S-neurons and not L-neurons are the source of ... - Springer Link

Joumal of Comparative Physiology A

J Comp Physiol A (1985) 157:779 788

Sensory, Neural, and Behavioral Physiology

9 Springer-Verlag 1985

S-neurons and not L-neurons are the source of GABAergic action in the ocellar retina Josef Ammermfiller and Reto Weiler Zoologisches Institut der Ludwig-Maximilians-Universit/it, Luisenstrasse 14, D-8000 Miinchen 2, Federal Republic of Germany Accepted September 24, 1985

Summary. Electrophysiological evidence obtained with current- and voltage clamp experiments from single L-neurons of the ocellar nerve of locust (Locusta migratoria) questions a direct synaptic feedback from these neurons onto the photoreceptors. The synaptic currents recorded under voltage clamp reflected the photoresponse of the L-neuron, despite the fact it developed no synaptic activity under these conditions. This result is contrary to GABAergic feedback models proposed in the literature. Electrophysiological recordings, as well as immunocytochemistry revealing GABA and glutamate decarboxylase, indicated a possible contribution of S-neurons in such a feedback system. A population of probable S-neurons whose somas were in the pars intercerebralis adjacent to the ocellar nerve tracts was heavely labelled. About 10 fibres entered each tract and formed a dense network of fine arborizations within the ocellar plexiform layer. L-neurons showed no GABA-immunoreactivity. Based on these data a new model for GABAergic feedback is proposed and discussed.

Introduction Synaptic feedback between photoreceptors and second order neurons in the vertebrate retina has received a great deal of attention since it was first described (Baylor et al. 1971). It can enhance the dynamic range of photoreceptors and horizontal cells (Lasater 1982) and is involved in color coding (Fuortes and Simon 1974; Stell and Lightfoot 1975). In addition, it shows plasticity correlated with the adaptive state of the retina (Weiler and Wagner 1984). The basic wiring diagram is well understood (Stell et al. 1975) and pharmacological experiments have shown GABA to be the most

likely candidate for the feedback transmitter (Murakami et al. 1982). The obvious importance of such a local retinal circuit has drawn attention to the arthropod ocellar system, since recordings from dragonfly photoreceptors and large second order neurons also suggest a possible feedback between these two neurons (Chappell and Dowling 1972; Klingman and Chappell 1978; Stone and Chappell 1981) with GABA as the underlying transmitter. Observations of reciprocal synapses between photoreceptors and second order neurons support this idea (Dowling and Chappell 1972). However, these findings were contradicted by results from a recent study involving simultaneous recordings from a photoreceptor and its postsynaptic neuron (Simmons 1982). In this study we have investigated the possible existence of a feedback between photoreceptors and L-neurons in the locust ocellar system. Previous electrophysiological experiments on this system have not adressed this question (Wilson 1978 a, b). We therefore combined our electrophysiological experiments with immunocytochemical localization of the putative transmitter GABA and its synthesising enzyme glutamic acid decarboxylase (GAD). Based on these data we can rule out any GABA-mediated feedback from L-neurons onto the photoreceptors and suggest that it is more likely that GABAergic S-neurons are involved in such a pathway.

Methods Electrophysiology. Experiments were performed on the median ocellus of adult locusts (Locusta migratoria L.), reared in the laboratory. For electrophysiological recording the dorsal third of the head, containing the brain, was cut from the body and fixed in a small, wax-coated chamber. The dorsal cuticula was cut away to enable access of the electrodes. The open headcapsule was filled with saline (retool/l: 170 NaCI; 5 KC1; 1.8

780 CaC12; 2 MgC12; 56 Cholin-Cl; 20 HEPES; pH 6.8) covering only the median ocellar nerve and ocellus. The saline could be exchanged with a push-pull syringe-system with an inlet and outlet just beneath the ocellar nerve. Special care was taken to leave the tracheal air supply intact. To improve air supply, the dorsal air sac was sometimes opened. To facilitate electrodepenetration, the nerve was superfused for 1 min with 1% protease (type V; Sigma) in saline, which softened the neural sheath. The whole preparation was surrounded by a moistchamber to prevent it from drying. Under these conditions recordings from L-neurons remained stable for periods in excess of 3 h. The light from a halogen lamp (100 W; 12 V) was focused by a lens onto one end of a light fibre. The other end was placed 2 cm in front of the headcapsule, stimulating the median ocellus and, by stray-light, the lateral ocelli and compound eyes. The intensity was attenuated by neutral density filters. Glass-microelectrodes were filled with 2 mol/1 potassium acetate or 4% Lucifer Yellow and had resistances of 30-70 M(2 and 300-600 Mf~, respectively. For current-clamp and voltage-clamp it was necessary to penetrate the same neuron with two electrodes. Three criteria were used for simultaneous recording from one neuron. 1 : Complete coincidence of cell noise. 2: The light responses recorded by the two electrodes were exactly the same. 3: A n input resistance of more than one Mf~, measured as voltage seen by one electrode, as a result of a small current pulse delivered through the other. Penetrations were performed near the ocellar neuropil at a distance of about 200 ~tm from the ocellar cup. In each of the ocelli studied one L-neuron was current- and voltage-clamped respectively. The amplifiers for current-clamp and voltage-clamp were conventionally designed, with a high-voltage operational amplifier (BB 3584) as the last stage in the current-pump. The current electrode was shielded with silver paint (Auromal 37 M, Doduco), which was isolated from the saline by coating with cyanoacrylat. Voltage was measured between the intracellular electrodes and a reference electrode outside the nerve, which was connected to the saline via an agar-bridge. Current was measured by a current-to-voltage converter, connected between an Ag/AgCl-electrode in the saline and the current amplifier. Current was injected via the current-pump through the second electrode. In the voltage-clamp mode, the gain was 8,000 x-30,000 x to improve speed and reliability. The seriesresistance, which was introduced by the neural sheath surrounding the ocellar nerve, was measured in current-clamp and never exceeded 70 kf2. In voltage-clamp this resistance was compensated up to 50% by a circuit, equivalent to the one introduced by Hodgkin et al. (1952). The input resistances of the L-neurons had a mean value of 1.6 Mr2 corresponding with measurements of Wilson (1978b). The electrical signals were stored on a F M tape-recorder (TEAC R 81) and later fed to a computer (Commodore 8032-SK with andy-system) for analysis and plotting. The voltage-clamp quality in the ocellar neuropil could not be tested directly, therefore some calculations about voltage decrement were performed. Wilson (1978 b) estimated the membrane resistivity Rm of L-neurons to range from 2,000 f2. cm 2 to 4,000 f2. cm a. This estimation was confirmed during analysis of the passive cable properties of L-neurons (unpublished results). Morphological measurements done with Lucifer Yellow stained L-neurons yielded a mean axon diameter of 20 gin. Together with the lower value of 2,000 s 2 for Rr, and an assumed intracellular resistivity of 50 s cm a minimal value of 0.14 cm for the length constant ,~ was calculated. To estimate the voltage-clamp quality we assumed the validity of the d 3/2law for the L-neuron arborizations. In this case the dendritic tree can be represented by an equivalent membrane cylinder whose length constant is equal to the one of the main trunk (Rall 1977). From morphological data a maximal distance of

J. Ammermiiller and R. Weiler : GABAergic action in the ocellus 0.06 cm from the point of electrode penetration to the most distal dendritic branch was determined (own measurements; G o o d m a n et al. 1979). The calculation about the voltage decrement was based on the equation of a semi-infinite fibre, voltage-clamped at a distance of x = x l from the sealed end (x = 0; Rall 1977). It results a ratio of the voltage at the sealed end to the voltage at the voltage-clamp point of 0.9. This means that the voltage at the distal end of the arborizations will not deviate more than 10% from the command voltage. Since 2 was calculated on very cautious assumptions we conclude that voltage-clamp quality would be acceptable even if the d3/2-1aw were not strictly fulfilled. Further evidence for acceptable clamp quality came from experiments where voltage steps were applied to the L-neurons. The charging currents at the beginning and the end of the voltage steps settled within 0.5 ms indicating that longitudinal current flow was negligible.

Immunocytochemistry. The head of a locust was fixed with 4% paraformaldehyde for one hour, washed in phosphate buffer and then immersed in 30% sucrose, before freezing under liquid nitrogen. The frozen tissue was cut on a cryostat and 30 gm sections were mounted on gelatine precoated slides. The section plane was either frontal or tangential. Sections were incubated overnight at 4 ~ with the first antibody, which was either an antibody against G A B A (IBL, Hamburg) or glutamic acid decarboxylase (GAD) (gift from Dr. C. Brandon). Both antibodies were raised in rabbit. Immunoreactivity was revealed either with the indirect fluorescent method using FITC labelled second antibodies (Sigma) or with methods involving H R P as a marker (peroxidase-antiperoxidase complex, Sigma; streptavidin-peroxidase complex, Amersham). The GABA-antibody was used in concentrations of 1:750 (PBS with 0.3% Triton X) for the fluorescent method and 1 : 5,000 for the peroxidase methods. The GAD-antibody was used in concentrations of 1 : 1,000 for the peroxidase method. The FITC-labelled second antibody as well as the link sera were used at concentrations of I : 200. The PAP-complex and the avidin-peroxidase complex

Curare

2rnin

6rain

8rain

Lm v 100 ms

Fig. 1. Effect of curare superfusion of the median ocellus on the light response of an L-neuron. The bar indicates duration of the light stimulus. Relative intensity was - 3 . 1 . The slow OFF-transient (SOT) was weak in this cell. Time indicates duration of superfusion

J. Ammermfiller and R. Weiler: GABAergic action in the ocellus were used at concentrations of 1:500. The incubation of all these substances was performed at room temperature and lasted about 11/2 h for each step. All steps were separated by two washing steps of 10 min in PBS. Animals that were used for electrophysiological experiments with subsequent Lucifer Yellow injection prior to immunocytochemistry were treated in a similar way. Only the incubation and washing periods were reduced to about half in order to keep possible loss of Lucifer Yellow as low as possible. Both antibodies showed high specificity in different neuronal tissues and preabsorbtion overnight with G A B A and GAD, respectively at 10 gmol/1 concentration prevented any immunocytochemical reaction.

Results

Electrophysiology Effects of curare. The effects of curare on L-neurons of the locust were tested by superfusing the median ocellar nerve with saline containing 10 mg/ ml d-tubocurarine (Sigma). The results are shown in Fig. 1. For the description of the L-neuron lightresponse the nomenclature introduced by Wilson (1978a) is used. Six min after adding curare, the amplitude of the ON-transient was reduced as was the level of the plateau which follows the ON-transient. The slow OFF-transient (SOT) which was not very prominent in this particular cell was drastically increased in size and duration. After 8 rain

1

781

of curare superfusion the amplitude of the ONtransient was very small and the plateau had become positive relative to the dark resting potential. The latter was shifted 3 mV in negative direction compared to the resting potential before curare superfusion. In other experiments positive shifts of the resting potential were observed too. The SOT remained a very prominent slow depolarization of about the same duration as after 6 min. The prominent SOT, which was recorded from all L-neurons after curare superfusion, depended on the intensity of the light flashes, indicating a light dependent input onto L-neurons which is not blocked by curare superfusion of the median ocellus. In the dragonfly, curare had similar effects on the light response of L-neurons. Acetylcholine was therefore considered the most likely photoreceptor transmitter (Klingman and Chappell 1978).

Reversal potentials of the light response. In order to elucidate possible different ionic mechanisms involved in the synaptic processes, we tried to estimate the reversal potentials of the different components of the light response. Two electrodes were inserted into the same L-neuron which was then current-clamped. The experiments yielded a dark resting poten-

._~ . . . . . . . . .

j' _ _

'

a

20 mV

~)0nA 50ms

J ,

r--

20mY

R_00nA 50ms

I lOmV "~

c

_ _

'~ _ _

r--1100

nA

50ms

Fig. 2a-c. Current-clamp of three different L-neurons to reveal the reversal potentials of the different components of the light response. Upper trace: the voltage in all recordings; lower trace: the current. Bars indicate light stimuli. a Left: normal light response; middle: depolarization increased amplitudes of the ON-transient and the plateau and decreased the amplitude of the SOT. The two fast OFF-transients disappeared; right: hyperpolarization decreased the ON-transient and the plateau has reached reversal. The SOT has decreased. 3 small anode-breakdown spikes were visible at the end of the current pulse. Relative light intensity in all cases - 2 . 7 . b Left: normal light response; middle: the ON-transient has reached reversal following hyperpolarization; right: with increasing hyperpolarization the plateau also reached reversal. Light intensity --3.1. c Left: normal light response with a high noise level; middle: the ON-transient and the plateau reached reversal at the same potential, the SOT has decreased; right: all three components of the light response have reversed at this negative potential. Relative light intensity - 2.7

782

tial of - 4 1 _ _ 1 0 m V (n=31) for the L-neurons. The reversal potential of the ON-transient was found to be - 86 _ 13 mV (n = 18), which is in good agreement with former results (Wilson 1978 b). The reversal potential of the plateau potential was in most cases different from that of the ON-transient. Values slightly more positive (Fig. 2a) or slightly more negative (Fig. 2 b) than the ON-transient reversal occurred frequently, but the difference was never more than 10 mV. In most cases it was not possible to reach a clear reversal potential for the SOT, but the amplitude of the SOT decreased with positive as well as negative potentials relative to the resting potential (Fig. 2 a). In Fig. 2c a rare example is shown, where an unequivocal reversal of the SOT was observed, being about 40 mV more negative than the dark resting potential. It was not possible to depolarize L-neurons very strongly due to their rectifying properties in positive direction. Therefore, we can not rule out an additional reversal potential more positive at potential than the one shown in Fig. 2 a. Such a value was extrapolated by Wilson (1978b). In fact, the data of Fig. 2a suggest such an additional value. Two different reversal potentials for the SOT, however, would indicate that two processes are involved in forming the SOT.

Voltage-clamp of the light response. We clamped 33 L-neurons at their resting potentials and delivered light stimuli to the median ocellus. In this situation voltage dependent conductance changes in the L-neuron clamped are avoided by keeping the voltage constant. Transient synaptic output from the clamped L-neuron to other neurons is consequently blocked, and the clamp current, necessary for keeping the membrane potential at resting value, simply reflects the sum of the synaptic currents seen by the L-neuron. Figure 3 shows typical results of such an experiment. The intensity and duration of the light stimuli were the same before and during voltage-clamp. The measured synaptic currents in the voltageclamp mode closely resembled the normal light responses with the exception of the fast OFF-transients, which are the result of voltage dependent membrane properties (Wilson 1978b; Ammermfiller 1984). The transient nature of the synaptic currents was even enhanced, compared to the voltage response (Fig. 3 b). This reflects the fact, that the membrane charge normally has to be changed by the synaptic currents reducing the amplitude of the fast ON-transient compared to the plateau phase. In the voltage-clamp mode, with a constant membrane potential, this charging is not necessary

J. Ammermfiller and R. Weiler: GABAergic action in the ocellus

mV nA -,

-60 l +200

'~'-'~ n

-20

.

b

.

.

.

%-------

-

-

L

-Ooz

50 ms Fig. 3a, b. Voltage-clamp of an L-neuron. The upper trace shows the voltage, the lower the current. Bars indicate the light stimuli, relative intensity was - 2 . 3 . Figures at the right are absolute values of membrane potential and clamp current, respectively, a Normal light response with prominent SOT shaped by three fast OFF-transients. Dark resting potential - 4 0 mV. b Membrane voltage-clamped at - 4 0 mV. The clamp current resembles the voltage response in (a) with the exception of the fast OFF-transients. The initial outward current reflecting the ON-transient is enhanced

~ ' ~ ' ~ ~

+40mV

~'-,----~',~

+20mV

d

RP

,J*~~.~,.

-20 mV

~'-~,'~,~'---~-~--~-~

- 4 0 mV

~q,~,~-~..~,~*~ 60mV , 110nA 50ms -

Fig. 4. Voltage-clamp of the light response of an L-neuron at different holding potentials relative to the resting potential (RP). Only current traces are shown. The clamp current of the light response reverses at - 4 0 mV relative to the resting potential. All components of the light response increase with clamp potentials more positive than RP. Bottom line indicates the light stimulus with a relative intensity of - 3 . 4

J. Ammermfiller and R. Weiler : GABAergic action in the ocellus

783

and the synaptic currents, responsible for the ONtransient, are enhanced compared to the currents responsible for the plateau. An inward current at the end of the light stimulus reflects the SOT, indicating that this phase of the light response is also mainly produced by changing synaptic action and not by voltage dependent conductance changes. Conductance measurements of Wilson (1978b) argue for an increased synaptic input at this phase of the light response. In some cases it was possible to shift the holding potential of the voltage-clamp from resting potential to more positive or negative values in order to check the reversal potentials in the voltageclamp mode and to verify the values obtained in the current-clamp. Figure 4 shows an example of such an experiment, which largely confirmed the results obtained in the current-clamp experiments. In this case, both ON-transient and plateau reversal occurred at the same potential around 40 mV more negative than the dark resting potential. The not very prominent SOT of this cell disappeared towards the negative and slightly increased towards positive values, indicating a reversal potential more negative than the resting potential.

Irnmunocytochemistry It has been shown that G A B A and its antagonists picrotoxin and bicuculline affect the light responses of L-neurons and photoreceptors, even if the ocellar nerve is cut and therefore isolated from the brain (Klingman and Chappell 1978; Stone and Chappell 1981). It follows that the GABAergic action is localized in the distal ocellar system. However, the above results do not favour the Lneurons as the corresponding feedback neurons. We therefore used immunocytochemistry to localize G A B A within neurons of the ocellar system. Figure 5 shows an example of an L-neuron injected with Lucifer Yellow, following electrophysiological recording. Subsequently the brain was then processed to reveal G A B A immunoreactivity. In sections of the brain with the ocellar tract, the Lucifer Yellow labelled soma of the L-neuron, as well as part of its neurite within the ocellar tract were brightly fluorescent under incident UV-light (390 nm) (Fig. 5a). The same section was photographed with transmission light to reveal immunoreactivity to G A B A (Fig. 5 b). No such immunoreactivity was detected in the Lucifer Yellow labelled soma of the L-neuron nor in its neurite and also not in the neurites of the other L-neurons in the ocellar tract. However, G A B A immunoreactivity was clearly visible in a cluster of cell somas adja-

Fig. 5 a, b. Micrographs of a horizontal section of the left lateral ocellar tract in the brain. The line indicates the dorsal midline of the brain. The sections have been immunocytochemicatly treated to reveal GABA following intracellular recording and staining with Lucifer Yellow of an L-neuron. In a the bright fluorescence of Lucifer YeIlow is visible in the soma of an Lneuron (L) which is a M L 1-2L cell based on the nomenclature of Goodman (1976). In the left ocetlar tract part of its axon is also visible (arrow). In b the same section is viewed with transmitting light to reveal the PAP-reaction product of GABA-immunocytochemistry. No label is visible in the stained L-neuron nor in its axon (L, arrow). GABA-immunoreactivity is visible in a cluster of small somas adjacent to the ocellar nerve tract (open arrows). Bar 100 gm

cent to the ocellar tract. These somas had diameters of about 15 gm and were located in the pars intercerebralis of the brain, near the large somas (diameters about 40 gm) of the L-neurons. This is also seen in a horizontal section of the brain, just dorsal to the median ocellar tract, with the two lateral tracts sectioned transversely (Fig. 6a). G A B A labelled somas sent fibres towards the ocel-

784

J. Ammermiiller and R. Weiler: GABAergic action in the ocellus

Fig. 6. a Horizontal section through the brain at the level of the median ocellar tract (MOT). GABAimmunoreactive somas (arrows) are adjacent to the lateral ocellar tracts (LOT). The small labelled somas send their processes into the lateral tracts (open arrows). Labelled profiles are also visible within the tracts (asteriks). Other labelled somas (arrowheads) do not contribute to the ocellar tracts. The unlabelled somas of 3 probable L-neurons are also visible (L). Bar 100 gm. b A labelled process (arrow) from a labelled soma enters the right LOT, bifurcates (small arrow) and winds along the axons of L-neurons (aL). Bar 50 ~tm

lar tracts and it seemed that the tracts were innervated by separate populations of GABAergic cells. The large somas of probable L-neurons seen inbetween the two lateral tracts were not labelled. In Fig. 6b a labelled thin fibre arising from an adjacent labelled soma is seen to enter the tract between two large axon profiles of L-neurons. These clus-

tered small GABAergic somas are most probably the only ones contributing with their processes to the ocellar nerves. Other GABAergic somas in the brain all lie in regions which are known not to contain neurons contributing to the ocellar nerves (Goodman and Williams 1976). The thin fibres entering the ocellar tracts could


785

Fig. 7. a Thin, GABA-immunoreactive fibres (arrows) within the median ocellar tract. Bar 50 gm. b Labelled profiles form a dense network in the ocellar plexiform layer. The fibres twist around the large profiles of the L-neuron axons (aL). They have a beaded appearance. The dark structures at the right (arrowheads) are cut trachea; P photoreceptor layer. Inset: profiles at higher magnification. Bars 50 gm and 10 ~tm

normally not be followed within the ocellar nerves due to their faint labelling. We therefore enhanced the GABA-content in GABAergic neurons by incubating the head in saline containing 100 ~tmol/1 G A B A and 100 ~tmol/1 Gabaculine (gift from Ciba-Geigy, Basel) prior to fixation. Gabaculine is a potent inhibitor of the enzyme GABA-transaminase responsible for the breakdown of G A B A (L6scher 1980). The GABA-labelling pattern of sections from the brain was identical to the one described above. Even after this treatment no GABA-immunoreactivity was detected in the somas nor the neurites of the L-neurons. However, it was now possible to detect fine fibres with diameters ranging from 1 to 5 ~m within the ocellar tracts and nerves that took a slender course inbetween the large axons of the L-neurons (Fig. 7 a). In the ocellar plexiform layers fine arborizations with fibre diameters less than 1 gm showed immunoreactivity to G A B A (Fig. 7b). Many of the fibres in the nerves and especially the arborizations in the plexiform layers of the ocelli showed regular

swellings. The overall morphological appearance of these GABAergic fibres closely resembled that of S-fibres, stained with cobalt in the locust (Goodman and Williams 1976) and in cockroach (Koontz and Edwards 1984). Their number within one ocellar nerve never exceeded 10 and there was no significant variation in number between median and lateral nerves. It was not possible to follow the course of the thin fibres back to their somas in the same preparation since immunoreactivity was not equally good throughout the whole tissue. However, only the small GABAergic somas in the pars intercerebralis adjacent to the ocellar tracts sent thin fibres into the ocellar nerves and their number of 20 to 30 is in good agreement with the total number of fibres in all three ocellar tracts. This supports the idea that the labelled fibres and somas represent a fraction of the S-neurons (Goodman and Williams 1976). GAD-immunocytochemistry showed essentially the same results, labelling the same small somas in the pars intercerebralis. However, recognition of the thin fibres was

786 not as good as with the GABA antibody probably due to the distribution of the G A D within the neurons.

Discussion

The electrophysiological and immunocytochemical data presented in this study taken together, exclude the possibility of a direct GABAergic feedback from L-neurons onto photoreceptors. They are therefore not consistent with the model introduced by Klingman and Chappell (1978) to explain the light response of L-neurons. They proposed a facilitatory GABAergic feedback from L-neuron dendrites onto photoreceptor terminals, responsible for the transient nature of the hyperpolarizing phase in the L-neuron light response and for the SOT after cessation of the light stimulus. The voltage-clamp experiments presented here (Figs. 3 and 4) show that there is most likely no relevant feedback from the L-neurons onto the photoreceptors. The synaptic currents measured in the voltageclamp mode closely resemble the normal light responses, while direct feedback from the L-neuron clamped is omitted in this situation. If the shape of the L-neuron light response were mainly the result of a photoreceptor-L-neuron feedback system, then the shape of the synaptic current in the voltage-clamp mode should be markedly different from the normal light response and should resemble the photoreceptor light response (Patterson and Goodman 1974). Of course we cannot exclude the possibility that non-clamped L-neurons feed back onto the same photoreceptors which synapse onto the clamped L-neuron and therefore the measured clamp currents reflect the feedback from the remaining Lneurons. However, two considerations argue against this possibility. From a morphological point of view it is very unlikely that the seven Lneurons in the median ocellus share input from the same 800-1,000 photoreceptors. In locusts, Goodman et al. (1979) showed that the dendritic arborizations of ML-type L-neurons are each largely confined to one of the subretinal halves of the ocellar cup. Thus L-neurons of this type receive input from different populations of photoreceptors. Even if feedback from non-clamped Lneurons were assumed, marked differences between the synaptic currents and the normal light responses should result. Since the large contribution of the L-neuron clamped is absent under voltage-clamp, the transient components of the synaptic currents should be largely reduced. In contrast

J. Ammermfillerand R. Weiler: GABAergicaction in the ocellus to that the transient components of the synaptic currents were enhanced in all experiments compared to the normal light response (Fig. 3). Finally, the subsequent immunocytochemical experiments demonstrating the lack of GABA immunoreactivity in the L-neurons exclude these neurons as sites of GABAergic feedback in locust ocetlus (Figs. 57). The question arises, whether dragonfly ocelli, on which Klingman and Chappell (1978) performed their experiments, and locust ocelli are comparable. There are good reasons to suppose they are. Pharmacological agents had similar effects on dragonfly and locust L-neurons (Klingman and Chappell 1978; Taylor 1981). The blocking effect of curare on the hyperpolarization of locust L-neurons (Fig. 1) is comparable to dragonfly, supporting the idea that acetylcholine is the transmitter released by the photoreceptor terminals in both species. Additional evidence comes from EM-studies, showing that the synaptic connectivity pattern in the two ocellar systems is essentially the same (Dowling and Chappell 1972; Goodman et al. 1979). Electrophysiological results obtained by Simmons (1982) with current injection into L-neurons and simultaneous recording from photoreceptors further support the physiological similarity and on the basis of his data he too questions the feedback model. On the other hand, GABA and its antagonists exerted a strong influence on the light responses of L-neurons and photoreceptors, even with the ocellar nerve cut (Klingman and Chappell 1978; Stone and Chappell 1981 ; Taylor 1981). Stone and Chappell (1981) showed in addition that antidromic stimulation of the whole ocellar nerve evoked hyperpolarizing responses in photoreceptors, indicating an input of second order cells onto photoreceptors. Our immunocytochemical results (Figs. 5-7) demonstrate that some of probable Sneurons are the most promising candidates for a GABAergic action within the ocellar plexiform layer and that L-neurons themselves are clearly not GABAergic. One plausible explanation of the observed pharmacological effects of GABA and its antagonists could involve a feedback system between photoreceptors and S-neurons. Indeed such a pathway has been anatomically demonstrated in locust (Mobbs 1978; Goodman et al. 1979; Goodman 1981). Electrophysiological recordings from assumed S-neurons are consistent with such an explanation. As it is shown in Fig. 8 the dependence of their spike-rate on light intensity showed ONOFF characteristics at medium intensities, while at low and high intensities the response pattern

J. Ammermtiller and R. Weiler: GABAergic action in the ocellus

.................. 2.7

13

L 0mv b

50 ms

Fig. 8a, b. Recordings from S-neurons. In a light responses to stimuli of different intensities, indicated at the right, are shown. In b light responses of a S-neuron and an L-neuron which were simultaneously recorded are compared. Relative light intensity - 2.1

was pure ON with a much longer spike train at high intensities. The latency decreased greatly with increasing intensity and the spike frequency became very phasic. The OFF-spikes at intensities of - 2 . 7 and - 1 . 5 coincided with the SOT of Lneurons (Fig. 2). One double recording from an L-neuron and an assumed S-neuron is shown in Fig. 8 b. In this case the plateau of the L-neuron and the spike frequency seemed to be correlated. Such recordings and further examples presented by Wilson (1978a) and Simmons (1982) demonstrate that S-neurons are plausible candidates for being involved in a feedback system within the ocellus. The consequences of such a feedback for the photoreceptor light response and the resulting transmitter release onto the second order neurons could be the same as in the model proposed by Klingman and Chappell (1978) although we do not know the mechanism at present. A model, where the S-neurons feed back onto the photoreceptors and where the L-neurons are only postsynaptic to the photoreceptors, is supported by the reversal potentials (Figs. 2 and 4). As the reversals of all phases of the L-neuron light response occur at approximately the same potential, it is likely that the same ionic mechanisms are involved. The simplest explanation for this is, that the same transmitter receptor system is responsible for all phases of the

787

L-neuron light response. We are aware that this simple model cannot explain the complete dynamic behaviour of the photoresponse of an L-neuron. In addition we will have to consider the mode of transmission between the photoreceptors and the L-neurons, the membrane properties of the L-neurons and additional local circuits within the ocellar plexiform layer, as well as efferent projections to it. Katz and Miledi (1971) showed, that the transmission at synapses in the squid stellate ganglion is markedly phasic, even with prolonged presynaptic depolarization. This would explain part of the cutback from the ON-transient to the plateau in the L-neuron light response. The L-neurons themselves show nonlinear and active membrane properties, includingnormal and anomalous rectification, as well as small action potentials, which play an important role in signal shaping (Wilson 1978b; Simmons 1982; Ammerm/iller 1984). Strong evidence for additional circuits comes from EM-studies where numerous synapses between Lneurones and S-neurones were observed in different species revealing separated zones of integration (Goodman et al. 1976; Goodman 1981; Toh and Sagara 1984). Physiological evidence for additional excitatory input onto L-neurons also comes from Fig. 1, where the hyperpolarizing phase of the light response is blocked by curare and a depolarizing influence, especially at light-off, becomes visible. Changing illumination of the three ocelli was found to have effects on the plateau and SOT in locust L-neurons (Taylor 1981). Simmons (1982) showed, that spikes in S-neurons can induce EPSPs in L-neurons. These additional inputs onto L-neurons could explain the slightly different reversal potentials of ON-transient and plateau (Fig. 2; Wilson 1978 b). The effects of S-neurons on photoreceptors and L-neuron light responses have been largely omitted in the literature, due to the difficulty of obtaining stable recordings from them. Rare examples of published S-neuron recordings show a great variability in their light responses, ranging from pure ON or OFF types to more complex ON-OFF responses (Fig. 8; Wilson 1978a; Simmons 1982). The demonstration of GABA in a fraction of probable S-neurons emphasizes their important role within the ocellar network and future analysis of signal transmission in this system should give them adequate attention. Acknowledgements. This work was supported by grants from the Deutsche Forschungsgemeinschaft to F. Zettler (Ze 115/9) and R. Weiler (SFB 220). We would like to t h a n k Dr. R. Douglas for critical reading of the manuscript.

788

References Ammermfiller J (1984) Membraneigenschaften nichtspikender L-Neurone im Ocellusnerv von Locusta migratoria L. Dissertation der Fakult/it ffir Biologic, Universit/it Mfinchen, Mtinchen Baylor DA, Fuortes MGF, O'Bryan PM (1971) Receptive fields of cones in the retina of the turtle. J Physiol 214:265-294 Chappell RL, Dowling JE (1972) Neural organization of the median ocellus of the dragonfly. I. Intracellular electrical activity. J Gen Physiol 60:121-147 Dowling JE, Chappell RL (1972) Neural organization of the median ocellus of the dragonfly. II. Synaptic structure. J Gen Physiol 60:148-161 Fuortes MGF, Simon EJ (1974) Interactions leading to horizontal cell response in the turtle retina. J Physiol 240:177-198 Goodman CS (1976) Anatomy of the ocellar interneurons of acridid grasshoppers. I. The large interneurons. Cell Tissue Res 175:183-202 Goodman CS, Williams JLD (1976) Anatomy of the ocellar interneurons of acridid grasshoppers. II. The small interneurons. Cell Tissue Res 175:203-225 Goodman LJ (1981) Organization and physiology of the insect dorsal ocellar system. In: Autrum H (ed) Vision in invertebrates. (Handbook of sensory physiology, vol VII/6C) Springer, Berlin Heidelberg New York, pp 201-286 Goodman LJ, Mobbs PG, Guy RG (1976) Information processing along the course of a visual interneuron. Experientia 33 : 748-750 Goodman LJ, Mobbs PG, Kirkham JB (1979) The fine structure of the ocelli of Sehistocerca gregaria: The neural organization of the synaptic plexus. Cell Tissue Res 196:487510 Hodgkin AL, Huxley AF, Katz B (1952) Measurements of current-voltage relations in the membrane of the giant axon of Loligo. J Physiol 116: 424-448 Katz B, Miledi R (1971) The effect of prolonged depolarizations on synaptic transfer in the stellate ganglion of the squid. J Physiol 216:503-512 Klingman A, Chappell RL (1978) Feedback synaptic interaction in the dragonfly ocellar retina. J Gen Physiol 71 : 157-175 Koontz MA, Edwards JS (1984) Central projections of firstorder ocellar interneurons in two orthopteroid insects


Acheta domesticus and Periplaneta americana. A comparative study. Cell Tissue Res 236:133 146 Lasater EM (1982) A white-noise analysis of responses and receptive fields of catfish cones. J Neurophysiol 47:1057-1068 L6scher W (1980) Comparative study of the inhibition of GABAaminotransferase by different anticonvulsant drugs. Arch Int Pharmacodyn 243:48-53 Mobbs PG (1978) Some aspects of the structure and development of the locust ocellus. PhD thesis, University of London, London Murakami M, Shimoda J, Nakatani K, Miyachi E, Watanabe S (1982) GABA mediated negative feedback and color opponency in carp retina. Jpn J Physiol 32:927-935 Patterson JA, Goodman LJ (1974) Intracellular responses of receptor cells and second-order cells in the ocelli of the desert locust Sehistocerca gregaria. J Comp Physio195:237-250 Rall W (1977) Core conductor theory and cable properties of neurons. In: Kandel ER (ed) The nervous system: Cellular biology of neurons (Handbook of physiology, sect I, part I, vol I) Am Physiol Soc, Bethesda, pp 39-98 Simmons PJ (1982) The operation of connections between photoreceptors and large second-order neurones in dragonfly ocelli. J Comp Physiol 149:389-398 Stell WK, Lightfoot DO (1975) Color specific interconnections of cones and horizontal cells in the retina of the goldfish. J Comp Neurol 159:473-502 Stell WK, Lightfoot DO, Wheeler TG, Leeper HF (1975) Goldfish retina: functional polarization of cone horizontal cell dendrites and synapses. Science 190:989-990 Stone SL, Chappell RL (1981) Synaptic feedback onto photoreceptors in the ocellar retina. Brain Res 221:374-381 Taylor CP (1981) Graded interactions between identified neurons from the simple eyes of an insect. Brain Res 215:382-387 Toh Y, Sagara H (1984) Dorsal ocellar system of the American cockroach. II. Structure of the ocellus and ocellar nerve. J Ultrastruct Res 86:119-134 Weiler R, Wagner HJ (1984) Light-dependent change of conehorizontal cell interactions in carp retina. Brain Res 298 : 1-9 Wilson M (1978 a) The functional organisation of locust ocelli. J Comp Physiol 124: 297-316 Wilson M (1978b) Generation of graded potential signals in the second order cells of locust ocellus. J Comp Physiol 124:317-331