Colocalization of substance P and γ-aminobutyric acid in amacrine cells of the cat retina

164

Brain Research, 447 (1988) 164-168 Elsevier

BRE 22851

Colocalization of substance P and y-aminobutyric acid in amacrine cells of the cat retina Roberta G. Pourcho and Dennis J. Goebel Department of Anatomy and Cell Biology, Wayne State University, Detroit, M148201 (U.S.A.) (Accepted 19 January 1988) Key words: Substance P; ),-Aminobutyric acid; Retina; Amacrine cell; Cat; lmmunocytochemistry; Colocalization

Substance P and ),-aminobutyric acid (GABA) were colocalized by immunocytochemistry in two subpopulations of amacrine cells in the cat retina. All of the cells which stained for substance P also showed GABA reactivity. However, there were many GABA-immunoreactive cells which did not stain for substance P. The presence of neuropeptides provides a basis for additional neurochemical characterization of the multiple populations of GABA immunoreaetive cells.

Coexistence of neuropeptides with conventional transmitters has been documented repeatedly throughout the central nervous system. The retina is no exception and a variety of peptide-transmitter 8' 19.20 and peptide-peptide 6"9 combinations have been reported. These combinations have led to the advance of several important hypotheses regarding the significance and possible function of such colocalizations. L a m e t al. 6 suggest that each subclass of neurons may contain a unique set of neuroactive substances which contributes to the characteristic physiology and connectivity of that cell type. Thus, the transmitter content of a cell would provide its unique neurochemical signature. These workers further suggest that the coexistence of neuroactive substances within a single neuron may contribute to the self-regulation of the synaptic function of that cell 7. If the signature hypothesis with its functional implications is to prove generally applicable, one would except to find multiple neuroactive substances in essentially all neuronal populations. In retina, colocalizations have been observed most frequently in teleost and avian species 6'8"9'19'2c~with little support in mammalian studies. One of the most extensively studied of the mammalian species is the cat where

Golgi studies have made it possible to identify individual subpopulations of retinal neurons on the basis of their characteristic appearance and branching patterns 5. Furthermore, considerable progress has been made in identifying those subpopulations of neurons, particularly amacrine cells, which may use the conventional amino acids, "y-aminobutyric acid ( G A B A ) and glycine, as neurotransmitters 3'13'14. The majority of peptide-transmitter colocalizations in other retinas have been with one or the other of these inhibitory amino acids. Among the neuropeptides most frequently found to be associated with other transmitters is substance P (SP). Immunocytochemical gtudies have demonstrated the presence of SP-like immunoreactivity in retinas from a wide variety of species 1. In most mammals, several subpopulations of SP-positive amacrine cells have been reported. Preliminary observations suggested that only a single type of SP cell was present in the cat retina ~. However, Vaney and Whitington TMhave reported the presence of SP immunoreactivity in both amacrine and displaced amacrine cells. Studies in our laboratory have extended these findings to describe two subpopulations of amacrine cells in the cat retina which exhibit SP-like immunoreac-

Correspondence: R. Pourcho, Department of Anatomy and Cell Biology, Wayne State University School of Medicin.e~ 54(! E: Canfield, Detroit, MI 48201, U.S.A. 0006-8993/88/$03.50© 1988 Elsevier Science Publishers B.V. (Biomedical Division)

165 tivity4a6. One cell type is a unistratified amacrine cell whose soma may be located either in the inner nuclear layer (1NL) or in the ganglion cell layer (GCL) but which ramifies narrowly in stratum 4 (s4) of the inner plexiform layer (IPL). The other amacrine is bistratified with processes in both sl and s4 and its soma is located exclusively within the INL. The present study was undertaken to determine whether GABA immunoreactivity is colocalized with SP in these cells. For localization of SP, a rat monoclonal antibody (PeIFreez Biologicals) was used. This antibody was developed by Cuello et ai. 2 and has been extensively characterized. The antiserum against GABA was produced in our laboratory using techniques introduced by Storm-Mathisen et al) 7 and applied previously in this laboratory 15. Briefly, rabbits were injected with 0.5 ml GABA conjugated via glutaraldehyde to bovine serum albumin (GABA-G-BSA) mixed with an equal amount of complete Freund's adjuvant. The animals were boosted at 2-6-week i."tervals with half the original dosage in incomplete adjuvant. Serum was withdrawn within 7-10 days after the third series of injections and purified by affinity chromatography. Specificity of the antiserum was confirmed by immunospot testing on nitrocellulose paper. The antiserum was reactive with GABA-GBSA at dilutions up to 1:10,000 but showed minimal cross-reactivity with conjugates of glycine or taurine. Tissue tests showed that all immunoreactivity could be abolished by preabsorption of the antiserum with an excess of glutaraldehyde-treated GABA. Immunocytochemistry was carried out with freshly dissected cat retinas which had been fixed for 10-12 hours in 4% paraformaldehyde with 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. The tissue was rinsed in buffer and immersed overnight in 30% sucrose. Pieces of retina were embedded in 30% gelatin and sectioned on a freezing microtome. The samples were treated with 1% sodium borohydride for 1 h to quench residual aldehyde activity and then washed in 0.1 M Tris saline (TS), pH 7.4. Tissue was preabsorbed in TS containing 1% BSA for 1 h and then incubated in a combination of 1:100 SP rat monoclonal and 1:100 GABA rabbit polyclonai antisera in TS with 1% BSA and 0.2% Triton X-100 for 4 days at 4 °C. Samples were washed in TS and reacted for 4 h at 37 °C in 1:50 rhodamine-conjugated rabbit

anti-rat lgG and 1:50 fluorescein-conjugated goat anti-rabbit lgG. After washing with TS, samples were mounted on slides and covered with glycerin in phosphate buffer. The sections were photographed using a Zeiss Axiophot microscope equipped for epifluorescence. Cat retinas showed the presence of SP-like immunoreactivity in cell bodies of a relatively small percentage of the amacrine cells (Fig. la). Delicate processes from these cells could be traced into the IPL. The same sections showed GABA immunoreactivity in a much greater number of amacrine cells (Fig. lb) with GABA-containing processes forming a dense plexus within the IPU Cell bodies showing colocalization of SP and GABA were seen in both the INL (Fig. 2a,b) and the GCL (Fig. 2c,d). Many of the double-labeled conventional amacrines could be identified as the bistratifed variety of the SP-immunoreactive cell with processes ramifying in sl and s4 (Fig. la). Since the bistratified SP cells have been found to have their somas located only within the INL, the displaced amacrines seen to label with both SP and GABA (Fig. 2c,d) were identified as belonging to the unistratified variety. All of the SP-positive amacrine cells were found to stain for GABA. However, a number of other cells which did not stain for SP were seen to exhibit GABA immunoreactivity. Autoradiographic studies have previously identified several subpopulations of amacfine cells in the cat retina which exhibit high-affinity uptake of [3H]GABA3 or the GABA agonist, [3H]muscimoll3. These cells include the A2, A!0, A13, A17, and A19 cells as described by Kolb et a l : as well as an interplexiform cell I°. The cells identified as SP-positive amacrines appeared different from the cells previously found to accumulate GABA or muscimol and were tentatively identified as the A12 and A15 amacrines H6. The present study indicates that these cells contain not only SP but also GABA, Since the autoradiographic studies were able to sample only selected amacrine cells and since the SP-positive cells comprise a relatively small percentage of the total GABA-containing population, it is not surprising to find additional subpopulations of cells, not previously identified, which contain GABA. The presence of SP in a subset of the GABA-containing cells provides a neurochemical basis for mak-

166 ing distinctions among the multiple populations of GABA-containing cells in the cat retina. Since

GABA has been observed to colocalize with a variety of peptides and transmitters, it is likely that addi-

Fig. I. Cat retina, photographed for both SP and G A B A immunofluorescenee, a: SP-like immunoreactivity can be seen in an amacrine cell with its soma in the INL. Processes from this cell extend into the IPL. b: GABA-like immunoreactivity was present in the same amacrine cell (arrow) shown in Fig. la. A number of other cells also exhibited G A B A reactivity and formed a dense plexus within the IPL. OS, outer segments: ONL, outer nuclear layer; OPL, ~uter plexiform layer: GCL, ganglion cell layer. Bar = 10!ira.

167

Fig. 2. Paired photographs of SP- (a) and GABA-like (b) immunoreactivity in an amacrine cell with its soma in the INL. A displaced

amacrine cell also shows both SP (c) and GABA (d) reactivity. Bar = 101ml. tional G A B A - p e p t i d e combinations may be found in other amacrine cell types. This co-localization of SP and G A B A provides, to our knowledge, the first demonstration of the coexistence of a peptide-transmitter combination in the cat retina. However. G A B A has been co-localized with enkephalin in avian and catfish retinas s'~'lL~'2t~and with scrotonin in the rabbit retina ~2. The findings of this study provide general support for the signature hypothesis put forward by Lam et

al. t'. It is of interest to note that there are two morphologically distinct subpopulations of amacrine cells in the cat retina which contain both SP and G A B A . The unique characteristics of these cells may depend upon the addition of a third neuroactive substance ~ or upon differences in their synaptic connectivity. The functional significance of SP in combination with G A B A in the retina also remains to be determined. Studies of retinas in which enkephalin and G A B A are colocalized suggest that cnkephalin serves to in-

168 hibit the release of G A B A , causing the synapses to be self-regulating7. Alternatively, the effect of SP may be primarily upon other neurons. In the case of rat olfactory bulb, there is evidence to indicate that SP exerts a depressant effect by stimulating the release of G A B A from other non-SP containing cells t.

1 Brecha, N.C., Eldred, W., Kuljis, R.O. and Karten, H.J., Identification and localization of biologically active peptides in the vertebrate retina. In N.N. Osborne and G.J. Chader (Eds.), Progress in Retinal Research, Vol. 3, Pergamon Oxford, 1984,pp. 185-226. 2 Cuello, A.C., Galfre, G. and Milstein, C., Detection of substance P in the central nervous system by a monoclonal antibody, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 3532-3536. 3 Freed, M.A., Nakamura, Y. and Sterling, P., Four types of amacrine in the cat retina that accumulate GABA, J. Comp. Neurol., 219 (1983)295-304. 4 Goebel. D. and Pourcho, R., Substance P-like immunoreactive amacrine cells in the cat retina, Invest. Ophthaltool. Vis. Sci., Suppl. 28 (1987) 350. 5 Kolb, H., Nelson, R. and Mariani, A., Amacrine cells, bipolar cells and ganglion cells of the cat retina, Vision Res., 21 (1981) 1081-1114. 6 Lain, D.M.-K., Li, H.-B., Su, Y.-Y.T. and Watt, C.B., The signature hypothesis: co-iocalizations of neuroactive substances as anatomical probes for circuitry analysis, Vision Res.. 10 (1985) 1353-1364. 7 Lain, D.M.-K., Su, Y.-Y.T. and Watt, C.B., The self-regulating synapse: a functional role for the co-existenceof neuroactive substances, Brain Res. Rev., 11 (1986) 249-257. 8 Li, H.-B., Watt, C.B. and Lain, D.M.-K., The presence of neurotensin in enkephalinergic and glycinergic amacrine cells in the chicken retina, Invest. Ophthalmol. Vis. Sci., Suppl. 26 (1985) 278. 9 Li, H.-B., Watt, C.B. and Lain, D.M.-K., The coexistence of two neuroactive peptides in a subpopulation of retinal amacrine cells, Brain Research, 345 (1985) 176-180. 10 Nakamura, Y., McOuire, B.A. and Sterling, P., Interplexiform cells in the cat retina: identification by uptake of y[3H]aminobutyric acid and serial reconstruction, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 658-661.

It will be important to determine the mechanisms which .re operative in the cat retina. This work was supported by NIH E Y 02267 and by Core Vision G r a n t EY 04068 from the National Eye Institute.

11 Olpe, H.R., Held, J., Bittiger, H. and Steinmann, M.W., Substance P depresses neuronal activity in the rat olfactory bulb in vitro and in vivo: possible mediation via 7-aminobutytic acid release, Brain Research, 412 (1987) 269-274. 12 Osborne, N.N. and Beaton, D.W., Direct histochemical localization of 5,7-dihydroxytryptamine and the uptake of serotonin by a subpopulation of GABA neurones in the rabbit retina, Brain Research, 382 (1986) 158-162. 13 Pourcho, R.G. and Goebel, D.J., Neuronal subpopulations in cat retina which accumulate the GABA agonist, [3H|muscimol: a combined Golgi and autoradiographic study, J. Comp. Neurol., 219 (1983) 25-35. 14 Pourcho, R.G. and Goebel, D.J., A combined Golgi and autoradiographic study of [3H]glycine-accumulating amacrine cells in the cat retina, J. Comp. Neurol., 233 (1985) 473-480. 15 Pourcho, R.G. and Goebel, D.J., Immunocytochemical demonstration of glycine in retina, Brain Research, 348 (1985) 339-342. 16 Pourcho, R.G. and Goebel, D.J., Substance P-like immunoreactive amacrine cells in cat retina, submitted. 17 Storm-Mathisen, J., Leknes, A.K., Bore, A.T., Vaaland, J.L., Edminson, P., Haug, F.-M.S. and Otterson, O.P., First visualization of glutamate and GABA in neurons by immunocytochemistry, Nature (Lond.), 301 (1983) 517-520. 18 Vaney, D.I. and Whitington, G.E., Substance P-immunoreactive amacrine cells in cat retina, Soc. Neurosci. Abstr., 12 (1986) 642. 19 Watt, C.B., Su, Y.-Y.T. and Lain, D.M.-K., Interactions between enkephalin and GABA in avian retina, Nature (Lond.), 311 (1984) 761-763. 20 Weiler, R. and Ball, A.K., Co-localization of neurotensinlike immunoreactivity and 3H-glycineuptake system in sustained amacrine cells of turtle retina, Nature (Lond.), 311 (1984) 759-761.