Thesis, University of California, Berkeley. McAllistair J. P. and Wells J. (1981) The structural organization of the ventral posterolateral nucleus in the rat. J. camp. Neural. 197, 271-301. Ohara P. T., Lieberman A. R., Hunt S.P. and Wu J.-Y. (1983) Neural elements containing glutamic acid decarboxylase (GAD) in the dorsal lateral geniculate nucleus of the rat: immunohist~hemical studies by light and electron microscopy. Neuroscience 8, 189-2 11. Ottersen 0. P. and Storm-Mathisen J. (1984) Glutamate- and GABA-containing neurons in the mouse and rat brain, as demonstrated with a new immunocytochemical technique. J. camp. Neural. 229, 374-392. Ottersen 0. L. and Storm-Mathisen J. (1984) GABA-containing neurons in the thalamus and pretectum of the rodent. Anat. Embryol. 170, 197-207. Pearson J. C. and Haines D. E. (1980) Somatosensory thalamus of a prosimian primate (Galago se~egulensis). II. An HRP and Golgi study of the ventral posterolateral nucleus (VPL). J. camp. Neurol. 190, 559-580. Pearson J. C., Norris J. R., Coccia M. R. and Mann B. L. (In press) A Golgi study of the opossum ventral basal complex. J. camp. Neurol.
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16. Penny G. R., Conley M., Schmechel D. E. and Diamond I. T. (1984) The distribution of glutamic acid dccarboxylase immunoreactivity in the diencephalon of the opossum and rabbit. J. camp. Neural. 228, 38-56. 17. Penny G. R., Fitzpatrick D., Schmechel D. E. and Diamond I. T. (1983) Glutamic acid decarboxylase-immunoreactive neurons and horseradish peroxidase-labeled projection neurons in the ventral posterior nucleus of the cat and Galago senegalensis. J. Neurosci. 3, 1868-I 887. 18. Peschanski M., Ralston H. J. and Roudier F. (1983) Reticularis thalami afferents to the ventrobasal complex of the rat thalamus: an electron microscope study. Brain Res. 270, 325-329. 19. Ramon y Cajal S. (1966) Studies on zhe Dieneepha~~n. E. Ram~n-Moliner (Transl.) Thomas, Springfield, IL. 20. Ralston H. J. III (1983) The synaptic organi~tion of the ventrobasal thalamus in the rat, cat, and monkey. In So~a~o~e~sor~~Integration in the Thalamus (eds Macchi G., Rustioni A. and Spreafico R.), pp, 24ll250. Elsevier. Amsterdam. 21. Ribak C. E., Vaughn J. E. and Saito K. (1978) Immunocytochemical localization of glutamic acid dccarboxylase in neuronal somata following colchicine inhibition of axonal transport. Brain Res. 140, 315-332. 22. Rustioni A., Schmechel D. E., Spreafico R., Cheema S. and Cuenod M. (1983) Excitatory and inhibitory amino acid putative neurotransmitters in the ventralis posterior complex: an autoradiographic and immunocytochemical study in rats and cats, In Somatosensory Integration in the Thalamus (eds Macchi G., Rustioni A. and Spreafico R.), pp. 365-383. Elsevier, Amsterdam. relay neurons in the rat VB complex studied by 23. Saporta S. and Kruger L. (1977) The organization of thal~ocortical the retrograde transport of horseradish peroxidase. J. camp. Neurof. 174, 187-208. 24. Scheibel M. E. and Scheibel A. B. (1966) Patterns of organization in specific and nonspecific thalamic fields. In The Thalamus (eds Purpura D. M. and Yahr M. D.), pp. 1346. Columbia University Press, New York. 2s. Somoygi P. and Takagi H. (1982) A note on the use of picric acid paraformaldehyde fixative for correlated light and electron microscopic immunocytochemistry. Neuroscience 7, 1179-l 183. 26. Spreafico R., Schmechel D. E., Ellis L. C. Jr and Rustioni A. (1983) Cortical relay neurons and interneurons in the n. ventralis posterolateralis of cats: a horseradish peroxidase, electron microscopic, Golgi and immunocytochemical study. Neuroscience 9, 491-509. 27. Sternberger L. A. (1979) ~mmunocyt~chemistry, pp. 104169. John Wiley, New York. 28. Sundsten J. W. and Prothero J. W. (1983) Thr~-dimensional r~onstruction from serial sections: II. A microcomputerbased facility for rapid data collection. Anat. Rec. 207, 665-671. 29. Tiimbiil T., Ungvary G., Hajdu F. and Modarasz M. (1969) Quantitative aspects of neuron arrangement in the specific thalamic nuclei. Acta Morphoi. Acad. Sci. Hung. 17, 2999313. 30. Weinberg R., Barbaresi P., Cheema S., Sprealico R. and Rustioni M. (1984) SI and SII projections from somatosensory thalamus of rats. Sot. Neurosci. Abstr. 10, 945. projection 31. Wells J., Mathews T. J. and Ariano M. A. (1982) Are there interneurons in the thalamic somatosensory nucleus
in the rat? Sot. Xeurosci. Abstr. 8, 37.
32. Woolsey T. A., Anderson J. R., Wann J. A. and Stanfield B. B. (1979) Effects of early vibrissa damage on neurons in the ventrobasal (VB) thalamus of the mouse. J. camp. Neuroi. 184, 363-380. 33. Yen C-T. and Jones E. G. (1983) Intracellular staining of physiologically identified neurons and axons in the somatosensory thalamus of the cat. Brain Rex 280, 148-154. (Accepted 5 August 1986)
0306-4522/87 $3.00 + 0.00 Pergamon JcwnaIs Ltd 0 1987 IBRO
LOCAL CIRCUIT NEURONS IN THE RAT VENTROBASAL THALAMUS-A GABA IMMUNOCYTOCHEMICAL STUDY ROGER M. HARRIS* and ANITA E. HENDRICKSON~ Departments of Biological Structure and tOphthalmology, University of Washington, School of Medicine, Seattle, WA 98195, U.S.A.
Abstract-The ventrobasal thalamus of seven rats was processed for immunocytochemistry using antisera to glutamate decarboxylase or y-aminobutyrate (GABA). Glutamate decarboxylase-stained sections showed a network of stained fibers and terminals but no stained cell bodies. GABA-stained sections had fewer stained fibers and terminals but did show a few stained cell bodies. Cell bodies were especially apparent when carbaxole was used for a chromogen for the ~roxida~anti~roxida~ visualization. The GABA-stained ‘cells were found to be distributed throughout the ventrobasal complex, to have smaller soma cross-sectional areas than most other cells (81 k 34 pm vs 105 i 36 pm for all cells) and to make up 0.4 +0.3% of the neuronal population of the ventrobasal complex. Injections of horseradish peroxidase into the somatosensory’ cortex (SI) retrogradely filled many neurons in the ventrobasal thalamus, but none of these labeled neurons were double labeled with GABA. These results indicate that the GABA-labeled cells probably represent a small population of local circuit neurons in the rat ventrobasal thalamus.
The neuronal
circuitry of the somatosensory thalamus follows a typical plan in most mammals. There are thalamocortical relay cells, often of several types, “,2gJ3local circuit neurons17,22,26and a neuropil containing complex synaptic glomeruli.20 This pattern has been found in cats,‘7*22~26 rabbits,16 Galago “~17and squirrel monkey. t6 The same structural organization has been found in other thalamic nuclei, including the lateral geniculate nucleus of all animals studied so far.’ The proportion of local circuit neurons in the somatosensory thalamus (the ventrobasal complex, VB) of the cat has been estimated at between 2022,26 and 30%.17 In the VB of the rat, however, there has been very little evidence for local circuit neurons. Golgi studies10*1v*24 have detailed the thalamo~ortical relay cells but have found no smaller Golgi type II cells. Large injections of horseradish peroxidase made into the somatosensory cortex have resulted in the labeling of almost all cells in the ventrobasal thalamus.g~23~31 In a careful study of this type, using thin plastic sections, Lee’ determined that less than 1% of thalamic neurons were unlabeled. A more direct search for local circuit neurons has been made possible by immun~ytffihemical methods. These studies depend on the assumption that local circuit neurons in the thalamus are inhibitory, and use y-aminobutyrate (GABA) as
*To whom correspondence should be addressed. Abbreviations: DAB, 3,3’-diaminobenzidine; GABA, y-aminobutyrate; GAD, glutamate decarboxylase; HRP, horseradish peroxidase; LCN, local circuit neuron; PBS, phosphate-buffe~d saline; VB, ventrobasal complex.
a neurotransmitter.11*17,*oMost immun~yt~hemical studies have been done by localizing glutamate decarboxylase (GAD), the rate-limiting enzyme for the synthesis of GABA. Such studies in the cat ventrobasal complex’7~22~26 and in the lateral geniculate nucleus of several mammalssJ’ have revealed sizeable numbers of GAD-stained neurons. No detailed studies of GAD-labeled cells in rat VB have been reported, but several authors have noted that such cells are very rare in this nucleus.1’~‘6~22 Recently, a more direct immunocytochemical approach has been developed using an antiserum to GABA itself.12 This antiserum appears to be more sensitive for demonstrating GABA-containing cell bodies than GAD antisera.6J2 A survey of the rat thalamus using the GABA anti~rum” showed very few stained neurons in the ventrobasal complex. In the present work, we have used the GABA antiserum in conjunction with horseradish peroxidase injections in the somatosensory cortex to locate putative GABA-containing local circuit neurons in the rat ventrobasal complex.
EXPERIMENTALPR~EDUR~ Eight Spragu*Dawley rats of about 25Og weight were used. Five rats were used for localization of neurons using antisera to GAD and GABA. These rats were perfused with one of the following fixatives in 0.05 M phosphate buffer, pH 7.4: 0.2% picric acid, 4% paraformaldehyde and 0.05% glutaraldehyde 25. , 4% paraformaldehyde, 6.05% sodium periodate and 0.34% lysine$ or 1%’ paraformaldehvde and 3% glutaraldehyde. After an 8 h waii for thorough hxation, the brains were postfixed with lo%, then 20% sucrose in phosphate buffer, removed and cryoprotected with 30% surose-phosphate buffer. Froxen sections 30 pm thick were cut through the thalamus.
229
ROGERM. HARRISand
230
Tissue sections were processed for immunocytochemistry using the standard peroxidase-antiperoxidase methods.5.‘2.27 Sections through different parts of the ventrobasal complex were incubated in either antiserum to GAD (obtained from J. Y. Wu, diluted 1:300) or to GABA (Immunonuclear, diluted 1:500 to I :3000). All serum dilutions were made with phosphate-buffe~d saline (PBS) containing 1%normal goat serum and 0.2% Triton X-100 detergent. Sections were incubated in primary antiserum at 4°C for 36 h. Following a 2-h rinse in PBS containing 0.1% T&on X-100, the sections were incubated in goat anti-rabbit IgG diluted I:30 for I h at 37”C, washed for 1 h, incubated in anti-rabbit peroxidase-antiperoxidase diluted I : 100 for I h at 37°C and rinsed first in PBS and then in Tris buffer. Sections were then reacted with 0.03% diamino~nzidine and 0.~6% H,O, in Tris buffer for 8min and rinsed with PBS. The reacted sections were mounted on slides, coverslipped and examined without counterstaining. Horseradish peroxidase injections
In three rats, GABA immunocytochemistry was combined with injections of horseradish peroxidase (HRP), using the protocol of Bowker ef al.’ In two rats, 40% HRP (Sigma type VI) in 2% dimethyl sulfoxide was injected stereotaxically into the somatosensory (SI) region of the cortex in one or two injections on each side. Each injection contained 0.5 ~1 of HRP and was pressure-injected using a 1 ~1 Hamilton syringe over 5 min. In one rat, 25% HRP in 2% dimethyl disulfoxide was iontophoretically injected into the ventrobasal complex of each side, using a ZpA, 50% on-off constant current source for 5 min. The rats were allowed to survive for 48 h and then perfused with 4% paraformaldehyde plus 0.1% glutaraldehyde in phosphate buffer, followed by 10% and then 20% sucrose in phosphate buffer. The brains were infiltrated in 30% sucrose buffer and then frozen-sectioned at 30pm. Every fourth section was processed for HRP visualization using the cobalt-intensified tetramethyl benzidine method,’ mounted on a subbed slide and counterstained with Cresyl Violet. Other sections in the same area were lirst reacted using the cobalt-intensified ~amino~nzidine method’ and then processed for immunocytochemistry using the antiserum to GABA. The immunocytochemical procedure was the same as that given above except that instead of using 3,3’-diaminobenzidine (DAB) in the final visuali~tion step, 0.02% 3-amino-9~thylcarbazole mixed with 5% dimethyl formamide in 0.05 M acetate buffer was used.3 This resulted in a red-colored reaction product easily distinguishable from the blue-black DAB reaction. These sections were mounted in glycerin, coverslipped and kept at 4°C. GABA ceil an&&v
Quantitative analysis of GABA-stained cells was carried out using the three double labeled animals. The GABAlabeled cells in VB were counted in four sections from each of two animals and their locations plotted using a 4 x objective and a camera lucida attachment. An approximate
ANITA
E. HENDIUCK~~R
overall distribution was obtained by plotting all eight sections on a single drawing of VB, adjusting the positions of the cells relative to the boundaries of VB to account for differences in shape of VB at different levels. Cell size distributions were determined by drawing each labeled cell in the above sections using a 100 x oil objective. These drawings were then measured to determine the somal cross-sectional area using a micr~omputer-driven tablet system.2* Similar measurements were made on two Nisslstained sections from two different rats. In these sections all cells visible within VB in several fields were drawn. Histograms of the cell size dist~bution for GABA-stained cells and for all Nissl-stained cells were prepared. To determine the proportion of GABA-containing cells, a count of all cells in eight Nissl-stained fields (average size: 0.29 mm*) was made using a 25 x objective. Two of these fields were areas retrogradely labeled by HRP where essentially every cell was HRP-labeled. The size of each field was measured using the tablet system, yielding a density of cells for each area. This was compared with the density of GABA-labeled cells, determined from the above counts and the area of VB in each section. Comparison of the average densities of GABA-labeled cells and all Nissl-stains cells gave the proportion of GABA-labeled cells in VB.
RESULTS
In GAD- or GABA-stained sections, the ventrobasal complex (VB) was easily distinguished by its reticulated appearance, due to numerous discrete bundles of unstained myelinated fibers which course through it (Fig. I). VB is bounded laterally by the external medullary lamina and ventrally by the medial lemniscus. The medial boundary was taken to be at the onset of the myelinated bundles. In optimal sections a thin interior lamina could be seen dividing ventral posterior medial from ventral posterior lateral nuclei. The VB region was diffusely stained a light brown by GAD or GABA, in distinction to the heavily stained thalamic reticular nucleus, which was seen laterally and ventrally to VB. Sections stained for GAD showed a dense network of stained fibers and puncta throughout VB (Fig. 2). These puncta, which probably represent axon terminals, were often found surrounding unstained cell bodies. There were no obviously GAD-stained cell bodies in VB, in contrast to cells of the tha~amic reticular nucleus in the same section which were heavily stained (Fig. 2). Thus, there was no positive evidence for GABA-containing neurons in VB using the GAD antiserum.
Fig. 1. Low-power view of the lateral thalamus double stained for GABA and HRP. The ventrobasal complex (VB) is the semicircular reticulated region, bound ventrally by the medial lemniscus (ML) and laterally by the heavily stained thalamic reticular nucleus (TRN). A small band of retrogradely labeled HRP cells is visible in the center of VB. Bar = 1 mm. Fig. 2. A section through the edge of VB stained with antiserum to GAD. Note the intense fiber and terminal labeling in VB in which cell bodies are outlined by stained terminals. The thalamic reticular nucleus (TRN), unlike VB, contains heavily labeled cell bodies. Bar = 100 pm. Fig. 3. A section through part of VB stained for GABA. There is some staining of fibers and terminals and one well-stained cell body (arrow). Bar = IOOpm. Fig. 4. A section through the lateral geniculate nucleus stained for GABA. Note the numerous well-stained cell bodies, unlike the sparse staining seen in VB. Bar = 100 {tm.
LCNs in rat ventrobasal thalamus
Figs
231
Figs 5-10.
232
233
LCNs in rat ventrobasal thalamus
GABA CELLS
ALL CELLS VPM \
Fig. 1I. Composite distribution of GABA-labeled cells from eight separate sections, all plotted on the same drawing. Fi, fimbria; Hi, hippocampus; IC, internal capsule; LD, lateral dorsal; ML, medial lemniscus; OT, optic tract; PO, posterior nucleus; ST, stria terminalis; TRN, thalamic reticular nucleus; VPL, ventral posterior lateral; VPM, ventral posterior medial; ZI, zona incerta.
Fig. 12. (A) Histogram of cross-sectional areas of GABAlabeled cells from eight sections through VB in two animals (64 cells). Mean (arrow) is 81 + 34pm*. (B) Histogram of cross-sectional areas of all cells in eight Nissl-stained fields from two animals (170 cells). Mean (arrow) is 105 f 36 pm*.
Fig. 5. A section through VB stained for GABA using carbazole as the chromogen. stained cell bodies are present. Bar = 1OOpm.
Two GABA-
Fig. 6. A GABA-stained
cell in VB showing staining of the proximal parts of two dendrites. Bar = 1OOym. Fig. 7. A section through VB double stained for GABA and HRP. HRP has been injected into the SI cortex, resulting in a band of retrogradely labeled cells in VB. A single GABA-stained cell, without HRP, is shown by the arrow. Bar = 100 pm. Fig. 8. A section through VB stained for GABA and HRP, following an HRP injection into the SI cortex. A number of HRP-labeled cells are present, as well as one prominent GABA-labeled cell (arrow). Bar = 100 pm. Fig. 9. A high-powered view of the GABA-labeled cell of Fig. 8. A second, larger cell was retrogradely labeled with HRP, as shown by the prominent HRP granules. Note the lack of HRP granules in the GABA-labeled cell. Bar = 10 pm. Fig. 10. High-power view of a double labeled neuron in the thalamic reticular nucleus (*). HRP has been injected in VB, resulting in the retrograde labeling of thalamic reticular cells. Note the reddish tinge, indicating the cell is GABAergic, and the prominent HRP granules. Bar = 10pm.
234
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The results using the GABA antiserum were somewhat different. The staining of fibers and terminals was less intense than with the GAD antiserum, particularly in those animals fixed with low levels of glutaraldehyde. There were occasional scattered cell bodies which were labeled (Fig. 3). This was particularly noticeable in the double labeled animals, where the GABA label was a red color due to use of carbazole as the chromogen (Fig. 5). The labeled cells tended to be small, round to oval shaped, and sparse in number. In a few cells, parts of the proximal dendrites were also labeled (Fig. 6). From the counts of eight sections through VB, the average number of labeled cells was 4.1 f 2.7 per section. This paucity of GABA cells in VB was not just an artefact of staining, because numerous GABA-stained cells were seen on the same sections in the dorsal lateral geniculate (Fig. 4), hippocampus and cerebral cortex. The distribution of GABA-labeled cells was determined by plotting their positions in eight sections. The results from all eight sections were consolidated and plotted on a single plot in Fig. 11. Labeled cells were scattered throughout both the lateral and medial subdivisions of VB. There were fewer labeled cells in the medial part of VB and more labeled cells along the lateral boundary. Some of the latter cells could have actually been cells of the thalamic reticular nucleus because the boundary between VB and the reticular nucleus was sometimes unclear. To determine whether some of these GABAlabeled cells could be thalamocortical relay cells projecting to the cortex, HRP injections were made in two rats into the SI somatosensory cortex. These resulted in bands of HRP-labeled cells in VB extending in a rostralcaudal direction. Within these bands, almost all cell bodies were retrogradely labeled with HRP (Fig. 7). After staining for GABA, using carbazole as the chromogen, occasional red GABAlabeled cell bodies were found in the vicinity of the HRP-labeled cells (Figs 7-9). No double labeled cells containing both HRP and GABA were seen. In order to be certain that our technique would detect a double labeled cell, a third rat was injected with HRP into VB itself. This resulted in the retrograde transport of HRP by cells in the thalamic reticular nucleus, which project their axons into VB and are known to contain GABA.‘* Double labeled neurons with red cytoplasm containing blue-black HRP granules were easily identified (Fig. IO). A comparison between these cells (Fig. 10) and the GABA-labeled and HRP-labeled cells (Fig. 9) in VB confirmed that none of the cells in the cortex-injected rats was double labeled. This provides evidence that the GABAlabeled cells do not project to the cerebral cortex and can thus be considered local circuit neurons. The distribution of soma cross-sectional areas for GABA-stained cells is shown in Fig. 12A and a similar distribution for all cells in Nissl-stained sections in Fig. 12B. The average cross-sectional area for GABA cells was 8 1 + 34 pm2 (population stan-
ANITA E. HENDRICKSON
dard deviation); for all cells, it was 105 & 36pm2. A r-test shows these averages to be significantly different P < 0.001). The ratio of the cross-sectional areas for GABA cells to all cells is 0.77 f 0.59. Thus the GABA-labeled cells tend to be somewhat smaller than the thalamocortical relay cells, although the distributions show there is considerable overlap. GABA staining was determined by measuring the density of GABA-labeled cells and the density of all cells in the Nissl-stained sections. The density of GABA-labeled cells over eight counted sections was 2.1 + 1.4cells/mm2, while the density of cells found in Nissl-stained sections was 495 + 183. The proportion of GABA cells in VB is then 0.4 + 0.3%.
DISCUSSION Our results confirm the claim by Otterson and Storm-Mathisen’2 that the GABA antiserum is more sensitive for staining cell bodies than GAD. It was difficult to find stained cell bodies in VB in our GAD-stained sections, perhaps because of the heavy staining of fibers and terminals in the neuropil or because colchicine pretreatment, which increases cell body staining by GAD,2’ was not used. Stained cells were easily seen using the GABA antiserum, particularly when carbazole was used as the chromogen. It appears that the GABA antiserum is more effective for demonstrating cell bodies and the GAD antiserum is more selective for axonal terminals. The major result of this work is to confirm that there is a small population of GABA-containing neurons in the ventrobasal thalamus of the rat. These neurons do not project to the somatosensory cortex as indicated by the lack of double labeled neurons in rats with HRP injected into the SI cortex. It is possible that the GABA cells could project specifically to the SII cortex and not SI, but this seems unlikely since the cells that project to SII have been located primarily in the posterior pole of VB.30 Thus, we may tentatively identify these GABA cells as a sparse population of local circuit neurons (LCNs). Several authors have suggested, on the basis of previous experiments using Golgi or retrograde HRP labeling techniques, that the rat VB may be devoid of LCNs.‘0,20.23.24,3’More recent immunocytochemical studies have suggested there may be a few.“.‘3s22 Our finding that 0.4 + 0.3% of VB cells are LCNs is consistent with the estimate made by Lee’ that there are less than 1% LCNs in this nucleus. These results are similar to those found in the opossum VB, in which only a few GAD-labeled cells are seen.“x” There is, however, a marked species difference between the rat and opossum and higher mammals such as the rabbit, cat or Galago, in which VB contains 2&30% LCNS.‘~.“.~~.~’ This may represent an evolutionary trend toward increasing numbers of LCNs in VB in higher mammals, as discussed
23.5
LCNs in rat ventrobasal thalamus by Penny et aLI It is interesting to note that the dorsal lateral geniculate nucleus .in our rats does contain numerous GAEA-stained neurons as seen in other studies,*~” so that there is a difference in circuitry between the two thalamic nuclei even in the same species. The cell dist~butions in our study indicate that the GABA-labeled cells are, on average, smaller than the thalamocortical relay cells. This is in agreement with studies of GAD-labeled neurons in cat and ~~~~~~ I1.22.26 and in rabbit and opossum.‘6 Large and small neurons were also found in Nissl-stained sections of the mouse VR3* The ratio of the average cross-sectional areas for GABA cells to all cells in our work was 0.77. This agrees with the value found in opossum, 0.7’9.16Animals which contain a significant number of LCNs were found to have a significantly lower ratio of cross-sectional areas (0.59 for rabbit, 0.55 for cat, 0.52 for Galago’6). Local circuit neurons in the rat and opossum are thus larger, relative to the thalamocortical relay cells, than LCNs in higher mammals. This may imply that there is less differentiation between the two cell types in these animals than in higher animals, and gives further support to the idea that the rat and opossum are early stages in the evolutionary development of the thalamus. The functional significance of these few LCNs in the neural circuitry of the rat VB is not at all clear. It is questionable whether there are enough of them
to play a major role, as LCNs do in other mammals. Possibly these cells are involved in the processing of one specific submodality of somatosensory sensation. The small number of LCNs does indicate that the rat VB has, for the most part, a very simple circuitry. At the ultrastructural level, there appear to be few, if any, of the synaptic CompIications seen in higher mammals, such as large synaptic glomeruli or dendrodendritic synapses. 9,20Furthermore, intracellular HRP injections in rat VB4 indicate that all thalamocortical relay cells are similar in structure. This homogeneous neuronal population and lack of synaptic complexity makes the rat VB an excellent model for the study of thalamic processing. CONCLUSiONS
The rat ventrobasal thalmus does appear to contain a small population of local circuit neurons, as indicated by labeling of cells by an antiserum to GABA. These cells, which make up 0.4 + 0.3% of the population of VB, are smaller on average than the relay cells, and are dist~but~ throu~out VB. The functional significance of these local circuit neurons is not yet clear. Aclcnowledgemerats-This work was supported by National Institutes of Health Grants NS-19073 to R.M.H. and EY-01208 and EY-04536 to A.E.H. We thank J. Y. Wu for a gift of GAD antisera. We would like to thank Haila Vickland and Bente Noble for technical assistance and Doris Ringer for typing the manuscript.
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