THE JOURNAL OF COMPARATIVE NEUROLOGY 322:167-180 (1992)

Cytoarchitectonic Heterogeneities in the Thalamic Reticular Nucleus of Cats and Ferrets ANNEMARIE E. CLEMENCE AND JOHN MITROFANIS Department of Human Anatomy, University of Oxford, OX1 3QX, United Kingdom

ABSTRACT The thalamic reticular nucleus has been classically defined as a group of cells surrounding most of the rostral and lateral surfaces of the dorsal thalamus, lateral to the fibres of the external medullary lamina and medial t o those of the internal capsule. With the use of Nissl staining and antibodies to y-aminobutyric acid (GABA),somatostatin, and parvalbumin, this study describes the cytoarchitecture of the thalamic reticular nucleus of cats and ferrets. In cats, three subdivisions of the nucleus are distinguished, two of which are distinct in ferrets also. First, the main body of the reticular nucleus lies lateral to the fibres of the external medullary lamina (except ventrally) and medial to those of the internal capsule. In both cats and ferrets, this structure is heterogeneous, consisting of distinct layers, the details of which vary along the dorsoventral axis. A prominent rostroventral portion of comparatively small rounded cells is also apparent within the main body. Most reticular cells in all areas of the main body are labelled with all of the above mentioned antibodies. Second, the inner small-celled region is a group of small cells located between the external medullary lamina (ventrally) and the medial margin of the ventral regions of the main body of the reticular nucleus: the inner small-celled region is clearly differentiated in cats only. Previous studies have referred to this area as being part of the main body of the reticular nucleus, but we suggest that it may form a separate subnucleus. For example, the inner small-celled region stands in striking contrast to the main body of the reticular nucleus in that none of its cells are GABA immunoreactive and only a small caudal subpopulation are parvalbumin immunoreactive. A very similar pattern of immunostaining is apparent for the cells in the zona incerta, although the latter contains a small rostral subpopulation of GABA immunoreactive cells, Furthermore, although morphologically distinct from the zona incerta, the inner small-celled region fuses with it ventrocaudally. We suggest that the inner small-celled region may constitute a previously undescribed dorsal extension of the zona incerta, rather than a subdivision of the reticular nucleus. Third, the perireticular nucleus, hitherto unidentified, is a discrete group of small cells lateral to the main body of the reticular nucleus and medial to the corpus striatum (globus pallidus and caudate-putamen). It is apparent throughout most of the dorsoventral extent of the main body of the reticular nucleus of cats and ferrets. Although continuous with the latter ventrocaudally, the perireticular nucleus is otherwise clearly separated from it, forming a sheet around the main body of the reticular nucleus. There are no immunocytochemical differences between the perireticular and reticular cells with the antibodies used to date. 0 1992 Wiley-Liss, Inc. Key words: entopeduncular nucleus, inner small cell region, GABA, globus pallidus, parvalbumin, perireticular nucleus, somatostatin, zona incerta

The thalamic reticular nucleus of mammals is a sheet of cells surrounding most of the rostral and lateral surfaces of the dorsal thalamus. It is situated lateral to the fibres ofthe external medullary lamina and medial to those of the internal capsule and is continuous ventrally with the zona 0

1992 WILEY-LISS, INC.

incerta and caudally with the ventral lateral geniculate nucleus (reviews by Berman and Jones, '82; Jones, '85). The reticular nucleus lies in the path of thalamocortical and Accepted April 17,199~.

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corticothalamic fibres and most of these give collaterals to the nucleus as they pass through it. Collaterals associated with a particular sensory modality terminate in a defined region, or sector, of the reticular nucleus (Carman et al., '64; Minderhoud, '71; Jones, '75; Montero et al., '77; Ohara et al., '80; Crabtree and Killackey, '89). For example, the same region of the reticular nucleus that receives axonal collaterals from cells of the dorsal lateral geniculate nucleus also receives axonal collaterals from cells in the primary visual cortex. These thalamic and cortical inputs are thought to be excitatory to the reticular cells (Schlag and Waslak, '71; Hale et al., '82). The major efferents of the reticular nucleus go to the dorsal thalamic nuclei on the ipsilateral side (Minderhoud, '71; Jones, '851, where most reticular axons form inhibitory synapses on the dendrites of thalamic relay cells. There are also reports of reticular axons terminating on local GABAergic interneurones (Ohara and Lieberman, '85; Montero and Scott, '81; Montero and Singer, '85).Axons of some reticular cells also project to the midbrain (Scheibel and Scheibel, '66; Parent and Steriade, '84; Rieck et al., '86): these cells are distinct from those that project to the dorsal thalamus (Parent and Steriade, '84). Most reticular cells are immunoreactive to antibodies to GABA (Otterson and Storm-Mathison, '84; De Biasi et al., '86) and to the enzyme that synthesises it, glutamic acid decarboxylase (Houser et al., '80; Hendrickson et al., '83; Jones, '85). GABA is regarded as a major inhibitory transmitter of the central nervous system and recording studies indicate that excitation of reticular cells induces a long term hyperpolarisation in dorsal thalamic relay cells (Hale et al., '82; Jones, '85). In cats (Graybiel and Elde, '83; Oertel et al., '83),mice (Forlini et al., '901, and monkeys (Graybiel and Elde, '83),most reticular cells are somatostatin immunoreactive. Surprisingly, however, antibodies to somatostatin do not label cells in the reticular nucleus of rats (Graybiel and Elde, '83; Johansson et al., '84; Jones, '85; Vincent et al., '85). Recent studies have indicated that the reticular cells are also parvalbumin immunoreactive (Jones and Hendry, '89; Celio, '90; Frassoni et al., '91). Parvalbumin is a calcium-binding protein associated with relay cells and GABAergic interneurones in the dorsal thalamus (Stichel et al., '88; Jones and Hendry, '89; Rausell and Jones, '91) and with subpopulations of GABAergic cells in the cerebral cortex (Blumcke et al., '90; Van Brederode et al., 'go), corpus striatum (Cowen et al., 'go), and hippocampal formation (Ribak et al., '90). The precise function of somatostatin and parvalbumin in reticular cells is not known. The thalamic reticular nucleus has been classically described as a non-specific thalamic nucleus, since its cortical and thalamic connections have been thought to be rather diffusely arranged (Jones, '75) and its constituent cells have been described as forming a homogeneous population (Ramon y Cajal, '11; Steriade and Deschenes, '84; Ohara and Lieberman, '85). Recent studies have shown, however, that the reticular nucleus contains accurate topographical maps of the cortical sheet and of the dorsal thalamus (Montero et al., '77; Crabtree and Killackey, '89; Crabtree, '91; Conley and Diamond, '90; Cucchiaro et al., '91; Harting et al., '91), raising the possibility that it may have a more specific function on the corticothalamic and thalamocortical pathway than previously thought. The present study has examined whether there are any cyto or chemoarchitectonic differences in the thalamic

A.E. CLEMENCE AND J. MITROFANIS reticular nucleus of two carnivores, cats and ferrets. Cats were chosen because there have been many previous studies on the connectivity and dendritic morphology of reticular cells (see Jones, '85 for review) and ferrets were chosen for comparative purposes. Nissl staining and antibodies to GABA, parvalbumin, and somatostatin were used. In cats, three distinct regions of the thalamic reticular nucleus are apparent, two of-whichare apparent in ferrets also.

MATERIALS AND METHODS Normally pigmented cats (n = 5) and ferrets (n = 9) of either sex were used in this study. For both Nissl (cresyl violet and neutral red) and immunocytochemical staining of brain sections, animals were anaesthetised with sodium pentobarbitone (60 mg/ml) and perfused transcardially with phosphate buffered saline (pH 7.4), followed by 4% buffered paraformaldehyde or formaldehyde (500 ml for ferrets and 1 litre for cats) at room temperature. Some animals were perfused with a fixative consisting of 4% buffered paraformaldehyde with the addition of 1-0.125% glutaraldehyde [for GABA immunostaining. There were no differences in the quality of GABA immunostaining with the varying concentrations of glutaraldehyde. The higher concentrations of glutaraldehyde (1%)did, however, lessen the quality of the parvalbumin and somatostatin immunostaining]. After perfusion, brains were quickly removed and immersed in a fresh solution of fixative for 1-2 hours. The surrounding cortex was trimmed away and the blocks containing the thalamus were placed in phosphate-buffered saline with the addition of 35% sucrose for about 4 days, or until the block sank. Blocks were frozen in dry ice and sectioned on a freezing microtome at thicknesses ranging from 25-50 Fm.

Nissl (cresyl violet) staining Sections were collected into 10% formalin in phosphatebuffered saline (pH 7.4). They were then mounted on 5% gelatinised slides and placed in a 40°C oven for about 48 hours. Next, sections were placed in xylene and hydrated with descending alcohols and left in 70% alcohol for 48 hours. They were then transferred to 70% alcohol with addition of 1%hydrochloric acid for 20 minutes and then for 1 hour in 0.3% cresyl violet (pH 3.5) at 60°C. Sections were then dehydrated in ascending alcohols and placed in xylene overnight. Finally, sections were differentiated the following day in 95% alcohol and coverslipped with DPX.

Immunocytochemistry Three different primary antibodies were used in this study: i) rabbit anti-GABA (Sera, 1:500), ii) mouse antiparvalbumin (Sigma Chemical Co., St. Louis, MO, 1:1000), and iii) rabbit anti-somatostatin (Dakopats, 1:400).All antibodies were diluted to working solution with phosphatebuffered saline with the addition of 0.5% bovine serum albumin (Sigma Chemical Co., St. Louis, MO). Sections were washed for about 30 minutes with phosphate-buffered saline with the addition of 1% Triton (Ajax) and then incubated with one of the primary antibodies for 24-48 hours at 4°C. Sections were subsequently washed with phosphate-buffered saline for about 1 hour and further incubated with either biotinylated anti-rabbit IgG (Vector Burlingame, CA, 1:200; for sections incubated with antiGABA and anti-somatostatin) or anti-mouse IgG (Vector Burlingame, CA, 1:200; for sections incubated with anti-

STRUCTURE OF CARNIVORE THALAMIC RETICULAR NUCLEUS parvalbumin) for 24 hours at 4°C or for 2 hours at 37°C. After washing, tissue was incubated with the avidin-biotinperoxidase complex (Vector Burlingame, CA, 1 : l O O ) for 1-2 hours at room temperature. Finally, sections were washed briefly in phosphate-buffered saline and then in a 0.5% Nickel-Tris base saline mixture (pH 7.4) for about 1hour. They were then immersed in a solution containing 3,3diaminobenzidine tetrahydrochloride (Sigma Chemical Co., St. Louis, MO) (40 mg), HzOz (5 p1 of 30% solution), and Nickel-Tris base saline (300 ml), for 5-10 minutes, or up until adequate staining was achieved. This method is a slight variant of that described previously by Adams ('81). Sections were then washed in distilled water, mounted onto gelatinised slides and air dried for about 12 hours. They were then dehydrated in ascending alcohols, cleared in xylene or Histoclear, and coverslipped with (DPX). For ferrets, every section was collected and adjacent sections were immunoreacted with antibodies to GABA, parvalbumin, and somatostatin: the fourth section in the series was Nissl (cresyl violet) stained. For cats, sections were processed as above, except that there was no series immunoreacted with the antibody to somatostatin: Graybiel and Elde ('83) have previously published a comprehensive description of somatostatin immunoreactivity in the reticular nucleus of cats. Most of the immunostained sections of both species were then counterstained with neutral red and subsequently dehydrated as above. For control experiments, the primary antibody was replaced with either normal rabbit or mouse sera, or phosphatebuffered saline with the addition of 0.5% bovine serum albumen and then reacted as above. Control sections showed no positive immunoreactivity for any of the antibodies used. In order to determine whether all regions of the reticular nucleus are immunostained with the above antibodies, thin (25-30 pm), free-floating thalamic sections were used. These were then counterstained with neutral red. The thinner sections enabled the antibodies to penetrate the full thickness of the sections, thus labelling all the cells containing the antigen. Thicker ( > 40 pm) sections, did not allow adequate antibody penetration, and it was often found that cells in the middle of these thicker sections were not immunostained: cells in the middle of the section, were however, stained with neutral red, thus giving the false result that not all the reticular cells were immunostained. This problem was overcome by using the thinner sections.

RESULTS With the use of Nissl staining and immunocytochemistry, three major subdivisions of the reticular nucleus are apparent. These include i) the main body of the reticular nucleus, which generally refers to the sheet of cells located between the external medullary lamina (except ventrally) and the internal capsule; ii) the inner small-celled region, a group of small cells located between the external medullary lamina (ventrally) and the inner margin of the ventral regions of the main body of the reticular nucleus and; and iii) the perireticular nucleus, a group of small cells located lateral to the main body of the reticular nucleus and medial to the corpus striatum. The main body of the reticular nucleus and perireticular nucleus are well defined in both species, whereas the inner small-celled region, is clearly distinguished in cats only. Each of the above structures is considered separately in cats and ferrets.

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In both species, there are no qualitative differences in the labelling of the reticular nucleus with any of the antibodies used (except were outlined below; for example, in the antibody labelling of the inner small-celled region), but sections immunostained for parvalbumin and then Nissl (neutral red) counterstained consistently gave more intense labelling of cells and a clearer differentiation of the reticular nucleus from its neighbouring structures. Consequently, most attention will be focused on these sections.

Cats Main body of the reticular nucleus. The main body of the reticular nucleus of cats consists of a sheet-like nuclear complex, surrounding most of the dorsal thalamus (Fig. 1). In many areas, the nucleus appears to consist of distinct layers, the details of which vary along the dorsoventral axis. In horizontal sections, for instance, the main body of the reticular nucleus at its most dorsal aspect consists of a single line of cells flanked by the dorsal thalamus medially and the stria terminalis laterally (Fig. 1A). Further ventrally, cells spread out somewhat, and at the level of the dorsal lateral geniculate nucleus, they appear to be arranged in three, sometimes four, rows or layers (Figs. 1B, ZA). This area of the main body of the reticular nucleus of cats has been shown to be principally involved with the visual pathways (see maps of Jones, '75). These layers are even more pronounced in saggital parvalbumin immunoreactive sections (arrows, Fig. 2B), which show that the structure and arrangement of cells in the different layers are distinct. For example, in the outermost layer, cells are loosely scattered and the neuropil is lightly stained (with parvalbumin immunolabelling, in particular), whereas in the adjacent inner layer, cells are closer packed, slightly larger, and the neuropil is darkly stained (Fig. 2B). Cells in the innermost layer are generally smaller and the neuropil is lightly stained (Fig. 2B). In horizontal sections, the caudal regions of the dorsal areas of the main body of the reticular nucleus appear continuous with a group of cells scattered throughout the optic radiation, and more caudally still, with the perigeniculate nucleus (Fig. 2A). At the level of the mid-dorsal lateral geniculate nucleus, horizontal sections indicate that the portion of the main body of the reticular nucleus adjacent to the dorsal lateral geniculate nucleus consists of a single line of cells which is continuous with the perigeniculate nucleus (Fig. 1C). Immediately ventral to the dorsal lateral geniculate nucleus, the structure and arrangement of the main body of the reticular nucleus changes. In horizontal parvalbumin immunoreactive/neutral red stained sections, throughout the mediolateral extent of central and caudal areas of the main body of the reticular nucleus, cells are closely packed, relatively large, oval and fusiform shaped, and the neuropil is darkly stained (Fig. 3A,B). This region of the main body of the reticular nucleus in cats has been shown to be principally involved with the somatosensory (centrally) and auditory (caudally) pathways (see maps of Jones, '75). Rostrally, at this level, the main body of the reticular nucleus broadens out into an approximately triangular zone (rostroventral portion) and two distinct layers of comparatively small rounded cells are apparent: an outer layer of loosely scattered cells and lightly stained neuropil, and an inner layer of closer packed cells and darkly stained neuropil. In progressively more ventral horizontal sections, the inner layer and in particular, the outer layer of the rostroventral portion become more prominent (Fig. 2C,D).

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DORSAL

Fig. 1. Schematic maps of horizontal sections of cat thalamus. Sections were irnmunostained with parvalbumin and then Nissl (neutral red) counterstained and sections from dorsal (A) to ventral (GI areas of thalamus are shown. The main body of the reticular nucleus is shaded black; zona incerta and inner small-celled region are the striped regions; perireticular cells are represented as black circles; scattered cells which link the reticular and perigeniculate nuclei are represented

as unfilled circles. AN, anterior nuclei; CP, caudate-putamen; dLGN, dorsal lateral geniculate nucleus; GP, globus pallidus; IC, internal capsule; LD, lateral dorsal nucleus; LP, lateral posterior nucleus; MGN, medial geniculate nucleus; OR, optic radiation; OT, optic tract; PG, perigeniculate nucleus; Pul, pulvinar; ST, stria terminalis; VA, ventral anterior nucleus; VB, ventrobasal complex; vLGN, ventral lateral geniculate nucleus; VL, ventral lateral nucleus; 21, zona incerta. x 30.

The central and caudal areas of the main body of the reticular nucleus thin and taper off, leaving the rostroventral portion as the most ventral region of the nucleus (Fig. 1Gj. Previous studies have shown that the rostroventral portion receives inputs from various motor (Jones, '75; Steriade and Deschenes, '84; Cornwall et al., '901, limbic (Jones, '75; Cornwall et al., '90), and brainstem (Cornwall et al., '90; Pare et al., '90) centres. Inner small-celled region. In cats, a distinct region of small cells, different in cytoarchitecture to the region of larger cells of the main body of the reticular nucleus (from Nissl staining), are apparent between the external medullary lamina and the innermost margin of the ventral regions of the main body of the reticular nucleus (on left side of Fig. 3C,D; arrow, Fig. 4A). These small cells comprise a sheet-like structure extending throughout most of the innermost margin of the ventral regions of the main body of the reticular nucleus, and in caudal regions, immediately ventral to the dorsal lateral geniculate nucleus, this group of cells expands to form a conspicuous nuclear mass (shaded area Fig. 1D-F; large arrows, Fig. 3A,B). From this caudal thickening of the nucleus, horizontal sections show a thin rostral extension of cells which fails to reach the rostral pole of the main body of the reticular nucleus (Fig. 1D-F). Further ventrally, the rostral extension of the inner small-celled region is not apparent and the large caudal mass of the inner small-celled region is continuous with the

zona incerta (Figs. lG, 4A). As with the main body of the reticular nucleus, the inner small-celled region is traversed by mylenated fibres, giving it a reticulated appearance (arrow, Fig. 4C). The soma1 shape of cells in the inner small-celled region in horizontal sections is diverse, with some somata being spindle shaped, orientated parallel to the main body of the reticular nucleus and others being rounded (Fig. 3C,D). The spindle-shaped cells appear more frequent dorsally and in the thin rostral extension of the inner small-celled region, whereas the rounded cells are more apparent ventrally. In coronal section, cells in the inner small-celled region are more or less rounded. In Nissl (cresyl violet or neutral red) stained coronal and horizontal sections, the cells of the inner small-celled region are smaller and more lightly stained than those in the zona incerta. Parvalbumin immunoreactivity is not apparent in most cells of the inner small-celled region. In parvalbumin immunoreactive/Nissl (neutral red) stained horizontal sections, most cells in the inner small-celled region are Nissl stained but not immunostained for parvalbumin, unlike those in the main body of the reticular nucleus (Fig. 3D). The parvalbumin immunoreactive cells are more frequent caudally in the inner small-celled region, just ventral to the dorsal lateral geniculate nucleus (arrow, Fig. 4C). In the zona incerta, as with the inner small-celled region, most cells are not parvalbumin immunoreactive. However, there

Fig. 2. Cat thalamus. Sections immunostained with parvalbumin and then Nissl (neutral red) counterstained (PVIN). All figures are horizontal sections, except for (B), which is a parasaggital section. In (A), (C), and (D), rostral is to top and medial to left. In (0,dorsal is to top and caudal to left, Small arrows in (B), (C), and (D) show layers in

the main body of the reticular nucleus. Unfilled arrow in (A) indicates perireticular nucleus. Arrowheads in (C) and (D) show the same blood vessel. dLGN, dorsal lateral geniculate nucleus; LP, lateral posterior nucleus; OR, optic radiation; PGN, perigeniculate nucleus; VB, ventrobasal complex; ZI, zona incerta. (A), (C) X 10; (B) x 100; (D) ~ 4 0 .

Fig. 3. Cat thalamus: inner small-celled region. (A), (B), and (D) are parvalbumin immunostained and Nissl (neutral red) counterstained (PVIN). (A) is a horizontal section, at level of medial geniculate nucleus. (B)is a higher magnification of caudal aspect of (A). Large arrows in (A) and (B) indicate inner small-celled region and small

arrows indicate same blood vessel. (C) is a Nissl (cresyl violet) stained section of comparable area to (D). In each figure, rostra1 is to top and medial to left. VB, ventrobasal complex; MGN, medial geniculate nucleus. (A) X10; (B) ~ 4 0 (0, ; (D) X400.

STRUCTURE OF CARNIVORE THALAMIC RETICULAR NUCLEUS

Fig. 4. Cat thalamus: inner small-celled region. Coronal sections. (A) is a Nissl (cresyl violet) stained section taken at level of mid-dorsal lateral geniculate nucleus. (B) GABA immunostained section: note that there are no labelled cells in inner small celled region. (C) parvalbumin immunostained section of similar area to (B): note the small group of

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labelled cells in the inner small-celled region. In each figure, dorsal is to top and lateral to left. dLGN, dorsal lateral geniculate nucleus; OT, optic tract; VB, ventrobasal complex; 21, zona incerta. PV, parvalbumin. (A) x 10; (B) x40; (C) x 100.

Fig. 5. Cat perireticular nucleus. (A) Nissl (cresyl violet) stained. (B) parvalbumin immunostained, Nissl (neutral red) counterstained (PV/N). (C) GABA immunostained. In each figure, lateral is to the top and rostral to the left. Arrows indicate perireticular cells, lateral to the main body of the reticular nucleus (bottom of figures). Unfilled arrows show parvalbumin labelled fibres. (A), (B) ~ 2 0 (C) 0 x 100.

is a small population of parvalbumin immunoreactive cells in the caudal regions of zona incerta, which are generally flanked dorsally and ventrally by adjacent bands of nonparvalbumin immunoreactive cells. In caudal coronal sections, these parvalbumin immunoreactive cells in the zona

incerta appear to form a contiguous band with the parvalbumin immunoreactive cells in the inner small-celledregion. GABA immunoreactivity is not apparent in the cells of the inner small-celledregion, unlike those in the main body of the reticular nucleus. Figure 4B is a coronal section

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Fig. 6. Cat thalamus. (A) parasaggital section after parvalbumin immunostaining. (B) is a higher magnification of the top (dorsal) part of (A). ( C )is a higher magnification of the bottom (ventral) part of (A). In each figure, dorsal is to top and caudal t o left. Arrows in (B) and (C) indicate the same cells in the perireticular nucleus shown in (A). dLGN, dorsal lateral geniculate nucleus; PGN, perigeniculate nucleus; TRN, thalamic reticular nucleus. (A) ~ 4 0(B), ; (C) x 100.

showing that the main body of the reticular nucleus is strongly GABA immunoreactive, but the inner small-celled region, immediately adjacent to the medial border of the main body of the reticular nucleus, is not GABA immunoreactive (arrow). In zona incerta, most cells are not GABA immunoreactive (Fig. 4B), except for a small group of cells which are located rostrally. These GABA immunoreactive cells are not coincident with the parvalbumin immunoreactive cells in this nucleus. Perireticular nucleus. In cats, there is a group of small neurones, the perireticular nucleus, located between the main body of the reticular nucleus and corpus striatum (arrow, Fig. 5A).Horizontal sections show that these cells are apparent throughout most of the dorsoventral extent of the main body of the reticular nucleus (black circles, Fig. 1) and that their somata appear generally rounded and smaller than those in the main body of the reticular nucleus (Fig. 5A). The perireticular cells, together with those of the reticular nucleus, are parvalbumin (Fig. 5B) and GABA immunoreactive (Fig. 5 0 . From saggital parvalbumin immunoreactive sections, it appears that the perireticular nucleus is continuous with the main body of the reticular nucleus ventrocaudally, forming a horseshoe-like structure (Fig. 6A,C).Dorsocau-

dally in horizontal sections, the perireticular cells are generally arranged in an ordered row, whereas rostroventrally, they are more scattered (Fig. 1).Very few perireticular cells are apparent in areas adjacent to the dorsal lateral geniculate nucleus (Fig. 1). The arrangement of the glia (in Nissl-stained horizontal sections) is distinct in regions immediately medial and lateral to the perireticular nucleus: laterally, the glia are scattered in a somewhat irregular arrangement, whereas medially, they are arranged in lines perpendicular to the reticular and perireticular nuclei (Fig. 5A).Parvalbumin immunoreactive fibres, some of which may be either the axons of corticothalamic and thalamocortical fibres, or the dendrites of the perireticular cells, follow these lines of glia into the main body of the reticular nucleus (unfilled arrows, Fig. 5B).

Ferrets Main body of the reticular nucleus. The main body of the reticular nucleus in ferrets, as in cats, consists of a sheet-like nuclear complex, surrounding most of the dorsal thalamus (Fig. 7). In many areas, the nucleus appears to consist of distinct layers, the details of which vary along the

STRUCTURE OF CARNIVORE THALAMIC RETICULAR NUCLEUS

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DORSAL

A I

"I'

VB

'ENTRAL

Fig. 7. Schematic maps of horizontal sections of ferret thalamus. Sections were immunostained with parvalbumin and Nissl (neutral red) counterstained (PVIN) and sections from dorsal (A) to ventral (HI areas of thalamus are shown. Reticular nucleus is shaded black; zona incerta is the striped region; cells of perireticular nucleus are represented as black circles; scattered cells that link the main body of the reticular and perigeniculate nuclei are represented as unfilled circles. AN, anterior nuclei; CP, caudate-putamen; dLGN, dorsal lateral genic-

d a t e nucleus; EPN, entopeduncular nucleus; GP, globus pallidus; IC, internal capsule; LD, lateral dorsal nucleus; LP, lateral posterior nucleus; MGN, medial geniculate nucleus; OR, optic radiation; OT, optic tract; PG, perigeniculate nucleus; Pul, pulvinar; ST, stria terminalis; VA, ventral anterior nucleus; VB, ventrobasal complex; vLGN, ventral lateral geniculate nucleus; VL, ventral lateral nucleus; ZI, zona incerta. x 30.

dorsoventral axis. In horizontal sections, the main body of the reticular nucleus at its most dorsal aspect consists of a single line of cells flanked by the dorsal thalamus medially and the stria terminalis laterally (Fig. 7A). Further ventrally, cells spread out somewhat, and at the level of the dorsal regions of the dorsal lateral geniculate nucleus, they appear to be arranged in three, sometimes four layers (arrows, Fig. 8A). In general, the structure and arrangement of these dorsal layers of the main body of the reticular nucleus of ferrets are similar to those described in cats, although they are less distinct. Cells in the outermost layer tend to be loosely scattered and the neuropil is lightly stained (with parvalbumin immunolabelling, in particular); cells in the adjacent inner layer, tend to be more closely packed, slightly larger, and the neuropil is darkly stained, and cells in the innermost layer are generally smaller and the neuropil is lightly stained. In horizontal sections, the dorsocaudal region of the main body of the reticular nucleus is continuous with a group of cells scattered throughout the optic radiation, and more caudally still, with the perigeniculate nucleus (Figs. 7B,C, 8A). It should be noted that in the somatostatin immunostained sections of ferrets, only a small proportion of the total population of cells in the perigeniculate nucleus are labelled: parvalbumin and GABA immunoreactivity, by contrast, is apparent in most perigeniculate cells. This finding corroborates the

results of Graybiel and Elde ('83) who report that somatostatin immunoreactivity in cats is apparent in a few perigeniculate cells only. Furthermore, Conley et al. ('91) have shown that somatostatin immunoreactivity is localised in only a small proportion of the total population of cells in the lateral regions of the visual sector of the main body of the reticular nucleus in galagos (cells in all other areas of the nucleus are somatostatin immunoreactive): this region of the nucleus in galagos appears to have a similar connectivity to the carnivore perigeniculate nucleus (see Conley and Diamond, '90). Immediately ventral to the dorsal lateral geniculate nucleus, the structure and arrangement of the main body of the reticular nucleus changes. In horizontal parvalbumin immunoreactive sections, throughout the mediolateral extent of the central and caudal regions of the main body of the reticular nucleus, cells are closely packed, relatively large, oval and fusiform shaped, and the neuropil is darkly stained (Fig. 8B,C). Rostrally, at this level, the main body of the reticular nucleus broadens out into a triangular rostroventral portion, which comprises of two layers of comparatively small rounded cells: an outer layer of loosely scattered cells and lightly stained neuropil, and an inner layer of closer packed cells and darkly stained neuropil (arrows Fig. 8C). In progressively more ventral horizontal sections, the outer layer of the rostroventral portion becomes more

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Fig. 8. Series of horizontal sections along dorsoventral axis of ferret thalamus: (A)is most dorsal section; (B), (C), and (D)are progressively more ventral. Sections were parvalbumin immunostained. In each figure, rostral is to top and medial left. Unfilled arrows in (A), (B), and (C) indicate perireticular nucleus. Arrowheads in (B) and (C) show

parvalbumin immunostained fibres running between perireticular and reticular nuclei. Arrows in (A) and (C) show layers of the main body of the reticular nucleus. AN, anterior nuclei; EPN, entopeduncular nucleus; GP, globus pallidus; VB, ventrobasal complex. X25.

prominent (Fig. 8D) and the central and caudal regions of the main body of the reticular nucleus, become thin and taper off (Fig. 7F-HI. Inner small-celled region. In ferrets, there is no structure comparable to the inner small-celled region of cats. Figure 9A,B,C are horizontal Nissl (cresyl violet) stained sections, which show that there is no clearly distinguishable inner small-celled region between the innermost region of the main body of the reticular nucleus and the external medullary lamina. However, lightly labelled small cells, generally spindle shaped, are occasionally apparent between the innermost region of the main body of the reticular nucleus and the external medullary lamina (ar-

rows, Fig. 9C), but they never form a continuous and distinct region. Furthermore, these cells appear to be GABA and parvalbumin immunoreactive. Perireticular nucleus. In ferrets, as in cats, there is a well-differentiated group of small neurones located between the main body of the reticular nucleus and corpus striatum (arrow, Fig. 10A). Horizontal sections show that these cells are apparent throughout most of the dorsoventral extent of the main body of the reticular nucleus (black circles, Fig. 7) and that their somata appear generally rounded and smaller than the reticular cells (Fig. 10A). The perireticular cells, together with those of the reticular nucleus, are parvalbumin (arrow Fig. lOB), GABA (arrow Fig. lOC), and somato-

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Fig. 9. Ferret thalamus. All figures are horizontal Nissl (cresyl violet) stained sections. In (At and (B),rostral is to top and medial to left. In (C), lateral is to top and rostral is to left. Unfilled arrows in (A) indicate perireticular nucleus. Small arrows in (C) show cells located

between external medullary lamina and inner margin of the main body of the reticular nucleus: these cells did not constitute a clearly differentiated nucleus as in cat. R, reticular nucleus; GP, globus pallidus; DT, dorsal thalamus. (A) X40 (B) x 100 (C) x 100 (D) ~ 2 0 0 .

statin immunoreactive (arrow, Fig. 10D). From horizontal parvalbumin immunoreactive sections, it is clear that the perireticular nucleus is continuous with the main body of the reticular nucleus ventrocaudally, forming a striking horseshoe-like structure (Figs. 7,8). From these horizontal sections, the perireticular cells appear to be arranged in an ordered row caudally, but rostrally they tend to be more scattered (Figs. 7,8). As in cats, the arrangement of the glia (in Nissl-stained horizontal sections) in ferrets is distinct in regions immediately medial and lateral to the perireticular nucleus: laterally, the glial are scattered somewhat, whereas medially, they are arranged in lines perpendicular to the main body of the reticular nucleus and perireticular nucleus (Figs. 9A, 10A).

It is not known if the cells in the different layers of the main body of the reticular nucleus have distinct dendritic morphologies. Several previous studies have reported that cells in the rostroventral portion generally have a multipolar dendritic arrangement (Scheibel and Scheibel, '72; Spreafico et al., '911, whereas cells elsewhere in the nucleus generally have dendrites orientated parallel to the plane of the nucleus (Scheibel and Scheibel, '72; Yen et al., '85; Spreafico et al., '91). A more rigourous examination of the dendritic morphology of reticular cells, in relation to the different layers, awaits investigation. The precise patterns of connectivity of the different layers of the main body of the reticular nucleus is not known. They may for example, connect to distinct areas of the cerebral cortex, the dorsal thalamus, and/or the midbrain. In rabbits, for instance, visual area 1 (Vl) of the cortex projects to the outer two thirds of the main body of the reticular nucleus, whilst V2 projects to the inner one third (Crabtree and Killackey, '89): it is possible that the outer layers of the dorsocaudal main body of cats are related to V1 whilst the innermost layers are related to V2. Furthermore, Cucchiaro et al. ('91) have shown that the perigeniculate nucleus, which is regarded as part of the visual reticular nucleus (Sanderson, '711, connects solely with the A laminae of the dorsal lateral geniculate nucleus: one or more of the dorsal layers of the main body of the reticular nucleus may thus project exclusively to other areas of the visual thalamus such as the C laminae of the dorsal lateral geniculate nucleus or the pulvinar for example (see also Conley et al., '91). Alternatively, the different layers of the reticular nucleus may represent distinct patterns of connectivity the nucleus has with the dorsal thalamus and with the midbrain. It has been shown, for example, that the reticular cells which project to the midbrain are distinct to those that project to the dorsal thalamus (Parent and Steriade, '84).The region of the reticular nucleus which is most involved with the midbrain

DISCUSSION The thalamic reticular nucleus has previously been defined as a sheet of cells that surround most of the rostral and lateral surfaces of the dorsal thalamus, are GABAergic, and lie lateral to the fibres of the external medullary lamina and medial to those of the internal capsule (see Berman and Jones, '82, and Jones, '85 for review). The present results show that there are cytoarchitectonic heterogeneities in the reticular nucleus of cats and ferrets. In cats, three subdivisions of the nucleus are distinguished, two of which are distinct in ferrets also.

Layers of the main body of the thalamic reticular nucleus This study has shown that the reticular nucleus of cats, and to a lesser extent ferrets, consists of distinct layers, the details of which varies along the dorsoventral axis. There is an outer layer of scattered cells, most distinct in the rostroventral portion, and an inner layer of generally larger, densely packed cells. Dorsal to the dorsal lateral geniculate nucleus, the main body of the reticular nucleus contains one, sometimes two additional layers.

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Fig. 10. Ferret perireticular nucleus. All figures are horizontal sections. (A)Nissl (cresyl violet) stained, (B) parvalbumin immunostained, (C) GABA immunostained, and (D) somatostatin immunostained. in each figure, lateral is to top and rostra1 to left. Arrows indicate cells in perireticular nucleus, lateral to the main body of the reticular nucleus (bottom of figures). x 100.

pathways is the rostroventral portion (see Cornwall et al., '90; Pare et al., '90): it may be that reticular cells in one of the two layers in this region of the nucleus are connected to the midbrain and cells in the other layer to the dorsal thalamus.

Inner small celled region: A dorsal extension of the zona incerta? This study reports on a group of cells (inner small-celled region) confined to the inner margin of the ventral regions

STRUCTURE OF CARNIVORE THALAMIC RETICULAR NUCLEUS of the main body of the reticular nucleus, yet outside of the external medullary lamina. Previous studies have referred to this area as part of the main body of the reticular nucleus (Berman and Jones, '82; Jones, '851, but we suggest that the inner small-celled region forms a separate subnucleus in its own right. This subnucleus is well differentiated in cats, but not in ferrets: the significance of this species difference is unclear. The inner small celled region consists of a group of small cells, somewhat separated from the main body of the reticular nucleus and continuous with the zona incerta ventrally. The cytoarchitecture of the cells of the inner small-celled region is clearly distinct from the cells of the reticular nucleus and from those of the zona incerta, being generally smaller and more lightly labelled. The cells of the inner small-celled region of cats appear distinct to the small f cells described in the medial part of the rat reticular nucleus (Spreafico et al., '91) for two major reasons. First, the cat inner small-celled region appears in a distinct compartment, somewhat separate from the reticular nucleus: the rat f cells are incorporated within the reticular nucleus. Second, the inner small-celled region is not GABA immunoreactive (see below): the rat f cells are immunostained for GABA. With respect to immunocytochemical character, the inner small-celled region is quite distinct from the main body of the reticular nucleus, but similar to the zona incerta. First, and perhaps most important, the inner small-celled region is not GABA immunoreactive: GABA and glutamic acid decarboxylase immunoreactivity characterise reticular cells (see Jones, '85). Furthermore, GABA immunoreactivity is not apparent in cells of the zona incerta, except for a small group rostrally in the nucleus (see also Jones, '85). Second, the majority of cells in the inner small-celled region, together with those in the zona incerta, are not parvalbumin immunoreactive: in both structures, there is a small group of labelled cells located caudally. Third, from the data of Graybiel and Elde ('83), most reticular cells are somatostatin immunoreactive, whereas cells in the vicinity of the inner small-celled region and in the zona incerta are not immunoreactive to the antibody to somatostatin (their Fig. 5 ) . The connectivity of the inner small celled region has not yet been fully examined. The recent results of Crabtree ('91), however, suggest that the somatosensory cortex of cats does not project to the inner small-celled region (as distinct to immediately adjacent areas of the main body of the reticular nucleus). Furthermore, the results of Royce et al. ('91) indicate that the centromedian and parafascicular nuclei of cats appear to receive projections from the rostroventral portion of the reticular nucleus (see also Steriade et al., '84),but also, in one particular experiment (their Fig. 2), in precisely the location of the inner small-celled region. In addition, from Royce and colleagues the results of labelled cells formed a contiguous line from the presumed inner small-celled region and into the zona incerta. These connectivity data, together with the findings that the inner small-celled region is spatially separate and cyto and chemoarchitectonically distinct from the main body of the reticular nucleus, suggest that it may constitute a previously undescribed, dorsal extension of the zona incerta.

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Perireticular nucleus: a newly described thalamic structure The present study has drawn attention to a group of small neurones located lateral to the main body of the reticular nucleus and medial to the corpus striatum. These small cells, or perireticular nucleus, have not been described previously (see Berman and Jones, '82; Jones, '85) and represent a distinct thalamic nucleus which itself constitutes a sheet-like structure surrounding the sheetlike reticular nucleus. The perireticular nucleus is in a distinct location among the fibres of the internal capsule adjacent to the thalamus. The cells appear to lie along the border where the characteristic fibre bundles of the internal capsule passing from the cerebral cortex to the cerebral peduncle are giving off fibres to the thalamus with a characteristic linear arrangement of the glia. Although the perireticular nucleus is morphologically distinct from the main body of the reticular nucleus, the two structures seem closely related since they are continuous ventrocaudally. Furthermore, the perireticular cells are labelled with all the immunocytochemical markers that characterise reticular cells. These include antibodies to parvalbumin, GABA, and somatostatin (the latter shown in ferrets only): no single group of cells in the corpus striatum is characterised by all three markers (Ottersen and StormMathison, '84; Smith et al., '87; Reiner and Anderson, '90). The connectivity of the perireticular cells has yet to be determined, but it is possible that they have a similar connectivity to reticular cells. That is, most afferents are in the form of collaterals from thalamocortical and corticothalamic fibres and most efferents go to the dorsal thalamus. Since the perireticular nucleus is a sheet-like nucleus surrounding most of the reticular nucleus, it crosses the various 'modality specific sectors' of the reticular nucleus and may itself therefore also contain sectors. Alternatively, the perireticular nucleus may not project to the dorsal thalamus, but solely to the reticular nucleus. The perireticular nucleus may thus form a GABAergic inhibitory input to the reticular nucleus.

ACKNOWLEDGMENTS We are much indebted to Ray Guillery for his patience, guidance and support throughout the entire course of this study. Much appreciation is extended also to Zillah Deussen, Mary Walker, Davina Hocking, Mohan Masih, Colin Beasley, and David Stroud for their exemplary and polite technical assistance. AEC is a Wellcome Trust research assistant and JM is an Endeavour Fellow of the Royal Society.

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