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Reptilian dorsal column nucleus lacks GAD immunoreactive neurons
Brain Research, 503 (1989) 175-179 Elsevier
175
BRES 23802
Reptilian dorsal column nucleus lacks GAD immunoreactive neurons Michael B. Pritz and Mark E. Stritzel Division of Neurological Surgery, California College of Medicine, University of Co.lifornia lrvine Medical Center, Orange, CA 92668 (U.S.A.)
(Accepted 25 July 1989) Key words: Dorsal column nucleus; Glutamic acid decarboxylase;Intrinsic neuron; Local circuit neuron; Reptile; Somatosensation
Brains of reptiles, Caiman crocodilus, were processedby standard immunocytochemicalmethodologyusing a polyclonaiantibodyto glutamic acid decarboxylase(GAD) as well as several monoclonalantibodies to GAD. No neuronsimmunoreactivefor GAD, GAD(+), were observed in the dorsal column nucleus, although GAD(+) puncta were seen. These findings suggest that in Caiman, the dorsal column nucleus, like the dorsal thalamus, lacks local circuit neurons. Reptiles, like mammals, contain dorsal thalamic nuclei that project to the telencephalon17. In one group of reptiles, Caiman crocodilus, data based on retrograde horseradish peroxidase (HRP) experiments9"l° and immunocytochemistryit suggest that all dorsal thalamic nuclei lack local circuit or intrinsic neurons and contain solely relay cells. While the percentage of local circuit neurons in the dorsal thalamus of mammals varies6, certain thalamic nuclei in some mammalian species likewise lack intrinsic cells. In rat ventrobasal complex for example, the percentage of local circuit neurons based on HRP 7, and immunocytochemical1"4 data is miniscule. However, the percentage of neurons immunoreactive for glutamic acid decarboxylase, GAD(+) cells, in rat dorsal column nucleus wvs substantial ~. This suggested that inhibition in this sensory system occurred in a lower order nucleus located more peripherally ~. We asked whether an analogous situation existed in Caiman to explain the lack of G A D ( + ) cells in the dorsal thalamus. The observations described here are based on experiments performed on 15 juvenile Caiman crocodilus. Snout-vent length varied from 11.0 to 26.7 cm. Animals weighed between 22 and 345 g. In 10 animals, a po|yclonal anti-GAD antibody (gift from D. Schmechel) was used. Eight animals received 10 #1 of a 1% (w/v) colchicine solution directly instilled into the thir¢l ventricle, while two animals did not receive colchicine. Freefloating sections cut at 30/zm on a sliding microtome were processed by the avidin-biotin complex (ABC) method. A description of this methodology is ava~l~able elsewhere n. In 5 cases, the primary antibody was pre-
incubated for 24-36 h with minced tissue from muscle, kidney, thymus, liver, and lung as described by others 15 to pre-bind non-specific antibodies before incubation with brain tissue in order to decrease background staining. Concentration of the primary antibody varied from 1/500 to 1/16000. The best results were obtained at dilutions of 1/1000 and 1/2000. Controls included substitution of pre-immune sheep serum at concentrations of 1/100 to 1/4000, and omission of one of the following reagents - - primary antibody, biotinylated secondary lgG, or ABC. In 5 animals, monoclonal antibodies to GAD isolates designated as, GAD-l, GAD-2, and GAD-53 (gift from D. Gottlieh), were used. Free floating sections cut at 30 /~m on a sliding microtome were processed by the ABC technique utilizing methodology described by others 3. In two cases, 10/zl of a 1% (w/v) solution of colchicine was injected directly into the third ventricle. Concentration of primary antibody to different monoclonal antibodies varied as follows: GAD-l, 1/1000-1/8000; GAD-2, 1/2000-1/16000; and GAD-5, 1/500-1/8000. Controls included substitution of normal mouse serum at concentrations of 1/500 to 1/2000, and omission of one of the following reagents - - primary antibody, biotinylated secondary lgG, or ABC from the reaction. An account of the surgical procedures for colchicine pre-treatment has been described previously H. Animals that received colchicine survived from 12 to 24 h at water temperatures of 23 to 37 °C. Similar criteria for immunoreactivity were used ~x. Colchicine had a profound effect on the staining in which the polyclonal anti-GAD antibody was used, but
Correspondence: M.B. Pritz, Divisionof NeurologicalSurgery,Universityof California Irvine Medical Center, EO. Box 14091, Orange, CA 92613-4091, U.S.A.
0006-8993/89/$03.50© 1989 Elsevier Science Publishers B.V. (Biomedical Division)
176 was more robust than with GAD-1. Immunoreactive puncta, but no immunoreactive cells, were seen in the dorsal column nucleus with GAD-1 and G A D - 2 regardless of increased primary antibody concentration (Fig. 2B,D). Immunoreactivity in cells as well as puncta were seen in the cochlear nucleus in the same histologic section (Fig. 2A,C). No neurons in either the cochlear nucleus or dorsal column nucleus were immunoreactive with the monoclonal antibody GAD-5, although puncta were stained. Background cross-reactivity with the monoclonal anti-GAD antibody was minimal. Colchicine pretreatment had no appreciable effect on the staining of any of the G A D monoclonal antibodies. Our data indicate that the complete moiety of the G A D enzyme present in Caiman is probably not entirely visualized by each or all
had no appreciable effect in any of the G A D monoclonal antibody experiments. Similar staining patterns were found in all colchicine-pretreated polyclonal anti-GAD antibody cases. Although G A D ( + ) puncta were observed, no G A D ( + ~ cells were seen in the dorsal column nucleus (Fig. 1B). However, G A D ( + ) cells and puncta in t. ------Ut . . . . . . . ! . . . . . t" *1~,~ . o m a hletrdnglr, t,~rtlnn were I.lll~, ~,Ut.tllt•,tl.l ttlw s.t . . . . . . . . . . . . . . . Ei~U~t.'I~.,U..= ~.~t
~aatv
stained including impregnation of secondary dendrites (Fig. IA). Similar concentrations of substituted preimmune serum failed to show any significant non-specific staining (Fig. 1C,D). Immunocytochemical observations with the monoclonal anti-GAD antibody using GAD-1 and G A D - 2 showed a qualitatively similar pattern of staining. However, at a similar dilution, immunoreactivity with G A D - 2
i
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C
---"
.f
D
Fig. 1. lmmunoreactivity in the cochlear nucleus and dorsal column nucleus using a polyclonal anti-GAD antibody. Photographs of the same sagittal section demonstrating GAD(+) neurons and puncta in the cochlear nucleus (A) and GAD(+) l~uncta, but nu GAD(+) cells, in the dorsal column nucleus (B). The concentration of sheep anti-GAD antibody in A and B was 1/2000 in these experiments in which colchicine pretreatment was used. Concentration of pro-immune sheep serum in control sections from the same case through the cochlear nucleus (C) and dorsal column nucleus (D) was !/2000. Arrow in A.points to visualization of a secondary dendrite. Bar = 50/~m in A and B and 200~m in C and D.
177
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Fig. 2. Immunoreactivity in the cochlear nucleus and the dorsal column nucleus using a monoclonal antibody to GAD. Photographs of the same sagittal section demonstrate G A D ( + ) neurons and puncta in the cochiear nucleus (A,C) and G A D ( + ) puncta, but no G A D ( + ) cells in the dorsal column nucleus (B,D) in which monoclonal antibodies GAD-I in A,B and GAD-2 in C,D were used. Concentrations of the primary antibody was 118000 in A,B and 1116000 in C,D. Concentration of normal mouse serum in control sections for the same case through the cochlear nucleus (E) and dorsal column nucleus (Fb ~as 1/2~00. Bar = 50/~m in A - D and 200 pm in E and E
178 with the presence of inhibitory intrinsic cells, then the dorsal column nucleus of Caiman lacks local circuit neurons. Therefore, the absence of G A D ( + ) cells in the dorsal thalamus of Caiman is not related to the presence of intrinsic neurons in this lower order nucleus located more peripherally H. The condition in Cain:an then differs from that in rat where the paucity of v~ntrobasal complex G A D ( + ) neurons TM may be explained by the significant numbers of G A D ( + ) cells in the dorsal column nucleus 1. A different mechanism must be invoked to explain the absence of local circuit neurons in the dorsal thalamus in these reptiles. In addition, G A D ( + ) cells and thus local circuit neurons are not a feature unique to rat dorsal column nucleus, but are present in cat 5"~4a9, and probably in many, if not all, mammals. Since similar G A D ( + ) cells are lacking in Caiman and perhaps in other reptiles as well, then the occurrence of local circuit neurons in the dorsal column nucleus may indeed be an important feature in the evolution of the somatosensory system. Although data to support such a hypothesis is scant, it is tempting to suggest that certain neuronal circuits may be linked to the development of intrinsic neurons in the dorsal column nucleus. Specifically, the dorsal column nucleus of Caiman lacks direct projections to the thalamus ~3 as well as direct input from the telencephalon (unpublished observations) which are prominent features in mammals2. Viewed in this way, the presence of local circuit neurons in the dorsal column nucleus of mammals may be related to the occurrence of these two important neural circuits.
of G A D monoclonal antibodies that we utilized. The polyclonal anti-GAD antibody despite its greater nonspecific staining seems to recognize more of the Caiman brain GAD. For example, certain G A D ( + ) olfactory bulb cells that were observed in a previous study in which a polyclonal anti-GAD antibody was used (see reg. 11, Fig. 2D,E) were not seen with any of the G A D monoclonal antibodies. No G A D ( + ) cells were observed over any dorsal thalamic nucleus with any GAD monoclonal antibody. The lack of neuronal immunoreactivity in the dorsal column nucleus of Caiman in these experiments is subject to a number of possible errors. These present significant interpretative difficulties are inherent to all immunocytochemical studies. These concerns, relevant to technical errors, non-specific staining, and cross-species immunoreactivity, have been discussed in detail previously 11. To circumvent some of these problems, sections were cut sagittally to include both the cochlear nucleus, which contains G A D ( + ) cells in birds s and mammals 16'1s, and the dorsal column nucleus in the same histologic section. Thus, the finding of G A D ( + ) cells in the cochlear nucleus and their absence in the dorsal column nucleus as well as the presence of G A D ( + ) puncta in the dorsal column nucleus would suggest that these possible sources of error do not explain the lack of G A D ( + ) dorsal column nucleus neurons in Caiman. In addition, we used several G A D monoclonal antibodies because of the possibility that certain G A D ( + ) neurons may not have been stained with the polyclonal anti-GAD antibody. GAD-1 and GAD-2 monoclonal antibodies showed similar dorsal column nucleus and cochlear nucleus staining patterns when compared to the polyclonal anti-GAD antibody. Since crocodilians are the reptilian group most closely related to birds 2°, we had hoped that the GAD-5 monoclonal antibody, since it is unique to chick 3, would be especially sensitive. However, this did not prove to be the case. Considering the above limitations, our results suggest the following. While G A D ( + ) puncta are present in the dorsal column nucleus, G A D ( + ) cells are not. If one equates neuronal immunoreactivity to the GAD antibody
gift of anti-GAD monoclonal antibodies and for his thoughtful suggestions regarding the immunocytochemical experiments. We also thank Dr. D. Schmechel for his gift of polyclonal anti-GAD antibody and pre-immune serum, K. Maskew for manuscript preparation, and L. Sutherland and the Department of Pathology for the use of photographic facilities. Supported by General Surgery Grant 119 and funds from the Blakely Compensation Plan. A preliminary account of some of these findings has been presented previously12.
1 Barabaresi, E, Spreafico, R., Frassoni, C. and Rustioni, A., GABAergic neurons are present in the dorsal column nuclei but not in the ventroposterior complex o[ rats, Brain Research, 382 (1986) 305-326. 2 Berkley, K.J., Budell, R.J., BIomqvist,A. and Bull, M., Output systems of the dorsal column nuclei in the cat, Brain Res. Rev., 11 (1986) 199-225. 3 Gonlieb, D.I., Chang, Y.-C. and Schwob, J.E., Monoclonal antibodies to glutamicacid decarboxylase,Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 8808-8812. 4 Harris, R.M. and Hendrickson, A.E., Local circuit neurons in the rat ventrobasal thalamus - - a GABA immunocytochemical study, Neuroscience, 21 (1987) 229-236.
5 Isomura, G. and H~imori, J., Three types of neurons in the medial euneate nucleus of the cat, Neurosci. Res., 5 (1988) 395-408. 6 Jones, EG., The Thalamus, Plenum, New York, 1985, 935 pp. 7 Lee, C.H., The Structural Organization of the Rat Ventrobasal Complex, Doctoral dissertation, University of Cafifornia, Berkeley, 1981. 8 Miiller, C.M., ~,-Aminobutyricacid immunoreactivity in brainstern auditory nuclei of the chicken, Neurosci. Lea., 77 (1987) 272-276. 9 Pritz, M.B. and Stritzel, M.E., Percentage of relay and intrinsic neurons in two sensory thalamic nuclei projecting to the non-cortical telencephalon in reptiles, Caiman crocodilus, Brain
We are particularly grateful to Prof. D. Gottlieb for his generous
179 Research, 376 (1986) 169-174. 10 Pritz, M.B. and Stritzel, M.E., Percentage of intrinsic and relay cells in a thalamic nucleus projecting to general cortex in reptiles, Caiman crocodilus, Brain Research, 409 (1987) 146150. II 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. 12 Pritz, M.B. and Stritzel, M.E., Cerebellar projecting and local circuit neurons of reptilian dorsal column nucleus, Soc. Neurosci. Abstr., 14 (1988) 55. 13 Pritz, M.B. and Stritzel, M.E., Reptilian somatosensory midbrain: identification based on input from the spinal cord and dorsal column nucleus, Brain Behav. Evol., 33 (1989) 1-14. 14 Rustioni, A., Sehmechel, D.E., Cheema, S. and Fitzpatrick, D., Glutamic acid decarboxylase-containing neurons in the dorsal column nuclei of the cat, Somatosens. Res., 1 (1984) 329-357. 15 Sehwob, J.E., Farber, N.B. and Gottlieb, D.I., Neurons of the
16 17 18
19
20
olfactory epithelium in adult rats contain vimentin, J. Neurosci., 6 (1986) 208-217. Thompson. G.C., Cortez, A.M. ~nd Lam, D.M.-K., Localization of GABA immunoreactivity in the auditory brainstem of guinea pigs, Brain Research, 359 (!985) 119-122. Ulinski, ES., Dorsal Ventricular Ridge: A Treatise on Forebrain Organization in Reptiles and Birds, Wiley, New York, 1983, 284 PP. Wenthold, R.J., Zempel, J.M., Parakkal, M.H., Reeks, K.A. and Altschuler, R.A., Immunocytochemical localization of GABA in the cochlear nucleus of the guinea pig, Brain Research, 380 (1986) 7-18. Westman, J., Blomqvist, A., K6hler, C. and Wu, J.-Y., Light and electron microscopic localization of glutamic acid decarboxylase and substance P in the dorsal column nuclei of the cat, Neurosci. Leu., 51 (1984)347-352. Whetstone, K.N. and Martin, L.D., New look at the origin of birds and crocodiles, Nature (Lond.), 279 (1979) 234-236.
175
BRES 23802
Reptilian dorsal column nucleus lacks GAD immunoreactive neurons Michael B. Pritz and Mark E. Stritzel Division of Neurological Surgery, California College of Medicine, University of Co.lifornia lrvine Medical Center, Orange, CA 92668 (U.S.A.)
(Accepted 25 July 1989) Key words: Dorsal column nucleus; Glutamic acid decarboxylase;Intrinsic neuron; Local circuit neuron; Reptile; Somatosensation
Brains of reptiles, Caiman crocodilus, were processedby standard immunocytochemicalmethodologyusing a polyclonaiantibodyto glutamic acid decarboxylase(GAD) as well as several monoclonalantibodies to GAD. No neuronsimmunoreactivefor GAD, GAD(+), were observed in the dorsal column nucleus, although GAD(+) puncta were seen. These findings suggest that in Caiman, the dorsal column nucleus, like the dorsal thalamus, lacks local circuit neurons. Reptiles, like mammals, contain dorsal thalamic nuclei that project to the telencephalon17. In one group of reptiles, Caiman crocodilus, data based on retrograde horseradish peroxidase (HRP) experiments9"l° and immunocytochemistryit suggest that all dorsal thalamic nuclei lack local circuit or intrinsic neurons and contain solely relay cells. While the percentage of local circuit neurons in the dorsal thalamus of mammals varies6, certain thalamic nuclei in some mammalian species likewise lack intrinsic cells. In rat ventrobasal complex for example, the percentage of local circuit neurons based on HRP 7, and immunocytochemical1"4 data is miniscule. However, the percentage of neurons immunoreactive for glutamic acid decarboxylase, GAD(+) cells, in rat dorsal column nucleus wvs substantial ~. This suggested that inhibition in this sensory system occurred in a lower order nucleus located more peripherally ~. We asked whether an analogous situation existed in Caiman to explain the lack of G A D ( + ) cells in the dorsal thalamus. The observations described here are based on experiments performed on 15 juvenile Caiman crocodilus. Snout-vent length varied from 11.0 to 26.7 cm. Animals weighed between 22 and 345 g. In 10 animals, a po|yclonal anti-GAD antibody (gift from D. Schmechel) was used. Eight animals received 10 #1 of a 1% (w/v) colchicine solution directly instilled into the thir¢l ventricle, while two animals did not receive colchicine. Freefloating sections cut at 30/zm on a sliding microtome were processed by the avidin-biotin complex (ABC) method. A description of this methodology is ava~l~able elsewhere n. In 5 cases, the primary antibody was pre-
incubated for 24-36 h with minced tissue from muscle, kidney, thymus, liver, and lung as described by others 15 to pre-bind non-specific antibodies before incubation with brain tissue in order to decrease background staining. Concentration of the primary antibody varied from 1/500 to 1/16000. The best results were obtained at dilutions of 1/1000 and 1/2000. Controls included substitution of pre-immune sheep serum at concentrations of 1/100 to 1/4000, and omission of one of the following reagents - - primary antibody, biotinylated secondary lgG, or ABC. In 5 animals, monoclonal antibodies to GAD isolates designated as, GAD-l, GAD-2, and GAD-53 (gift from D. Gottlieh), were used. Free floating sections cut at 30 /~m on a sliding microtome were processed by the ABC technique utilizing methodology described by others 3. In two cases, 10/zl of a 1% (w/v) solution of colchicine was injected directly into the third ventricle. Concentration of primary antibody to different monoclonal antibodies varied as follows: GAD-l, 1/1000-1/8000; GAD-2, 1/2000-1/16000; and GAD-5, 1/500-1/8000. Controls included substitution of normal mouse serum at concentrations of 1/500 to 1/2000, and omission of one of the following reagents - - primary antibody, biotinylated secondary lgG, or ABC from the reaction. An account of the surgical procedures for colchicine pre-treatment has been described previously H. Animals that received colchicine survived from 12 to 24 h at water temperatures of 23 to 37 °C. Similar criteria for immunoreactivity were used ~x. Colchicine had a profound effect on the staining in which the polyclonal anti-GAD antibody was used, but
Correspondence: M.B. Pritz, Divisionof NeurologicalSurgery,Universityof California Irvine Medical Center, EO. Box 14091, Orange, CA 92613-4091, U.S.A.
0006-8993/89/$03.50© 1989 Elsevier Science Publishers B.V. (Biomedical Division)
176 was more robust than with GAD-1. Immunoreactive puncta, but no immunoreactive cells, were seen in the dorsal column nucleus with GAD-1 and G A D - 2 regardless of increased primary antibody concentration (Fig. 2B,D). Immunoreactivity in cells as well as puncta were seen in the cochlear nucleus in the same histologic section (Fig. 2A,C). No neurons in either the cochlear nucleus or dorsal column nucleus were immunoreactive with the monoclonal antibody GAD-5, although puncta were stained. Background cross-reactivity with the monoclonal anti-GAD antibody was minimal. Colchicine pretreatment had no appreciable effect on the staining of any of the G A D monoclonal antibodies. Our data indicate that the complete moiety of the G A D enzyme present in Caiman is probably not entirely visualized by each or all
had no appreciable effect in any of the G A D monoclonal antibody experiments. Similar staining patterns were found in all colchicine-pretreated polyclonal anti-GAD antibody cases. Although G A D ( + ) puncta were observed, no G A D ( + ~ cells were seen in the dorsal column nucleus (Fig. 1B). However, G A D ( + ) cells and puncta in t. ------Ut . . . . . . . ! . . . . . t" *1~,~ . o m a hletrdnglr, t,~rtlnn were I.lll~, ~,Ut.tllt•,tl.l ttlw s.t . . . . . . . . . . . . . . . Ei~U~t.'I~.,U..= ~.~t
~aatv
stained including impregnation of secondary dendrites (Fig. IA). Similar concentrations of substituted preimmune serum failed to show any significant non-specific staining (Fig. 1C,D). Immunocytochemical observations with the monoclonal anti-GAD antibody using GAD-1 and G A D - 2 showed a qualitatively similar pattern of staining. However, at a similar dilution, immunoreactivity with G A D - 2
i
j
C
---"
.f
D
Fig. 1. lmmunoreactivity in the cochlear nucleus and dorsal column nucleus using a polyclonal anti-GAD antibody. Photographs of the same sagittal section demonstrating GAD(+) neurons and puncta in the cochlear nucleus (A) and GAD(+) l~uncta, but nu GAD(+) cells, in the dorsal column nucleus (B). The concentration of sheep anti-GAD antibody in A and B was 1/2000 in these experiments in which colchicine pretreatment was used. Concentration of pro-immune sheep serum in control sections from the same case through the cochlear nucleus (C) and dorsal column nucleus (D) was !/2000. Arrow in A.points to visualization of a secondary dendrite. Bar = 50/~m in A and B and 200~m in C and D.
177
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Fig. 2. Immunoreactivity in the cochlear nucleus and the dorsal column nucleus using a monoclonal antibody to GAD. Photographs of the same sagittal section demonstrate G A D ( + ) neurons and puncta in the cochiear nucleus (A,C) and G A D ( + ) puncta, but no G A D ( + ) cells in the dorsal column nucleus (B,D) in which monoclonal antibodies GAD-I in A,B and GAD-2 in C,D were used. Concentrations of the primary antibody was 118000 in A,B and 1116000 in C,D. Concentration of normal mouse serum in control sections for the same case through the cochlear nucleus (E) and dorsal column nucleus (Fb ~as 1/2~00. Bar = 50/~m in A - D and 200 pm in E and E
178 with the presence of inhibitory intrinsic cells, then the dorsal column nucleus of Caiman lacks local circuit neurons. Therefore, the absence of G A D ( + ) cells in the dorsal thalamus of Caiman is not related to the presence of intrinsic neurons in this lower order nucleus located more peripherally H. The condition in Cain:an then differs from that in rat where the paucity of v~ntrobasal complex G A D ( + ) neurons TM may be explained by the significant numbers of G A D ( + ) cells in the dorsal column nucleus 1. A different mechanism must be invoked to explain the absence of local circuit neurons in the dorsal thalamus in these reptiles. In addition, G A D ( + ) cells and thus local circuit neurons are not a feature unique to rat dorsal column nucleus, but are present in cat 5"~4a9, and probably in many, if not all, mammals. Since similar G A D ( + ) cells are lacking in Caiman and perhaps in other reptiles as well, then the occurrence of local circuit neurons in the dorsal column nucleus may indeed be an important feature in the evolution of the somatosensory system. Although data to support such a hypothesis is scant, it is tempting to suggest that certain neuronal circuits may be linked to the development of intrinsic neurons in the dorsal column nucleus. Specifically, the dorsal column nucleus of Caiman lacks direct projections to the thalamus ~3 as well as direct input from the telencephalon (unpublished observations) which are prominent features in mammals2. Viewed in this way, the presence of local circuit neurons in the dorsal column nucleus of mammals may be related to the occurrence of these two important neural circuits.
of G A D monoclonal antibodies that we utilized. The polyclonal anti-GAD antibody despite its greater nonspecific staining seems to recognize more of the Caiman brain GAD. For example, certain G A D ( + ) olfactory bulb cells that were observed in a previous study in which a polyclonal anti-GAD antibody was used (see reg. 11, Fig. 2D,E) were not seen with any of the G A D monoclonal antibodies. No G A D ( + ) cells were observed over any dorsal thalamic nucleus with any GAD monoclonal antibody. The lack of neuronal immunoreactivity in the dorsal column nucleus of Caiman in these experiments is subject to a number of possible errors. These present significant interpretative difficulties are inherent to all immunocytochemical studies. These concerns, relevant to technical errors, non-specific staining, and cross-species immunoreactivity, have been discussed in detail previously 11. To circumvent some of these problems, sections were cut sagittally to include both the cochlear nucleus, which contains G A D ( + ) cells in birds s and mammals 16'1s, and the dorsal column nucleus in the same histologic section. Thus, the finding of G A D ( + ) cells in the cochlear nucleus and their absence in the dorsal column nucleus as well as the presence of G A D ( + ) puncta in the dorsal column nucleus would suggest that these possible sources of error do not explain the lack of G A D ( + ) dorsal column nucleus neurons in Caiman. In addition, we used several G A D monoclonal antibodies because of the possibility that certain G A D ( + ) neurons may not have been stained with the polyclonal anti-GAD antibody. GAD-1 and GAD-2 monoclonal antibodies showed similar dorsal column nucleus and cochlear nucleus staining patterns when compared to the polyclonal anti-GAD antibody. Since crocodilians are the reptilian group most closely related to birds 2°, we had hoped that the GAD-5 monoclonal antibody, since it is unique to chick 3, would be especially sensitive. However, this did not prove to be the case. Considering the above limitations, our results suggest the following. While G A D ( + ) puncta are present in the dorsal column nucleus, G A D ( + ) cells are not. If one equates neuronal immunoreactivity to the GAD antibody
gift of anti-GAD monoclonal antibodies and for his thoughtful suggestions regarding the immunocytochemical experiments. We also thank Dr. D. Schmechel for his gift of polyclonal anti-GAD antibody and pre-immune serum, K. Maskew for manuscript preparation, and L. Sutherland and the Department of Pathology for the use of photographic facilities. Supported by General Surgery Grant 119 and funds from the Blakely Compensation Plan. A preliminary account of some of these findings has been presented previously12.
1 Barabaresi, E, Spreafico, R., Frassoni, C. and Rustioni, A., GABAergic neurons are present in the dorsal column nuclei but not in the ventroposterior complex o[ rats, Brain Research, 382 (1986) 305-326. 2 Berkley, K.J., Budell, R.J., BIomqvist,A. and Bull, M., Output systems of the dorsal column nuclei in the cat, Brain Res. Rev., 11 (1986) 199-225. 3 Gonlieb, D.I., Chang, Y.-C. and Schwob, J.E., Monoclonal antibodies to glutamicacid decarboxylase,Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 8808-8812. 4 Harris, R.M. and Hendrickson, A.E., Local circuit neurons in the rat ventrobasal thalamus - - a GABA immunocytochemical study, Neuroscience, 21 (1987) 229-236.
5 Isomura, G. and H~imori, J., Three types of neurons in the medial euneate nucleus of the cat, Neurosci. Res., 5 (1988) 395-408. 6 Jones, EG., The Thalamus, Plenum, New York, 1985, 935 pp. 7 Lee, C.H., The Structural Organization of the Rat Ventrobasal Complex, Doctoral dissertation, University of Cafifornia, Berkeley, 1981. 8 Miiller, C.M., ~,-Aminobutyricacid immunoreactivity in brainstern auditory nuclei of the chicken, Neurosci. Lea., 77 (1987) 272-276. 9 Pritz, M.B. and Stritzel, M.E., Percentage of relay and intrinsic neurons in two sensory thalamic nuclei projecting to the non-cortical telencephalon in reptiles, Caiman crocodilus, Brain
We are particularly grateful to Prof. D. Gottlieb for his generous
179 Research, 376 (1986) 169-174. 10 Pritz, M.B. and Stritzel, M.E., Percentage of intrinsic and relay cells in a thalamic nucleus projecting to general cortex in reptiles, Caiman crocodilus, Brain Research, 409 (1987) 146150. II 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. 12 Pritz, M.B. and Stritzel, M.E., Cerebellar projecting and local circuit neurons of reptilian dorsal column nucleus, Soc. Neurosci. Abstr., 14 (1988) 55. 13 Pritz, M.B. and Stritzel, M.E., Reptilian somatosensory midbrain: identification based on input from the spinal cord and dorsal column nucleus, Brain Behav. Evol., 33 (1989) 1-14. 14 Rustioni, A., Sehmechel, D.E., Cheema, S. and Fitzpatrick, D., Glutamic acid decarboxylase-containing neurons in the dorsal column nuclei of the cat, Somatosens. Res., 1 (1984) 329-357. 15 Sehwob, J.E., Farber, N.B. and Gottlieb, D.I., Neurons of the
16 17 18
19
20
olfactory epithelium in adult rats contain vimentin, J. Neurosci., 6 (1986) 208-217. Thompson. G.C., Cortez, A.M. ~nd Lam, D.M.-K., Localization of GABA immunoreactivity in the auditory brainstem of guinea pigs, Brain Research, 359 (!985) 119-122. Ulinski, ES., Dorsal Ventricular Ridge: A Treatise on Forebrain Organization in Reptiles and Birds, Wiley, New York, 1983, 284 PP. Wenthold, R.J., Zempel, J.M., Parakkal, M.H., Reeks, K.A. and Altschuler, R.A., Immunocytochemical localization of GABA in the cochlear nucleus of the guinea pig, Brain Research, 380 (1986) 7-18. Westman, J., Blomqvist, A., K6hler, C. and Wu, J.-Y., Light and electron microscopic localization of glutamic acid decarboxylase and substance P in the dorsal column nuclei of the cat, Neurosci. Leu., 51 (1984)347-352. Whetstone, K.N. and Martin, L.D., New look at the origin of birds and crocodiles, Nature (Lond.), 279 (1979) 234-236.