II4
\,~.'..s~wmc L¢/~, i (1~;92~[14 I . : t992 Elsevier Scientil]c Publishers Ireland Lid. All rights reserxed tB(t4-3940i~2i3 {~5.0i)
NSL 08476
Cat retinal ganglion cells show transient responses to acetylcholine and sustained responses to L-glutamate* J.E.G. Downing** and A. Kaneko Department of lnjbrmatkm Physiology, National Institutefor Physiological Science.s, Okazaki (Japan) (Received 26 April 1991 : Revised version received 16 December 1991 ; Accepted 20 December 1991 )
Key words. Acute dissociation; Adult cat; Retinal ganglion cell subtype; Receptor desensitization The neuropharmacological basis for the different receptive field properties of cat retinal ganglion cells was investigated using whole cell voltageclamp recordings from acutely dissociated adult tissue. Subclasses of physiologically characterised ganglion cells were determined on the basis of (i) their soma diameters and (ii) their projection to central visual nuclei (identified by microinjection of fluorescent dyes into the lateral geniculate and/or superior colliculus). The sensitivities of all categories of ganglion cells, prepared from peripheral retina were found to be similar for ;v-amino butyric acid, glycine, acetylcholine and glutamate. The kinetics of desensitisation differed among receptor subtypes, revealing possible physiologically significant molecular specialisations that could be involved in shaping synaptic transmission.
The physiology of vertebrate retinal neurones is comparatively well documented from studies using isolated and in vivo preparations. However, the stability of intracellular recordings is limited and extensive cellular interactions retained within intact tissue complicate the interpretation of direct synaptic transmission, independent of feedback loops or modulation. Recent advances in cell isolation techniques and the application of 'giga-seal' suction electrode voltage-clamp have optimised conditions for measuring the chemosensitivity of retinal neurones. Initial studies on the mammalian retina have made use of rodent species [1, 10, 11, 14]. We have chosen to study ganglion cells (GCs) from the adult cat in order to link biophysical measurements of membrane properties to what is known about their different light evoked responses. Whole cell voltage-clamp recordings were obtained using methodologies detailed elsewhere [6, 10, 14]. Briefly, recordings were made at -60 mV holding poten*Presented at the 19th Annual Meeting of the Society for Neuroscience, Phoenix, Arizona, USA (November 19891, and to the British Photobiology Society, Kings College, London, UK (December, 1989). *'Present address: Neuroscience Laboratory, Department of Biology; Imperial College, London SW7 2BB. UK.
Correspondence: J.E.G. Downing, Neuroscience Laboratory, Department of Biology, Imperial College, Prince Consort Road, London SW7 2BB, UK.
tial, in superfusate containing in mM: NaC1 135, KCI 5, CaC12 2, MgC12 1, HEPES 5, glucose 10 (pH 7.4, adjusted with NaOH). Patch electrodes were filled with a solution containing in mM: KC1 120, CaC12 0.5, EGTA 5, HEPES 10 (pH 7.2 adjusted with KOH). Recordings were taken from subtypes of retinal GCs, identified within mixed cell dissociates. Isolated cells were prepared using enzyme digestion (papa/n) and mechanical action [10]. Using these methods the complete range of cells types was separated from retinas of adult (~1 kg) cats. Cells retaining a high degree of morphological differentiation were consistently prepared. Bipolar cells [10] and horizontal cells (not shown) could be recognised among the dissociates, on morphological criteria alone. Solitary cell bodies, denuded of all but their primary dendrites were used in the present study. These were identified as GCs by retrograde labelling (Fig. 1) with 1, l'-dioctadecyl- 3,3,3',3'-tetramethylindocarbocyanine dye (D/I: 25-50/.tl; 2.5 mg/ml in 100% ethanol) injected into the superior colliculus (SC), and/or 4,6-diamidino-2phenylindole (DAPI: 25-50 p1; 20/.tg/ml aq.) into the lateral geniculate nucleus (LGN), 3 days before enucleat/on. Soma size and projection pattern were used to further categorise GC subclasses. Hence, physiological experimentation was performed on two very different populations of GC, identified by their staining from injections made either into the SC or the LGN. Stereotaxic placement of fluorescent dye was achieved under anaesthesia induced by injection of sodium pentobarbi-
115 5It
El
4tl ~'
30
E
2O
O m Fig. l. Prior to electrophysiological examination. GCs were positively identified within mixed cell cultures by fluorescence imaging of retrogradely transported dye from the SC or LGN. A: phase contrast image: B: fluorescence image of the same cell retrogradely labelled with DAPI from the SC. Note that the bright staining of the nucleus by DAP[ was easily contrasted with plasma naembraneous staining of cells by' Dil (not shown).
tal (i.p,), followed by inhalation of a mixture of 70% nitrous oxide and 30% oxygen and 0.5 1.5% halothane. For enucleation, animals were anaethetised using halothane prior to cervical dislocation. Morphological identification of GC suboz~es. In the cat, GC cell body size and their central projection patterns has been linked to their physiological grouping [3, 5]: cells having large (~), medium ~ ) and small (}') soma sizes correspond to brisk transient (Y) cells, brisk sustained (X) cells and sluggish (W) cells, respectively. The size distribution of cat GCs encountered in the present study is presented in Fig. 2. The sample (,=285) includes some unstained GCs identified on the basis of morphological and functional queues (prominent nucleolus [8], and spike activity during seal formation). Cell body diameter alone was not sufficient to distinguish between GC types in dissociated form, since there was considerable overlap between the diameters of those cells labelled by retrograde tracer placed within either LGN or SC. Although our procedures were not known to be selective in any way for different GC types, it cannot be guaranteed that all GC classes were included in our large sample of cells. For instance recordings from large (c~ type) cells were apparently not encountered based on size criteria, perhaps resulting from their relatively low abundance. Cells labelled from the SC comprised a majority of small (mode for soma diameter: 12/am), }'-like GCs, but also contained a number of medium-sized soma. We therefore determined this population to contain large numbers of the sluggish W-type GCs, having complex receptive field properties (including those requiring elaborate trigger features, such as motion and orientational queues). Cells labelled from the LGN are likely to include many functional types. However, since double labelled cells
10 0 |0
20
30
Soma Diameter t ~tm~ Fig. 2. GC soma size frequency plot. Size distribution of isolated cells staincd by dye injection into SC (open squares) and LGN (iilled squares). The combined population of stained and unstained cells (open circles) identified as GCs by other criteria (see text). Cell bod3 size mcasurelnents are consistent with the pharmacological study having included different functional classes o1"GC.
(retaining dye from both LGN and SC injection sites) were removed from this population, and pooled in the former group of SC projecting cells (in cat no brisk sustained, X-type cells project to the SC), and also because superficial injections of the LGN were typically achieved, a preponderance of X-type GCs is likely in this sample. As expected, this population included cells having mostly medium (mode for soma diameter: 15/am), /3-like soma sizes. Tratlsmitter sensitivities qf GCs. To discover the molecular bases for the receptive field properties of retinal output, we characterised the transmitter-gated conductances of different ganglion cell subtypes, from the periphery. The present pharmacological study was performed on cells having different cell body sizes and central projection specificity (,=324). Therefore, the cells used presumably include both ON and OFF forms of sluggish and brisk physiological groupings. Patch-clamp recordings [6, 10, 14] made in the whole cell configuration from these cells are summarised in Fig. 3. Data are presented from a screen including }'-amino butyric acid (GABA), glycine, acetylcholine (ACh) and glutamate, it should be noted that cells were maintained at -60 mV holding potential, and in the presence of magnesium, therefore eliminating the possible influence of N-methylD-aspartate (NMDA)-specific glutamate responses. Drugs were applied at known concentrations, during approximately 10-s intervals, to a continuous stream of superfusate flowing over cells from which recordings were being made. Solution changes were rapid, in the order of 10 ms [14]. A consistent finding was that the sensitivities of all categories of GC were comparable. All cells, whatever
116
A
GABA -
"--
Gly -
/
B
ACh
"~'l~ ~'~¢~
7
,/ 300pM
'°°1
,
,/
'°°1 .
.~
Glut
-"
S~uM
~O00~M
100pM
i
'~
'/ lO~M
C
'
1,25 JJM
3o~M
loo~M
,o,-W. = pA
/
t ~
3F~M
'
10pM
S sec
0"3 tJM
oA I
30]JM
I 0 sec
Fig. 3. Whole cell voltage-clamp recordings from identified cat retinal GCs, highlighting differences in dose dependence of desensitisation properties. A: strongly dose-dependent desensitisation of responses to GABA and glycine; B: transient cholinergic responses, demonstrating rapid desensitisation at all doses; C: sustained responses to glutamate. Responses were elicited by approximately 10-s applications of known concentrations of either of the 4 transmitter agonists tested: GABA, gtycine, ACh or glutamate. Data is presented from different cells. However, all categories of GC demonstrated sensitivity to each transmitter. Responses to the inhibitory transmitters are shown at threshold, half maximal and saturating doses. Responses to ACh are taken from near threshold doses. Glutamate-induced currents are in response to moderate or saturating doses. Records are made in the presence of magnesium and at a holding potential of -60 inV. Traces are from a pen recorder with frequency response bandwidth of DC 140 Hz.
(%)
loo
80" LO "O m v O3 O
~
ACh
Gly 60-
GABA
Q 40
20 Glut 0 10 0
,3 10 ~
1'02
Dose
103
[JJM]
Fig. 4. Comparison of agonist-induced receptor desensitisation to GABA, glycine,ACh and glutamate. The fractional current decay at 5 s from peak are plotted (Id5, as percent) against agonist concentration. Data is from responses of the same cell to the 4 different neurotransmitters, but is representative of all GC types tested. Desensitisation of responses to inhibitory amino acids was dose dependent. Cholinergic responses decayed rapidly over all effective concentrations, including threshold doses. The sustained currents induced by glutamate underwent no appreciable desensitisation over this time course.
their s o m a size g r o u p , o r specificity o f central p r o j e c t i o n were sensitive to all 4 transmitters, with similar thresholds, kinetics a n d ranges o f effective doses. It s h o u l d be stressed however, t h a t the usefulness o f the t r a n s m i t t e r screen as a d i s c r i m i n a t o r y index was limited, since it includes the two m o s t p r e v a l e n t i n h i b i t o r y t r a n s m i t t e r c a n d i d a t e s ( G A B A a n d glycine). A l s o included is g l u t a m a t e , the p u t a t i v e t r a n s m i t t e r o f all b i p o l a r cells, a n d A C h , k n o w n f r o m in vivo studies to influence the activity o f all G C s , to s o m e extent [2, 9]. T h e r e f o r e the existence o f m o r e select p h a r m a c o l o g i c a l specificities with which G C s can be 'finger p r i n t e d ' is n o t excluded by the present study. Also, estimates o f c u r r e n t densities were n o t a t t e m p t e d . E x a m i n a t i o n o f the shape o f these t r a n s m i t t e r - g a t e d currents revealed that while the p e a k a m p l i t u d e o f the currents are all d o s e - d e p e n d e n t relationships (defining the m o l e c u l a r specificity o f l i g a n d for receptor), t h e rates o f c u r r e n t d e c a y in the c o n t i n u e d presence o f agonist ( r e c e p t o r desensitisation) are related to the a g o n i s t conc e n t r a t i o n in ways t h a t characterise the different receptors. These r e c e p t o r p r o p e r t i e s can be classified as follows: (a) d o s e - d e p e n d e n t desensitisation, the case for G A B A a n d glycine; (b) transient cholinergic responses, u n d e r g o i n g r a p i d desensitisation at all doses; (c) sustained responses to glutamate. T h e different kinetics o f a g o n i s t - i n d u c e d r e c e p t o r de-
117
sensitisation that were observed are summarised in Fig. 4. Multiple applications of test substances were possible. Trials were separated by 4-5-min intervals. The entire dose-response (current decay) for all transmitters collected for one cell is presented in the figure, demonstrating the stability of the recording electrode during drug application trials. The desensitisation rate of evoked currents was simplified to the current decay at 5 s (ldS) expressed as a percentage of the peak current, or its estimate in the case of high doses of glutamate (see Fig. 3). In contrast to results for the inhibitory transmitters, desensitisation of the current responses to ACh and glutamate were significantly less dose-dependent. The former underwent rapid desensitisation, even at threshold doses. The latter was characterised by sustained inward currents, even in the presence of millimolar glutamate. However, occasionally at these doses of glutamate, transient currents were elicited, superimposed on the sustained components (Fig. 3, response to 1000 ,aM glutamate). This observation may represent recruitment of lower affinity sites. (In the case of such transient responses, peak-sustained current estimates were obtained by extrapolation for the purposes of evaluating the decay rate for the sustained component.) Synaptic transmission involves multiple pre- and postsynaptic mechanisms, which determine the activity of the follower cell. These include presynaptic control of transmitter release, the spatio-temporal arrangement of inputs and outputs (feed-back), and the intrinsic properties of the cell. It is clearly possible that significant specialisation of synaptic function could be achieved through the kinetic parameters of postsynaptic receptors. Therefore, the different desensitisation characteristics of transmitter receptors outlined in this report may contribute to the shaping of sustained and transient retinal signals at the level of ganglion cells. in the case of cholinergic transmission, transient signalling may appropriately serve such visual activities as motion and/or direction sensitivity. As to which other functions may be subserved by the presence of these rapidly desensitising cholinergic receptors, found as they were on all GCs tested, is unclear. In the case of the putative bipolar cell (and photoreceptor) transmitter candidate, glutamate, a tonic response, accurately reflecting the level of illumination or activation of bipolar cell input could be of clear value in sustaining the perception of maintained stimuli, such as intensity or colour. Developmental, phylogenetic and central vs. peripheral nervous specialisation may underlie marked differences in the rates of cholinergic desensitisation. In contrast to the high rates of current decay, and relatively weak dose-dependence of desensitisation that we have recorded in adult mammalian retinal GCs (including
mouse and rabbit, data not shown), significantly lower rates of desensitisation for cholinergic responses have been recorded from post-natal rat retinal GCs (maximal rate of around 30% Id5 for 5/aM dose, at -80 mV holding potential (ref. 11, Fig. 2B)). Rates of nicotinic receptor desensitistion measured for embryonic chick sympathetic neurones are also notably more dose-dependent [4] (unpublished observations). To what extent these differences might relate to physiological functions remains to be answered. It could be speculated that different molecular forms of postsynaptic receptors can be "selected' to suit specialised, and indeed changing, operating requirements. Molecular alterations, including sub-unit substitution [13] and post-translational modification by phosphorylation [4. 7] may ultimately determine these operational differences. Release of ACh in the retina has been shown to be transient [12]. This presynaptic characteristic and the postsynaptic feature of cholinergic receptor function reported in the present study leads to the notion that ACh is mediating phasic signalling within the inner plexiform layer, while glutamate is transmitting tonic signals to GC membranes. This work was supported by an award to J.E.G.D. from the Japanese Ministry of Science Education and Culture, and contributions from the British Council.
1 Aizenman. E., Frosch, M.P. and Lipton, S.A., Responses mediated by excitatory amino acid receptors in solitary retinal ganglion cells flom the rat, J. Ph34iol.. 396 (1988l 75 91. 2 Ariel, M. and Daw, N.W., Effects of cholinergic drugs on receptive field properties of rabbit retinal ganglion cell';, J. Physiol., 324 11982) 135 160. 3 Bo3,coU, B.B. and Wassle, H.. The morphological types of ganglion cells of the domestic cat's retina, J. Physiol., 240 {1974) 397 419. 4 Downing. J.E.G. and Rolc. L.W., Activators of protein kinase C enhance acctylcholme receptor desensitization in sympathetic ganglion neurons. Proc. Natl. Acad. Sci. t .S.A., 84 (1987) 7739 7743. 5 Fukuda. Y. and Stone, J.. Retinal distribution and central projeclions of Y-, X- and W-cells of the cat's relina. J. Nenrophysiol.. 37 11974) 749 772. 6 Hamill. O.R. Marry, A., Neher, E., Sakmann, B. and Sigworth, F.J., hnproved patch-clamp techrdques for high-resolution current recording from cells and cell-free membrane palches, Pll%ers Arch..391 (1981)85 100. 7 Huganir, R.L. and Greengard, P.. Regulation of ncurotransmiUer receptor desensitization by protein phosphorylation. Neuron, 5 (1990) 555 567. 8 Ishida. A.T. and Cohen, B.N., (iABA-activated wholc-cell currents in isolaled retinal ganglion cells. J. Neurophysiol. 60 (1988) 381 396. 9 lkeda, H., Transmitter action at cat retinal ganglion cells, Prog. Retin. Res., 4 (19841 I 32.
118 10 Kaneko, A., Pinto, L.H. and Tachibana, M., Transient calcium current of retinal bipolar cells of the mouse, J. Physiol., 410 (1989) 613-629. 1 l Lipton, S.A., Aizenman, E. and Loring, R.A., Neural nicotinic acetylcholine responses in solitary mammalian retinal ganglion cells, Eur. J. Physiol., 410 (1987) 37-43. 12 Masland, R.H., Mills, J.W. and Cassidy, C., The functions of acetylcholine in the rabbit retina, Proc. R. Soc. London. Set. B, 223 (1984) 121-139.
13 Schuetze, S,M. and Role, L.W.. Developmental regulation ol nicotinic acetylcholine receptors, Annu. Rev. Neurosci., 10 (1987i 403 457. 14 Suzuki, S., Tachibana, M. and Kaneko, A., Effects of glycine and GABA on isolated bipolar cells of the mouse retina, J. Physiol., 421 (1990) 645 662.
\,~.'..s~wmc L¢/~, i (1~;92~[14 I . : t992 Elsevier Scientil]c Publishers Ireland Lid. All rights reserxed tB(t4-3940i~2i3 {~5.0i)
NSL 08476
Cat retinal ganglion cells show transient responses to acetylcholine and sustained responses to L-glutamate* J.E.G. Downing** and A. Kaneko Department of lnjbrmatkm Physiology, National Institutefor Physiological Science.s, Okazaki (Japan) (Received 26 April 1991 : Revised version received 16 December 1991 ; Accepted 20 December 1991 )
Key words. Acute dissociation; Adult cat; Retinal ganglion cell subtype; Receptor desensitization The neuropharmacological basis for the different receptive field properties of cat retinal ganglion cells was investigated using whole cell voltageclamp recordings from acutely dissociated adult tissue. Subclasses of physiologically characterised ganglion cells were determined on the basis of (i) their soma diameters and (ii) their projection to central visual nuclei (identified by microinjection of fluorescent dyes into the lateral geniculate and/or superior colliculus). The sensitivities of all categories of ganglion cells, prepared from peripheral retina were found to be similar for ;v-amino butyric acid, glycine, acetylcholine and glutamate. The kinetics of desensitisation differed among receptor subtypes, revealing possible physiologically significant molecular specialisations that could be involved in shaping synaptic transmission.
The physiology of vertebrate retinal neurones is comparatively well documented from studies using isolated and in vivo preparations. However, the stability of intracellular recordings is limited and extensive cellular interactions retained within intact tissue complicate the interpretation of direct synaptic transmission, independent of feedback loops or modulation. Recent advances in cell isolation techniques and the application of 'giga-seal' suction electrode voltage-clamp have optimised conditions for measuring the chemosensitivity of retinal neurones. Initial studies on the mammalian retina have made use of rodent species [1, 10, 11, 14]. We have chosen to study ganglion cells (GCs) from the adult cat in order to link biophysical measurements of membrane properties to what is known about their different light evoked responses. Whole cell voltage-clamp recordings were obtained using methodologies detailed elsewhere [6, 10, 14]. Briefly, recordings were made at -60 mV holding poten*Presented at the 19th Annual Meeting of the Society for Neuroscience, Phoenix, Arizona, USA (November 19891, and to the British Photobiology Society, Kings College, London, UK (December, 1989). *'Present address: Neuroscience Laboratory, Department of Biology; Imperial College, London SW7 2BB. UK.
Correspondence: J.E.G. Downing, Neuroscience Laboratory, Department of Biology, Imperial College, Prince Consort Road, London SW7 2BB, UK.
tial, in superfusate containing in mM: NaC1 135, KCI 5, CaC12 2, MgC12 1, HEPES 5, glucose 10 (pH 7.4, adjusted with NaOH). Patch electrodes were filled with a solution containing in mM: KC1 120, CaC12 0.5, EGTA 5, HEPES 10 (pH 7.2 adjusted with KOH). Recordings were taken from subtypes of retinal GCs, identified within mixed cell dissociates. Isolated cells were prepared using enzyme digestion (papa/n) and mechanical action [10]. Using these methods the complete range of cells types was separated from retinas of adult (~1 kg) cats. Cells retaining a high degree of morphological differentiation were consistently prepared. Bipolar cells [10] and horizontal cells (not shown) could be recognised among the dissociates, on morphological criteria alone. Solitary cell bodies, denuded of all but their primary dendrites were used in the present study. These were identified as GCs by retrograde labelling (Fig. 1) with 1, l'-dioctadecyl- 3,3,3',3'-tetramethylindocarbocyanine dye (D/I: 25-50/.tl; 2.5 mg/ml in 100% ethanol) injected into the superior colliculus (SC), and/or 4,6-diamidino-2phenylindole (DAPI: 25-50 p1; 20/.tg/ml aq.) into the lateral geniculate nucleus (LGN), 3 days before enucleat/on. Soma size and projection pattern were used to further categorise GC subclasses. Hence, physiological experimentation was performed on two very different populations of GC, identified by their staining from injections made either into the SC or the LGN. Stereotaxic placement of fluorescent dye was achieved under anaesthesia induced by injection of sodium pentobarbi-
115 5It
El
4tl ~'
30
E
2O
O m Fig. l. Prior to electrophysiological examination. GCs were positively identified within mixed cell cultures by fluorescence imaging of retrogradely transported dye from the SC or LGN. A: phase contrast image: B: fluorescence image of the same cell retrogradely labelled with DAPI from the SC. Note that the bright staining of the nucleus by DAP[ was easily contrasted with plasma naembraneous staining of cells by' Dil (not shown).
tal (i.p,), followed by inhalation of a mixture of 70% nitrous oxide and 30% oxygen and 0.5 1.5% halothane. For enucleation, animals were anaethetised using halothane prior to cervical dislocation. Morphological identification of GC suboz~es. In the cat, GC cell body size and their central projection patterns has been linked to their physiological grouping [3, 5]: cells having large (~), medium ~ ) and small (}') soma sizes correspond to brisk transient (Y) cells, brisk sustained (X) cells and sluggish (W) cells, respectively. The size distribution of cat GCs encountered in the present study is presented in Fig. 2. The sample (,=285) includes some unstained GCs identified on the basis of morphological and functional queues (prominent nucleolus [8], and spike activity during seal formation). Cell body diameter alone was not sufficient to distinguish between GC types in dissociated form, since there was considerable overlap between the diameters of those cells labelled by retrograde tracer placed within either LGN or SC. Although our procedures were not known to be selective in any way for different GC types, it cannot be guaranteed that all GC classes were included in our large sample of cells. For instance recordings from large (c~ type) cells were apparently not encountered based on size criteria, perhaps resulting from their relatively low abundance. Cells labelled from the SC comprised a majority of small (mode for soma diameter: 12/am), }'-like GCs, but also contained a number of medium-sized soma. We therefore determined this population to contain large numbers of the sluggish W-type GCs, having complex receptive field properties (including those requiring elaborate trigger features, such as motion and orientational queues). Cells labelled from the LGN are likely to include many functional types. However, since double labelled cells
10 0 |0
20
30
Soma Diameter t ~tm~ Fig. 2. GC soma size frequency plot. Size distribution of isolated cells staincd by dye injection into SC (open squares) and LGN (iilled squares). The combined population of stained and unstained cells (open circles) identified as GCs by other criteria (see text). Cell bod3 size mcasurelnents are consistent with the pharmacological study having included different functional classes o1"GC.
(retaining dye from both LGN and SC injection sites) were removed from this population, and pooled in the former group of SC projecting cells (in cat no brisk sustained, X-type cells project to the SC), and also because superficial injections of the LGN were typically achieved, a preponderance of X-type GCs is likely in this sample. As expected, this population included cells having mostly medium (mode for soma diameter: 15/am), /3-like soma sizes. Tratlsmitter sensitivities qf GCs. To discover the molecular bases for the receptive field properties of retinal output, we characterised the transmitter-gated conductances of different ganglion cell subtypes, from the periphery. The present pharmacological study was performed on cells having different cell body sizes and central projection specificity (,=324). Therefore, the cells used presumably include both ON and OFF forms of sluggish and brisk physiological groupings. Patch-clamp recordings [6, 10, 14] made in the whole cell configuration from these cells are summarised in Fig. 3. Data are presented from a screen including }'-amino butyric acid (GABA), glycine, acetylcholine (ACh) and glutamate, it should be noted that cells were maintained at -60 mV holding potential, and in the presence of magnesium, therefore eliminating the possible influence of N-methylD-aspartate (NMDA)-specific glutamate responses. Drugs were applied at known concentrations, during approximately 10-s intervals, to a continuous stream of superfusate flowing over cells from which recordings were being made. Solution changes were rapid, in the order of 10 ms [14]. A consistent finding was that the sensitivities of all categories of GC were comparable. All cells, whatever
116
A
GABA -
"--
Gly -
/
B
ACh
"~'l~ ~'~¢~
7
,/ 300pM
'°°1
,
,/
'°°1 .
.~
Glut
-"
S~uM
~O00~M
100pM
i
'~
'/ lO~M
C
'
1,25 JJM
3o~M
loo~M
,o,-W. = pA
/
t ~
3F~M
'
10pM
S sec
0"3 tJM
oA I
30]JM
I 0 sec
Fig. 3. Whole cell voltage-clamp recordings from identified cat retinal GCs, highlighting differences in dose dependence of desensitisation properties. A: strongly dose-dependent desensitisation of responses to GABA and glycine; B: transient cholinergic responses, demonstrating rapid desensitisation at all doses; C: sustained responses to glutamate. Responses were elicited by approximately 10-s applications of known concentrations of either of the 4 transmitter agonists tested: GABA, gtycine, ACh or glutamate. Data is presented from different cells. However, all categories of GC demonstrated sensitivity to each transmitter. Responses to the inhibitory transmitters are shown at threshold, half maximal and saturating doses. Responses to ACh are taken from near threshold doses. Glutamate-induced currents are in response to moderate or saturating doses. Records are made in the presence of magnesium and at a holding potential of -60 inV. Traces are from a pen recorder with frequency response bandwidth of DC 140 Hz.
(%)
loo
80" LO "O m v O3 O
~
ACh
Gly 60-
GABA
Q 40
20 Glut 0 10 0
,3 10 ~
1'02
Dose
103
[JJM]
Fig. 4. Comparison of agonist-induced receptor desensitisation to GABA, glycine,ACh and glutamate. The fractional current decay at 5 s from peak are plotted (Id5, as percent) against agonist concentration. Data is from responses of the same cell to the 4 different neurotransmitters, but is representative of all GC types tested. Desensitisation of responses to inhibitory amino acids was dose dependent. Cholinergic responses decayed rapidly over all effective concentrations, including threshold doses. The sustained currents induced by glutamate underwent no appreciable desensitisation over this time course.
their s o m a size g r o u p , o r specificity o f central p r o j e c t i o n were sensitive to all 4 transmitters, with similar thresholds, kinetics a n d ranges o f effective doses. It s h o u l d be stressed however, t h a t the usefulness o f the t r a n s m i t t e r screen as a d i s c r i m i n a t o r y index was limited, since it includes the two m o s t p r e v a l e n t i n h i b i t o r y t r a n s m i t t e r c a n d i d a t e s ( G A B A a n d glycine). A l s o included is g l u t a m a t e , the p u t a t i v e t r a n s m i t t e r o f all b i p o l a r cells, a n d A C h , k n o w n f r o m in vivo studies to influence the activity o f all G C s , to s o m e extent [2, 9]. T h e r e f o r e the existence o f m o r e select p h a r m a c o l o g i c a l specificities with which G C s can be 'finger p r i n t e d ' is n o t excluded by the present study. Also, estimates o f c u r r e n t densities were n o t a t t e m p t e d . E x a m i n a t i o n o f the shape o f these t r a n s m i t t e r - g a t e d currents revealed that while the p e a k a m p l i t u d e o f the currents are all d o s e - d e p e n d e n t relationships (defining the m o l e c u l a r specificity o f l i g a n d for receptor), t h e rates o f c u r r e n t d e c a y in the c o n t i n u e d presence o f agonist ( r e c e p t o r desensitisation) are related to the a g o n i s t conc e n t r a t i o n in ways t h a t characterise the different receptors. These r e c e p t o r p r o p e r t i e s can be classified as follows: (a) d o s e - d e p e n d e n t desensitisation, the case for G A B A a n d glycine; (b) transient cholinergic responses, u n d e r g o i n g r a p i d desensitisation at all doses; (c) sustained responses to glutamate. T h e different kinetics o f a g o n i s t - i n d u c e d r e c e p t o r de-
117
sensitisation that were observed are summarised in Fig. 4. Multiple applications of test substances were possible. Trials were separated by 4-5-min intervals. The entire dose-response (current decay) for all transmitters collected for one cell is presented in the figure, demonstrating the stability of the recording electrode during drug application trials. The desensitisation rate of evoked currents was simplified to the current decay at 5 s (ldS) expressed as a percentage of the peak current, or its estimate in the case of high doses of glutamate (see Fig. 3). In contrast to results for the inhibitory transmitters, desensitisation of the current responses to ACh and glutamate were significantly less dose-dependent. The former underwent rapid desensitisation, even at threshold doses. The latter was characterised by sustained inward currents, even in the presence of millimolar glutamate. However, occasionally at these doses of glutamate, transient currents were elicited, superimposed on the sustained components (Fig. 3, response to 1000 ,aM glutamate). This observation may represent recruitment of lower affinity sites. (In the case of such transient responses, peak-sustained current estimates were obtained by extrapolation for the purposes of evaluating the decay rate for the sustained component.) Synaptic transmission involves multiple pre- and postsynaptic mechanisms, which determine the activity of the follower cell. These include presynaptic control of transmitter release, the spatio-temporal arrangement of inputs and outputs (feed-back), and the intrinsic properties of the cell. It is clearly possible that significant specialisation of synaptic function could be achieved through the kinetic parameters of postsynaptic receptors. Therefore, the different desensitisation characteristics of transmitter receptors outlined in this report may contribute to the shaping of sustained and transient retinal signals at the level of ganglion cells. in the case of cholinergic transmission, transient signalling may appropriately serve such visual activities as motion and/or direction sensitivity. As to which other functions may be subserved by the presence of these rapidly desensitising cholinergic receptors, found as they were on all GCs tested, is unclear. In the case of the putative bipolar cell (and photoreceptor) transmitter candidate, glutamate, a tonic response, accurately reflecting the level of illumination or activation of bipolar cell input could be of clear value in sustaining the perception of maintained stimuli, such as intensity or colour. Developmental, phylogenetic and central vs. peripheral nervous specialisation may underlie marked differences in the rates of cholinergic desensitisation. In contrast to the high rates of current decay, and relatively weak dose-dependence of desensitisation that we have recorded in adult mammalian retinal GCs (including
mouse and rabbit, data not shown), significantly lower rates of desensitisation for cholinergic responses have been recorded from post-natal rat retinal GCs (maximal rate of around 30% Id5 for 5/aM dose, at -80 mV holding potential (ref. 11, Fig. 2B)). Rates of nicotinic receptor desensitistion measured for embryonic chick sympathetic neurones are also notably more dose-dependent [4] (unpublished observations). To what extent these differences might relate to physiological functions remains to be answered. It could be speculated that different molecular forms of postsynaptic receptors can be "selected' to suit specialised, and indeed changing, operating requirements. Molecular alterations, including sub-unit substitution [13] and post-translational modification by phosphorylation [4. 7] may ultimately determine these operational differences. Release of ACh in the retina has been shown to be transient [12]. This presynaptic characteristic and the postsynaptic feature of cholinergic receptor function reported in the present study leads to the notion that ACh is mediating phasic signalling within the inner plexiform layer, while glutamate is transmitting tonic signals to GC membranes. This work was supported by an award to J.E.G.D. from the Japanese Ministry of Science Education and Culture, and contributions from the British Council.
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13 Schuetze, S,M. and Role, L.W.. Developmental regulation ol nicotinic acetylcholine receptors, Annu. Rev. Neurosci., 10 (1987i 403 457. 14 Suzuki, S., Tachibana, M. and Kaneko, A., Effects of glycine and GABA on isolated bipolar cells of the mouse retina, J. Physiol., 421 (1990) 645 662.
