Neuroscience Letters, 163 (1993) 71-73 71 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940193/$ 06.00

NSL 09999

Cholinergic amacrine cells of the rat retina express the -subunit of the GABAA-receptor

Ursula Gre fe ra th a, Ulr ike Grf iner t a, Hans M6hle r b, Heinz WS.ss le a'*

aMax-Planck-lnstitut fiir Hirnforsehung, Deutschordenstrasse 46, D-60528, Frankfurt am Main, FRG bInstitut fiir Pharmakologie, ETH und Universitgit Ziirich, Ziirich, Switzerland

(Accepted 2 September 1993)

Key words." GABAA-receptor; ~-Subunit; Cholinergic amacrine cell; Immunocytochemical localization

Antibodies directed against the ~-subunit of the GABA~,-receptor were applied to cryostat sections of rat retinae. Two narrow bands of the inner plexiform layer were strongly immunoreactive. Some cell bodies in both the amacrine- and ganglion-cell layer were weakly immunoreactive. The position of the labelled bands and the distribution of the cell bodies was strongly reminiscent of the cholinergic amacrine cells. In order to show directly that cholinergic amacrine cells express the 6-subunit of the GABAA-receptor, double immunofluorescence with an antibody against choline acetyltransferase (CHAT) and with antibodies against the ~-subunit was performed on the same cryostat sections. This showed the labelled cells to be cholinergic amacrine cells.

Molecular cloning has revealed a structural heteroge- neity of GABA A receptors, comprised of at least six dif- ferent ~-subunits, three fl-subunits, three y-subunits and one 6-subunit [14, 15, 22]. A further subunit, p l , has been described in the retina [4]. The different subunits show different patterns of expression in the brain [11, 23] as well as in the retina [2,8], and exhibit different pharma- cological properties [9, 12, 22].

Cholinergic amacrine cells are found in all mammalian retinae and play an important role in the directionally selective circuitry of the retina [13, 17, 21]. Acetylcholine and GABA are colocalized in these cells [3, 10, 18], and both transmitters are also involved in the generation of directionally selective responses [24]. However, precise synaptic details of directionally selective circuits are still missing [6]. Here, we report that cholinergic amacrine cells exhibit a GABAA receptor subunit composition dif- ferent from other retinal neurons.

Adult albino rats were deeply anesthesized with ha- lothane and decapitated. The eyes were quickly enu- cleated and dissected. The retinae were immersion fixed in 4% paraformaldehyde in sodium phosphate buffer, pH 7.4 (PB) for 3 h at room temperature. After several washes in PB, pieces of the retina were immersed in 30% sucrose in PB overnight. Horizontal and vertical sections

*Corresponding author.

were cut on a cryostat; details of the immunostaining are given in Greferath et al. [7, 8].

Rabbit polyclonal antibodies against a 6-subunit spe- cific peptide (6 1 17) were used at a dilution of 1:100 [1]. They were visualized with secondary antibodies coupled to CY3 (carboxymethylindocyanine, Dianova, Ham- burg, FRG). A rat monoclonal antibody against ChAT (Boehringer, Mannheim, FRG) was used to im- munostain cholinergic amacrine cells [20]. FITC (fluo- rescein isothiocyanate)-coupled secondary antibodies (Sigma, Deisenhofen, FRG) were used to visualize ChAT immunoreactivity.

Figs. 1A and 1B compare the staining with the two antibodies in the same cryostat section. Fig. 1A shows the ChAT-immunoreactivity visualized with FITC fluo- rescence (arrows point to nonspecific fluorescence of blood vessels). The cell bodies of four cholinergic amac- rine cells in the inner nuclear layer (INL) and of two displaced cholinergic amacrine cells in the ganglion cell layer (GCL) are labelled. Two narrow bands of choliner- gic processes are labelled in the inner plexiform layer (IPL). This is the well known staining pattern of cholin- ergic amacrine cells of the rat retina [20] and of the mam- malian retina in general. Fig. 1B shows the localization of the ~-subunit of the GABAA receptor in the same sec- tion visualized with CY3 fluorescence. Two narrow hori- zontal bands in the IPL are strongly immunoreactive. The band closer to the GCL often showed slightly

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Fig. 1. A: vertical section of a rat retina labelled with an antibody against ChAT (FITC immunofluorescence; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer). The small arrows point to pieces of blood vessels which show non-specific fluorescence. The two horizontal lines mark the thickness of the IPL. B: same section as in A, labelled with antibodies against the ~-subunit of the GABAA receptor (CY3 immunofluo- rescence; bar = 50/2m (A,B) and 70/2m (C,D). C: vertical section of a rat retina labelled with an antibody against ChAT (FITC-irnmunoftuorescence; ONL, outer nuclear layer; OPL, outer plexiform layer; other conventions as in A). The arrowhead marks the labelled cell body of a cholinergic amacrine cell. D: same section as in C, labelled with antibodies against the ~-subunit of the GABAA receptor (CY3 immunofluorescence). The

arrowhead marks the weakly labelled cell body of the cholinergic amacrine cell shown in C.

weaker labelling than the band closer to the INL. Com- parison with Fig. 1A shows that the labelled bands are identical and, allowing for some differences in intensity, agree in practically all spatial details. Close inspection of Fig. 1B also shows very faint labelling of some of the six cell bodies of Fig. 1A. This is demonstrated more con- vincingly by another section shown in Fig. 1C,D. The ChAT-immunoreactive cell body and the two bands in Fig. 1C are also immunoreactive for the ~-subunit in Fig. 1D. Some blood vessels are stained non specifically in Fig, 1A and, since they are not visible in Fig. 1B, leakage of ChAT-fluorescence through the red barrier-filter is ex- cluded and therefore cannot artifactually produce the double labelling. Although the two bands of labelling in Figs. 1A and B are congruent, there is still the possibility that closely adjacent but not identical dendrites might be labelled. Dendrites of direction selective ganglion cells and of s-ganglion cells are known to costratify with cholinergic amacrine cells [6, 19]. To gain better resolu- tion. o f the dendritic network, horizontal sections were

cut and double-immunostained for ChAT and the 8-sub- unit (not shown). The dendritic networks were identical. We therefore conclude that cholinergic amacrine cells of the rat retina express the 8-subunit of the GABAA recep- tor. The strong labelling intensity of the two bands in the IPL and the weak labelling of the cell bodies suggest a concentration of the 8-subunits on the dendrites.

In the brain, the 8-subunit protein is most prominently expressed in the cerebellar granular layer and less promi- nently in the thalamus and in the hippocampus [1, 23]. There, it seems to colocalize with areas of low benzodiaz- epine (BZ) binding, and it has been suggested that GABAA receptors which contain the 8-subunit lack BZ modulat ion [23]. BZ binding is high in the IPL of the mammal ian retina, however, f rom the published autora- diographs it is difficult to recognize the precise pattern of lamination [25]. We also looked for the localization of GABA A receptor subunits other than the 8-subunit. Im- munocytochemical staining with antibodies against the a2 and fl2/3 subunits respectively, also revealed the two

cho l ine rg i c s t r a t a o f the IPL . In si tu h y b r i d i z a t i o n w i t h

p r o b e s specif ic fo r the ~4 s u b u n i t l abe l led a sparse p o p u -

l a t ion o f a m a c r i n e and d i sp l aced a m a c r i n e s , r e m i n i s c e n t

o f the cho l ine rg i c cells [8]. H e n c e , it is poss ib le t ha t

cho l ine rg i c a m a c r i n e cells express u n u s u a l c o m p o s i t i o n s

o f G A B A A recep tors . C h o l i n e r g i c a m a c r i n e cells h a v e

a lso been s h o w n to c o n t a i n G A B A i m m u n o r e a c t i v i t y [3,

10, 18], and t h e r e f o r e the ~ - s u b u n i t m a y be pa r t o f an

a u t o r e c e p t o r for G A B A .

T h e f u n c t i o n a l s igni f icance o f the exp re s s ion o f d - sub-

uni ts is still a m a t t e r o f d e b a t e [1, 9, 23]. It has been

sugges ted [16] tha t the d - s u b u n i t is m o s t p r o m i n e n t l y ex-

pressed in n e u r o n s w h o s e d e n d r i t i c p rocesses exis t in

c o m p l e x synap t i c a r r a n g e m e n t s w i th o t h e r neu rons .

C h o l i n e r g i c a m a c r i n e cells a n d the i r synap t i c c i r cu i t ry

ce r t a in ly b e l o n g to tha t c a t e g o r y [5], a n d it w o u l d be

exc i t ing i f the exp re s s ion o f the d - s u b u n i t is c o r r e l a t e d

wi th s o m e f u n c t i o n m e d i a t i n g d i r e c t i o n a l se lect ive re-

sponses .

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