Brain Research, 554 (1991) 325-328 (~) 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 A D ONIS 0006899391247397
325
BRES 24739
Calcium binding protein immunoreactivity in a reptilian thalamic reticular nucleus Michael B. Pritz and Mark E. Stritzel Division of Neurological Surgery, California College of Medicine, University of California lrvine Medical Center, Orange, CA 92668 (U.S.A.)
(Accepted 9 April 1991) Key words: Calbindin; Calcitonin gene-related peptide; Evolution; Parvalbumin; Reptile; Thalamic reticular nucleus
|mmunoreactivity to antisera directed against a variety of calcium binding proteins was investigated in the thalamic reticular nucleus of one species of reptiles, Caiman crocodilus. Cells and fibers were immunoreactive for parvalbumin whereas fibers but not neurons were immunoreactive for calbindin and calcitonin gene related peptide. These data support the observations that Caiman thalamic reticular nucleus contains at least two neuronal populations. One group, whose cells are immunoreactive for parvalbumin, forms at least part of the projection to the dorsal thalamus. The other group, which contains qualitatively different and sparser number of neurons, is immunoreactive for glutamic acid decarboxylase.
Recent experiments in one group of reptiles, Caiman crocodilus, have identified a nucleus that shares several features commonly attributed to mammalian thalamic reticular nucleus. These include location in the ventral thalamus with cells embedded in the exiting thalamotelencephalic fibers and a projection to the dorsal thalamus. Because of these similarities, we renamed this area, the nucleus of the dorsal peduncle of the lateral forebrain bundle, as the thalamic reticular nucleus 1°. However, significant differences between mammalian and Caiman thalamic reticular nucleus exist. In mammals, the reticular nucleus is composed of a single population of neurons that projects to the dorsal thalamus 4 and contains neurons immunoreactive for ~-aminobutyric acid ( G A B A ) 2, and its synthesizing enzyme, glutamic acid decarboxylase ( G A D ) 3,6. While some neurons scattered throughout the anterior portion of Caiman thalamic reticular nucleus are immunoreactive for G A D , G A D ( + ) , those cells projecting to the dorsal thalamus are not ~°. Since recent studies investigating calcium binding protein distribution in mammalian thalamic nuclei have identified parvalbumin immunoreactivity co-localized with G A B A in cells of the thalamic reticular nucleus 1,5, we asked whether such a population of neurons was present in Caiman as well. Experiments were performed on 12 juvenile Caiman crocodilus that ranged in snout-vent length from 14.6 to 17.0 cm. Although immunoreactivity to a variety of
calcium binding proteins was investigated 9, the following primary antibodies gave the best results after pretreatment with intraventricular injection of 10 or 20/A of a 1% solution (w/v) of colchicine 7. These included: sheep anti-parvalbumin antisera made against H P L C purified protein derived from mouse muscle and confirmed by Western blot technique; rabbit anti-calbindin antisera made against H P L C purified 28 k D a calbindin isolated from chicken intestine that will also recognize calretinin; and goat anti-calcitonin gene related peptide ( C G R P ) antisera. Antibodies to parvalbumin and calbindin were antisera of P.C. Emson and gift from E.G. Jones while the antibody to C G R P was a gift from Arnel Laboratories. Perfusion and tissue processing was similar to that described previously s except that the concentration of paraformaldehyde was 2% and a Vectastain elite A B C kit (Vector Laboratories, Burlingame, C A ) was used. Controls included substitution of normal serum at concentrations equal to or greater than that of the primary antibody and use of the biotinylated secondary antibody alone or in combination with avidin-biotin complex. Parvalbumin immunoreactivity was observed in Caiman thalamic reticular nucleus (Fig. 1). Fibers, puncta, as well as immunoreactive neurons were seen (Fig. 1B and C) throughout the extent of this nucleus. When staining of processes was visualized, neuronal elements interpreted to be dendrites were oriented parallel to the disposition of fibers within the thalamic reticular nucleus.
Correspondence: M.B. Pritz, Division of Neurological Surgery, University of California Irvine Medical Center, P.O. Box 14091, Orange, CA 92613-4091, U.S.A.
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Fig. 1. Parvalbumin immunoreactivity in Caiman thalamic reticular nucleus. A: line drawing that illustrates the location of the low-power photomicrograph in B (boxed area). C: a higher-power photo of B in which arrows (B and C) mark blood vessels that serve for orientation. Concentration of primary antibody in B and C was 1/8000 while the concentration of sheep serum in the control photo (D) was 1/4000. dp, dorsal peduncle of the lateral forebrain bundle; FB, forebrain bundles; H, hypothalamus; Hb, habenula; OT, optic tract; Rt, nucleus rotundus; SM, stria meduUaris. Bars B,C,D = 100 ~um.
Fig. 2. Calbindin immunoreactivity in Caiman thalamic reticular nucleus. A: line drawing that illustrates the location of the photomicrograph in B (boxed area). Concentration of primary antibody in B was 1/32,000 while the concentration of rabbit serum in the control photo (C) was 1/4000. Note the presence of calbindin immunoreactive fibers and absence of immunoreactive cells in B. dp, dorsal peduncle of the lateral forebrain bundle; Dla, nucleus dorsolateralis anterior; Dma, nucleus dorsomedialis anterior; FB, forebrain bundles; H, hypothalamus; Hb, habenula; OT, optic tract; SM, stria medullaris. Bars B,C = 100/~m.
327 Although quantitative measurements were not made, comparison with prior analyses suggested that these parvalbumin immunoreactive cells were of similar size and shape as those thalamic reticular neurons that projected to the dorsal thalamus and were larger than the G A D ( + ) cells described previously 1°. Control sections with normal serum substituted at concentrations twice that used for the primary parvalbumin antibody showed no evidence of significant non-specific immunoreactivity (Fig. 1D). Fibers immunoreactive for calbindin (Fig. 2B) and CGRP (Fig. 3B) were demonstrated in the dorsal peduncle of the lateral forebrain bundle. However, no immunoreactive neurons were identified in this nucleus despite the presence of labeled cells in nuclear groups of the dorsal thalamus in the same histologic section 9. Considering the general pitfalls and limitations intrinsic to immunocytochemical experiments 7'8 and those possible problems specific to the use of calcium binding protein antisera 5, the present study has identified a population of neurons in Caiman thalamic reticular nucleus that are immunoreactive for parvalbumin, parvalbumin(+). These parvalbumin(+) cells share several morphologic features in common with neurons of the thalamic reticular nucleus that projected to the dorsal thalamus 1°. These similarities include: cells soma size, disposition of neuronal processes parallel to the fibers of the lateral forebrain bundle, and distribution throughout the extent of the thalamic reticular nucleus. These same anatomical features distinguish parvalbumin(+) cells from G A D ( + ) neurons of the thalamic reticular nucleus. G A D ( + ) cells are smaller, do not have processes primarily oriented parallel to the fibers of the lateral forebrain bundle, and are located in the anterior portion of the thalamic reticular nucleus 1°. Taken together, these data suggest that Caiman thalamic reticular nucleus contains at least two neuronal populations. One group which projects to the dorsal thalamus, is immunoreactive for parvalbumin. The other group, which does not send axons to terminate in the dorsal thalamus and is presumably intrinsic, is immunoreactive for GAD. These findings are at variance with recent observations in mammals. Neurons of mammalian thalamic reticular nucleus co-localize parvalbumin and G A B A 1'5 and project to the dorsal thalamus 4. Interestingly, in mammals processes of thalamic reticular nucleus neurons are oriented at right angles to the fibers of the internal capsule 4. On the other hand, in Caiman thalamic reticular nucleus, the parvalbumin(+), dorsal thalamic projecting cells are oriented parallel to the fibers in the lateral forebrain bundle whereas at least some G A D ( + ) cells are aligned perpendicular to this fiber bundle interconnecting the thalamus with the telencephalon.
The present experiments, which demonstrate additional differences between Caiman and mammalian tha-
A
Dla
Imm
B
e
C
Fig. 3. CGRP immunoreactivity in Caiman thalamic reticular nucleus. A: line drawing that illustrates the location of the photomicrograph in B (boxed area). Concentration of primary antibody in B was 1/8000 while the concentration of goat serum in the control photo (C) was 1/2000. Note the presence of CGRP immunoreactive fibers and absence of immunoreactive cells in B. Abbreviations as in Fig. 2. Bars B,C = 100/zm.
328 lamic o r g a n i z a t i o n , l e a v e several q u e s t i o n s u n a n s w e r e d .
of t h a l a m i c o r g a n i z a t i o n in Caiman, are the subject of
First, are all dorsal t h a l a m i c p r o j e c t i n g n e u r o n s of the
future studies.
Caiman t h a l a m i c r e t i c u l a r nucleus p a r v a l b u m i n ( + ) ? Seco n d , are s u b p o p u l a t i o n s of p a r v a l b u m i n ( + ) cells p r e s e n t in Caiman t h a l a m i c r e t i c u l a r nucleus as has b e e n sugg e s t e d in m a m m a l s 1 ? A n s w e r s to these questions, as well as the biologic and e v o l u t i o n a r y significance of this type
1 Celio, M.R., Calbindin D-28k and parvalbumin in the rat nervous system, Neuroscience, 35 (1990) 375-475. 2 de Biasi, S., Frassoni, C. and Spreafico, R., GABA immunoreactivity in the thalamic reticular nucleus of the rat. A light and electron microscopical study, Brain Research, 399 (1986) 143147. 3 Houser, C.R., Vaughn, J.E., Barker, R.P. and Roberts, E., GABA neurons are the major cell type of the nucleus reticularis thalami, Brain Research, 200 (1980) 341-354. 4 Jones, E.G., The Thalamus, Plenum, New York, 1985, 935 pp. 5 Jones, E.G. and Hendry, S.H.C., Differential calcium binding protein immunoreactivity distinguishes classes of relay neurons in monkey thalamic nuclei, Eur. J. Neurosci., 1 (1989) 222-246. 6 Oertel, W.H., Graybiel, A.M., Mugnaini, E., Elde, R.P., Schmechel, D.E. and Kopin, I.J., Coexistence of glutamic acid
We are grateful to Prof. E.G. Jones for the gift of parvalbumin and calbindin antibodies and Arnel Laboratories for the CGRP antibody. We thank K. Maskew for manuscript preparation and L. Sutherland and the Department of Pathology for the use of photographic facilities.
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decarboxylase- and somatostatin-like immunoreactivity in neurons of the feline nucleus reticularis thalami, J. Neurosci., 3 (1983) 1322-1332. Pritz, M.B. and Stritzel, M.E., Thalamic nuclei that project to reptilian telencephalon lack GABA and GAD immunoreactive neurons and puncta, Brain Research, 457 (1988) 154-159. Pritz, M.B. and Stritzel, M.E., Reptilian dorsal column nucleus lacks GAD immunoreactive neurons, Brain Research, 503 (1989) 175-179. Pritz, M.B. and Stritzel, M.E., Calcium binding protein immunoreactivity in reptilian dorsal thalamus, Soc. Neurosci. Abstr., 16 (1990) 245. Pritz, M.B. and Stritzel, M.E., A different type of vertebrate thalamic organization, Brain Research, 525 (1990) 330-334.
325
BRES 24739
Calcium binding protein immunoreactivity in a reptilian thalamic reticular nucleus Michael B. Pritz and Mark E. Stritzel Division of Neurological Surgery, California College of Medicine, University of California lrvine Medical Center, Orange, CA 92668 (U.S.A.)
(Accepted 9 April 1991) Key words: Calbindin; Calcitonin gene-related peptide; Evolution; Parvalbumin; Reptile; Thalamic reticular nucleus
|mmunoreactivity to antisera directed against a variety of calcium binding proteins was investigated in the thalamic reticular nucleus of one species of reptiles, Caiman crocodilus. Cells and fibers were immunoreactive for parvalbumin whereas fibers but not neurons were immunoreactive for calbindin and calcitonin gene related peptide. These data support the observations that Caiman thalamic reticular nucleus contains at least two neuronal populations. One group, whose cells are immunoreactive for parvalbumin, forms at least part of the projection to the dorsal thalamus. The other group, which contains qualitatively different and sparser number of neurons, is immunoreactive for glutamic acid decarboxylase.
Recent experiments in one group of reptiles, Caiman crocodilus, have identified a nucleus that shares several features commonly attributed to mammalian thalamic reticular nucleus. These include location in the ventral thalamus with cells embedded in the exiting thalamotelencephalic fibers and a projection to the dorsal thalamus. Because of these similarities, we renamed this area, the nucleus of the dorsal peduncle of the lateral forebrain bundle, as the thalamic reticular nucleus 1°. However, significant differences between mammalian and Caiman thalamic reticular nucleus exist. In mammals, the reticular nucleus is composed of a single population of neurons that projects to the dorsal thalamus 4 and contains neurons immunoreactive for ~-aminobutyric acid ( G A B A ) 2, and its synthesizing enzyme, glutamic acid decarboxylase ( G A D ) 3,6. While some neurons scattered throughout the anterior portion of Caiman thalamic reticular nucleus are immunoreactive for G A D , G A D ( + ) , those cells projecting to the dorsal thalamus are not ~°. Since recent studies investigating calcium binding protein distribution in mammalian thalamic nuclei have identified parvalbumin immunoreactivity co-localized with G A B A in cells of the thalamic reticular nucleus 1,5, we asked whether such a population of neurons was present in Caiman as well. Experiments were performed on 12 juvenile Caiman crocodilus that ranged in snout-vent length from 14.6 to 17.0 cm. Although immunoreactivity to a variety of
calcium binding proteins was investigated 9, the following primary antibodies gave the best results after pretreatment with intraventricular injection of 10 or 20/A of a 1% solution (w/v) of colchicine 7. These included: sheep anti-parvalbumin antisera made against H P L C purified protein derived from mouse muscle and confirmed by Western blot technique; rabbit anti-calbindin antisera made against H P L C purified 28 k D a calbindin isolated from chicken intestine that will also recognize calretinin; and goat anti-calcitonin gene related peptide ( C G R P ) antisera. Antibodies to parvalbumin and calbindin were antisera of P.C. Emson and gift from E.G. Jones while the antibody to C G R P was a gift from Arnel Laboratories. Perfusion and tissue processing was similar to that described previously s except that the concentration of paraformaldehyde was 2% and a Vectastain elite A B C kit (Vector Laboratories, Burlingame, C A ) was used. Controls included substitution of normal serum at concentrations equal to or greater than that of the primary antibody and use of the biotinylated secondary antibody alone or in combination with avidin-biotin complex. Parvalbumin immunoreactivity was observed in Caiman thalamic reticular nucleus (Fig. 1). Fibers, puncta, as well as immunoreactive neurons were seen (Fig. 1B and C) throughout the extent of this nucleus. When staining of processes was visualized, neuronal elements interpreted to be dendrites were oriented parallel to the disposition of fibers within the thalamic reticular nucleus.
Correspondence: M.B. Pritz, Division of Neurological Surgery, University of California Irvine Medical Center, P.O. Box 14091, Orange, CA 92613-4091, U.S.A.
326
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Fig. 1. Parvalbumin immunoreactivity in Caiman thalamic reticular nucleus. A: line drawing that illustrates the location of the low-power photomicrograph in B (boxed area). C: a higher-power photo of B in which arrows (B and C) mark blood vessels that serve for orientation. Concentration of primary antibody in B and C was 1/8000 while the concentration of sheep serum in the control photo (D) was 1/4000. dp, dorsal peduncle of the lateral forebrain bundle; FB, forebrain bundles; H, hypothalamus; Hb, habenula; OT, optic tract; Rt, nucleus rotundus; SM, stria meduUaris. Bars B,C,D = 100 ~um.
Fig. 2. Calbindin immunoreactivity in Caiman thalamic reticular nucleus. A: line drawing that illustrates the location of the photomicrograph in B (boxed area). Concentration of primary antibody in B was 1/32,000 while the concentration of rabbit serum in the control photo (C) was 1/4000. Note the presence of calbindin immunoreactive fibers and absence of immunoreactive cells in B. dp, dorsal peduncle of the lateral forebrain bundle; Dla, nucleus dorsolateralis anterior; Dma, nucleus dorsomedialis anterior; FB, forebrain bundles; H, hypothalamus; Hb, habenula; OT, optic tract; SM, stria medullaris. Bars B,C = 100/~m.
327 Although quantitative measurements were not made, comparison with prior analyses suggested that these parvalbumin immunoreactive cells were of similar size and shape as those thalamic reticular neurons that projected to the dorsal thalamus and were larger than the G A D ( + ) cells described previously 1°. Control sections with normal serum substituted at concentrations twice that used for the primary parvalbumin antibody showed no evidence of significant non-specific immunoreactivity (Fig. 1D). Fibers immunoreactive for calbindin (Fig. 2B) and CGRP (Fig. 3B) were demonstrated in the dorsal peduncle of the lateral forebrain bundle. However, no immunoreactive neurons were identified in this nucleus despite the presence of labeled cells in nuclear groups of the dorsal thalamus in the same histologic section 9. Considering the general pitfalls and limitations intrinsic to immunocytochemical experiments 7'8 and those possible problems specific to the use of calcium binding protein antisera 5, the present study has identified a population of neurons in Caiman thalamic reticular nucleus that are immunoreactive for parvalbumin, parvalbumin(+). These parvalbumin(+) cells share several morphologic features in common with neurons of the thalamic reticular nucleus that projected to the dorsal thalamus 1°. These similarities include: cells soma size, disposition of neuronal processes parallel to the fibers of the lateral forebrain bundle, and distribution throughout the extent of the thalamic reticular nucleus. These same anatomical features distinguish parvalbumin(+) cells from G A D ( + ) neurons of the thalamic reticular nucleus. G A D ( + ) cells are smaller, do not have processes primarily oriented parallel to the fibers of the lateral forebrain bundle, and are located in the anterior portion of the thalamic reticular nucleus 1°. Taken together, these data suggest that Caiman thalamic reticular nucleus contains at least two neuronal populations. One group which projects to the dorsal thalamus, is immunoreactive for parvalbumin. The other group, which does not send axons to terminate in the dorsal thalamus and is presumably intrinsic, is immunoreactive for GAD. These findings are at variance with recent observations in mammals. Neurons of mammalian thalamic reticular nucleus co-localize parvalbumin and G A B A 1'5 and project to the dorsal thalamus 4. Interestingly, in mammals processes of thalamic reticular nucleus neurons are oriented at right angles to the fibers of the internal capsule 4. On the other hand, in Caiman thalamic reticular nucleus, the parvalbumin(+), dorsal thalamic projecting cells are oriented parallel to the fibers in the lateral forebrain bundle whereas at least some G A D ( + ) cells are aligned perpendicular to this fiber bundle interconnecting the thalamus with the telencephalon.
The present experiments, which demonstrate additional differences between Caiman and mammalian tha-
A
Dla
Imm
B
e
C
Fig. 3. CGRP immunoreactivity in Caiman thalamic reticular nucleus. A: line drawing that illustrates the location of the photomicrograph in B (boxed area). Concentration of primary antibody in B was 1/8000 while the concentration of goat serum in the control photo (C) was 1/2000. Note the presence of CGRP immunoreactive fibers and absence of immunoreactive cells in B. Abbreviations as in Fig. 2. Bars B,C = 100/zm.
328 lamic o r g a n i z a t i o n , l e a v e several q u e s t i o n s u n a n s w e r e d .
of t h a l a m i c o r g a n i z a t i o n in Caiman, are the subject of
First, are all dorsal t h a l a m i c p r o j e c t i n g n e u r o n s of the
future studies.
Caiman t h a l a m i c r e t i c u l a r nucleus p a r v a l b u m i n ( + ) ? Seco n d , are s u b p o p u l a t i o n s of p a r v a l b u m i n ( + ) cells p r e s e n t in Caiman t h a l a m i c r e t i c u l a r nucleus as has b e e n sugg e s t e d in m a m m a l s 1 ? A n s w e r s to these questions, as well as the biologic and e v o l u t i o n a r y significance of this type
1 Celio, M.R., Calbindin D-28k and parvalbumin in the rat nervous system, Neuroscience, 35 (1990) 375-475. 2 de Biasi, S., Frassoni, C. and Spreafico, R., GABA immunoreactivity in the thalamic reticular nucleus of the rat. A light and electron microscopical study, Brain Research, 399 (1986) 143147. 3 Houser, C.R., Vaughn, J.E., Barker, R.P. and Roberts, E., GABA neurons are the major cell type of the nucleus reticularis thalami, Brain Research, 200 (1980) 341-354. 4 Jones, E.G., The Thalamus, Plenum, New York, 1985, 935 pp. 5 Jones, E.G. and Hendry, S.H.C., Differential calcium binding protein immunoreactivity distinguishes classes of relay neurons in monkey thalamic nuclei, Eur. J. Neurosci., 1 (1989) 222-246. 6 Oertel, W.H., Graybiel, A.M., Mugnaini, E., Elde, R.P., Schmechel, D.E. and Kopin, I.J., Coexistence of glutamic acid
We are grateful to Prof. E.G. Jones for the gift of parvalbumin and calbindin antibodies and Arnel Laboratories for the CGRP antibody. We thank K. Maskew for manuscript preparation and L. Sutherland and the Department of Pathology for the use of photographic facilities.
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decarboxylase- and somatostatin-like immunoreactivity in neurons of the feline nucleus reticularis thalami, J. Neurosci., 3 (1983) 1322-1332. Pritz, M.B. and Stritzel, M.E., Thalamic nuclei that project to reptilian telencephalon lack GABA and GAD immunoreactive neurons and puncta, Brain Research, 457 (1988) 154-159. Pritz, M.B. and Stritzel, M.E., Reptilian dorsal column nucleus lacks GAD immunoreactive neurons, Brain Research, 503 (1989) 175-179. Pritz, M.B. and Stritzel, M.E., Calcium binding protein immunoreactivity in reptilian dorsal thalamus, Soc. Neurosci. Abstr., 16 (1990) 245. Pritz, M.B. and Stritzel, M.E., A different type of vertebrate thalamic organization, Brain Research, 525 (1990) 330-334.
