Morphological and GAD immunocytochemical properties of the dorsal lateral geniculate nucleus in a reptile

Brain Research Bulletin, Vol. 34, No. 1, pp. 723-726, 1994 Copyright Q 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0361-9230/94 $6.00 + .OO

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Morphological and GAD Immunocytochemical Properties of the Dorsal Lateral Geniculate Nucleus in a Reptile MICHAEL Department

B. PRITZ’

AND

MARK

E. STRITZEL

of Neurological Surgery, California College of Medicine, Irvine Medical Center, Orange, CA 92668 Received

22 July 1993; Accepted

27 October

University

of California

1993

PRITZ, M. B. AND M. E. STRITZEL. Morphological and GAD immunocytochemicalproperties of the dorsal lateral geniculate nucleus in a reptile. BRAIN RBS BULL 33(6) 723-726, 1994.-Morphological and glutamic acid decarboxylase (GAD) immunocytochemical properties of the dorsal part of the lateral geniculate nucleus were investigated in reptiles, Caiman crocodilus. GAD immunoreactive neurons, although sparse, were found in the dorsal part of the lateral geniculate nucleus. In addition, puncta and fibers immunoreactive for GAD were also observed in this same nucleus. These results in Cairnan utilizing GAD are similar to observations on the dorsal part of the lateral geniculate nucleus of other reptiles in which antisera to gamma amino butyric acid (GABA) were used. Dorsal geniculate

nucleus

Evolution

Inhibition

Local circuit neuron

Reptiles

Thalamus

in this nucleus in Caiman (12). To reconcile these apparent differences, we investigated the GAD immunoreactivity in the dorsal part of the lateral geniculate nucleus in Caiman.

IN reptiles, nuclei that comprise the dorsal thalamus have been defined as those neuronal aggregates that project to the telencephalon (14). In one group of reptiles, Cairnan crocodilus, seven thalamic nuclei have so far been found to send axons to the telencephalon. These nuclei are: nucleus dorsolateralis anterior (11), nucleus dorsomedialis anterior (4), nucleus rotundus (9), nucleus reuniens pars centralis (8), nucleus reuniens pars diffusa (15), the medialis complex (10,16), and nucleus diagonalis (4). Neurons immunoreactive to gamma aminobutyric acid (GABA), or glutamic acid decarboxylase (GAD), were not identified in any of these seven dorsal thalamic nuclei in Cuiman (12). However, this experiment (12) specifically did not investigate GABA/GAD immunoreactivity in the dorsal part of the lateral geniculate nucleus in Caiman. Recently, others (1,2,18) have investigated GABA immunoreactivity in the thalamus of several reptilian species other than Caiman. Some of these studies in other reptiles (1,2,18) misinterpreted our observations (12) and concluded that the dorsal part of the lateral geniculate nucleus in Caiman did not contain GABA immunoreactive neurons (1,2,18) or puncta (18) when, in fact, we had not examined GAD or GABA immunoreactivity

METHOD

Descriptive observations detailed in this report are based on five juvenile Caiman brains in which transverse sections, 25 or 50 pm in thickness, were stained with cresyl violet. Immunocytochemical experiments utilized a polyclonal antibody to GAD (gift from D. Schmechel) or a monoclonal antibody to GAD isolates designated as GAD-l, GAD-2, and GAD-5 (6) (gift from D. Gottlieb). Details of the methodology and interpretation of immunocytochemical experiments are available elsewhere (12,13). RESULTS

A lateral geniculate nucleus and nucleus ovalis have been identified in a morphological study in a closely related species, Alligator mississippiensis (7). However, this report did not divide the lateral geniculate into dorsal and ventral components. Others

’ Requests for reprints should be addressed to M. B. Pritz, M.D., Ph.D., Section of Neurological 545 Barnhill Drive, Indianapolis, IN 46202-5124.

723

Surgery,

Indiana

University

School of Medicine,

724

who have investigated retinal projections in Cairnan (3,517) have divided the lateral geniculate nucleus into dorsal and ventral portions (3,17) and have differentiated between a nucleus ovalis and the dorsal part of the lateral geniculate nucleus (17). However, anatomical descriptions and morphological documentation to distinguish these neuronal aggregates in C&man are limited. In Cairnan, the lateral geniculate lies just medial to the optic tract and extends throughout all but the most rostra1 portion of the thalamus. This neuronal aggregate begins at the level of caudal pole of nucleus rotundus and continues as far rostra1 as the caudal end of nucleus dorsolateralis anterior. A separation between a smaller, rounded dorsal portion and a larger, conical ventral part can be distinguished in cell-stained preparations. This distinction becomes clearer rostrally (Fig. 1A and E). The ventral geniculate

PRITZ

AND S’I’RITZEi.

can be further subdivided into a medial cell plate region that contains a dark band of densely packed neurons oriented vertically and a more laterally located cell poor region (Fig. 1E and F). Immediately rostra1 and in the same topographic location in the thalamus as the lateral geniculate nucleus lies nucleus ovalis (Fig. IH). These morphological features and topographic relationships allow identification of, and differentiation between. the dorsal and ventral parts of the lateral geniculate nucleus and nucleus ovalis. Having identified the dorsal part of the lateral geniculate nucleus in descriptive material, immunocytochemical preparations utilizing polyclonal and monoclonal antibodies to GAD were scrutinized. In material stained with a polyclonal antibody to GAD, many immunoreactive fibers and puncta were seen (Fig.

A

FIG. 1. Cytoarchitecture of the lateral geniculate nucleus and nucleus ovalis. Photographs through the central part of the lateral geniculate nucleus (A, E), the most rostra1 part of the lateral geniculate nucleus (B, F), the junction between the lateral geniculate nucleus and nucleus ovalis (C, G), and the most caudal portion of nucleus ovalis (D, H) are shown. Photos A-D are lower power views of the diencephalon while E-H are higher magnification photographs from the respective framed areas in A-D. The distance between the sections in photos A and B is 150 pm while the sections illustrated in B-D were adjacent to each other. The black arrowhead in E and F marks the division between the dorsal and ventral parts of the lateral geniculate nucleus. The white arrow in E and F points to the cell plate of the ventral part of the lateral geniculate nucleus. The asterisk in G indicates the iunction between the most rostra1 part of the lateral geniculate nucleus and the most caudal portion of nucleus ovalis. Dla, nucleus dorsolateralis anterior; Dma, nucleus dorsomedialis-anterior; dp, dorsaipeduncle of the lateral forebrain bundle; Gd, dorsal part of the lateral geniculate nucleus; Gv, ventral part of the lateral geniculate nucleus; H, hypothalamus; Hb, habenula; MC, medialis complex; OT, optic tract; ov, nucleus ovalis; Rc, nucleus reuniens pars centralis; Rd, nucleus reuniens pars diffusa; Rt, nucleus rotundus; SM, stria medullaris. Bar A-D = 400 ym; Bar E-H = 200 /Lm.

725

FIG. 2. GAD ~mmnnorea~tivity in Cukuzn dorsal g~niculat~ nucleus. Neurons (arrows), puncta, and fibers immnnoreactive for GAD were visualized by bri~ht~~~ld illum~ation by a ~ly~~onal antibody (A). Note the staining of neuronal processes (arrowheads) of one of the GAD(+) c&s {arrows) (A). Puncta but not neurons were seen under Noma& optics when mon~~onal isohtes GAD-1 CC), GAD-2 (III), and GADS (E) were employed. No ~mmnnorea~~~ity was observed in control sections that utilized preimmnne serum (B) or normal mouse serum (Q Co~~~tr~t~o~of primary antibody was: ~/~~ for the ~o~y~iona1 GAD antisera (A); 1/2~ for GAD-~ (C); XI4HMfor GAD2 @); and 112000 for GAD-5 (E). Con~o~~a~ion of preimmune serum (B) was 11500while the concentration of normal mouse serum (F) was 112000. Bars in B (for photos A and B) and F (for pfioros C-F) = 25 grn.

2A). GAD(+) neurons were identified (Fig. 2A) but their occurfence was rare. Nevertheless, GAD i~unorea~ti~ity visualized neuronal processes (Fig,. 2A). In experiments that utilized monoclonaf ~tjb~ies to GAD isolates designated as GAD-I, GAD2, and GAD-5 {6), pnncta but not neurons ~unoreacti~e to GAD were seen (Fig. 2C-E). Controls in these ~~~nocyt~-

chemical experiments were of !wo types. One included sections incubated with normal (Fig. 2F) or preimmune (Fig. 33) serum substituted for the primary antibody at concentrations equal to or greater than that used for the primary antibody. The other utilized sections that were processed with the omission of one of the following reagents: primary antibody, b~~t~~y~atedsecondary an-

PRITZ

726

tibody, or avidin-biotin imal (Fig. 2B and F).

complex.

Nonspecific

staining was min-

DISCUSSION

A comparison between these observations in Caiman and similar ones in other reptiles is hampered by differences in anatomical terminology as well as by incomplete knowledge of neural circuitry and morphologic features of the dorsal part of the lateral geniculate nucleus in these other reptilian species (1,2,18) in which GABA immunoreactivity in the thalamus has been investigated. Nevertheless, the presence of GAD(+) neurons and puncta in the dorsal part of the lateral geniculate nucleus of Caiman is similar to the observations of GABA immunoreactivity in a retinal recipient nucleus of the same name and topographical location found in several other reptiles: chameleon (2), turtles (1,18), snakes (18), and lizards (18). However, the occurrence of GAD(+) cells, is less frequent in Caiman than in these other reptiles. The present study clarifies the literature in regards to the GABA/GAD immunocytochemical properties of the dorsal part of the lateral geniculate nucleus in Caiman which have not been described until now. Contrary to the reports of others (1,2,18), the dorsal part of the lateral geniculate in Caiman contains GAB(+) cells, fibers, and puncta. These observations are in

AND

STRITZEL

agreement with GABA immunocytochemical studies performed in similarly labeled nuclei in other reptiles (1.2,18). Furthermore. the presence of GAD(+) cells in the dorsal part of lateral geniculate nucleus in Caiman contrasts with the absence of GAD(+ ) neurons in other thalamic nuclei (dorsolateralis anterior. dorsomedialis anterior, reuniens, rotundus, diagonalis, medialis complex). This pattern of immunoreactivity is not unique to Caiman. Assuming that similarly named nuclei in certain other reptiles (1,2,18) are homologous to Caiman thalamic nuclei and that neurons scattered at the periphery of certain thalamic nuclei in turtles (1) are not considered part of that nucleus, the following thalamic nuclei in other reptiles also lack GABA cells: rotundus (1,2,18), reuniens (1,18), dorsolateralis anterior (1,18), and dorsomedialis anterior (1,18). Taken together, these findings suggest a similar pattern of GABA/GALI immunoreactivity in the thalamus of all reptiles so far examined.

ACKNOWLEDGEMENTS

We are particularly grateful to D. Gottlieb for his gift of monoclonal GAD antiserum and to D. Schmechel for his gift of polyclonal GAD antiserum and preimmune serum. We also thank J. Jackson and C. Mugerditchian for manuscript preparation and L. Sutherland and the Department of Pathology, University of California Irvine, for the use of photographic facilities.

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