Nudibranch Mollusc Hermissenda Crassicornis Research Articles

6-bromohypaphorine from marine nudibranch mollusk Hermissenda crassicornis is an agonist of human α7 nicotinic acetylcholine receptor.

6-Bromohypaphorine (6-BHP) has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR). Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of chicken α7 ligand-binding and glycine receptor transmembrane domains) or on rat α4β2 nAChR expressed in Xenopus oocytes revealed no action of 6-BHP. However, in radioligand analysis, 6-BHP competed with radioiodinated α-bungarotoxin for binding to human α7 nAChR expressed in GH4C1 cells (IC50 23 ± 1 μM), but showed no competition on muscle-type nAChR from Torpedo californica. In Ca2+-imaging experiments on the human α7 nAChR expressed in the Neuro2a cells, 6-BHP in the presence of PNU120596 behaved as an agonist (EC50 ~80 μM). To the best of our knowledge, 6-BHP is the first low-molecular weight compound from marine source which is an agonist of the nAChR subtype. This may have physiological importance because H. crassicornis, with its simple and tractable nervous system, is a convenient model system for studying the learning and memory processes.

PP1 inhibitors depolarize Hermissenda photoreceptors and reduce K+ currents.

Previous research indicates that activation of protein kinase C (PKC) plays a critical role in the induction and maintenance of memory-related changes in neural excitability of Type B photoreceptors in the eyes of nudibranch mollusk Hermissenda crassicornis (H.c.). The enhanced excitability of B cells is due in part to PKC-mediated reduction in somatic K+ currents. Here we examined the effects of protein phosphatase inhibitors on Type B photoreceptor excitability and K+ currents to determine the role(s) of protein phosphatases on memory formation in Hermissenda. Using electrophysiological and pharmacological methods, we found that the PP1 inhibitors calyculin A and inhibitor-2 depolarized Type B photoreceptors by 20-30 mV. A broad-spectrum kinase inhibitor, H7, blocked this effect. The depolarization induced by PP1 inhibition occluded that produced by an in vitro associative conditioning procedure. Calyculin and inhibitor-2 reduced the same B cell K+ currents (I(A) and I(delayed)) that are reduced by in vitro and behavioral conditioning. H7 blocked the reductions. Cantharidic acid (PP2A inhibitor) and cyclosporin (PP2B inhibitor) had negligible effects on B cell resting membrane potential, K+ currents, and in vitro conditioning-produced cumulative depolarization of B cells. These results suggest that the functional activity of K+ channels in B cells is sustained by basal activity of PP1. Inhibiting PP1 appears to allow one or more constitutively active kinase(s) to reduce K+ channel activity and thus mimic the effects of conditioning. Our results suggest that PP1 may oppose and/or constrain the extent of learning-produced changes in B cell excitability.

Competence and metamorphosis in the long-term planktotrophic larvae of the nudibranch mollusc Hermissenda crassicornis (Eschscholtz, 1831)

Hermissenda crassicornis is a eurytrophic carnivorous opisthobranch mollusc that possesses long-term planktotrophic larvae. No morphological criteria for determining larval competence in this slug are reliable; thus competence has been established by inducing metamorphosis with the hydroid Tubularia crocea. The competence period lasts approximately three weeks at 12–17°C, the best time for inducing metamorphosis being from days 31–50 within a given egg mass, and more precisely from day 41 to 50, as concluded from several egg masses. Within-clutch and between-clutch variability exists in both Tubularia-induced and spontaneous metamorphosis. Variability within egg masses in Hermissenda is probably due to the gradual acquisition of metamorphic competence. Spontaneous metamorphosis increases significantly as larvae age within a given egg mass. Metamorphosis is non-specific, since the larvae are able to metamorphose with several natural inducers, such as other hydroids ( Pennaria sp.) and anemones ( Metridium senile and Haliplanella luciae). Combinations of Tubularia with glutamate at 10 −4 M or with Mg 2+ at 20 mM significantly increased the metamorphic yields, while NH 4 + was ineffective. K + at 30 mM and Cs + at 1 mM also increased the metamorphic yields, although Cs + at 30 mM was toxic. Several combinations of the natural inducers and some cations also produced high metamorphic yields, up to 25%. The effect of epinephrine at 10 −5 M in increasing the metamorphic yields, when used in combination with Tubularia, was equal to that obtained when using aged epinephrine, indicating that the oxidized products of epinephrine are probably responsible for this activity. Increasing the temperature from 12°C up to 17°C also had a significant effect in increasing the metamorphic yields with several inducers. The extracts of Tubularia with several organic solvents did not have an inducing effect. The natural inducer from Tubularia seems to be a water-soluble compound. Aqueous extracts of Tubularia at high concentrations (about eight-times the normal amounts) produced yields up to 50±8.3% metamorphosis, i.e., five-times the maximum yields ever reported for Hermissenda.

CHEMOTAXIS IN THE NUDIBRANCH HERMISSENDA CRASSICORNIS: DOES INGESTIVE CONDITIONING INFLUENCE ITS BEHAVIOUR IN A Y-MAZE?

The nudibranch mollusc Hermissenda crassicornis can be cultured easily in the laboratory and has been used as a biomedical model for learning and memory studies. Cultured animals whose only prey was Tubularia crocea were tested for responses to other prey. Naive slugs were conditioned with six different diets and tested for behaviour in a Y-maze. H. crassicornis was able to detect chemotactically food items which it had never been in contact with (such as Pennaria), but it did not detect some of the conditioning diets (e.g. Metridium). At least three hydroid species induce a chemotactic behaviour in the slug. It is shown that ingestive conditioning does affect their response in single-choice and double-choice experiments. As food items other than cnidarians are also detected, it is likely that different Wairomones are responsible for the nudibranch's behaviour.

Biological factors affecting larval growth in the nudibranch mollusc Hermissenda crassicornis (Eschscholtz, 1831)

The nudibranch mollusc Hermissenda crassicornis is used as a biomedical model for studying learning and memory. It is an eurytrophic benthic species with long-term, planktotrophic larvae, and it has been cultured in our laboratory for several years. This paper reports the effects of some dietary factors on H. crassicornis larvae that were investigated in order to establish the conditions for optimal larval growth. Of the several treatments tested, densities of 1–4 larvae ml −1, a diet of Isochrysis galbana Parke and Rhodomonas salina (Wislouch) Butcher at a 1:1 mixture, and an algal density of 10–25×10 3 cells ml −1 yielded the largest, healthiest larvae, that underwent metamorphosis. Both diet quantity and quality, as well as larval density, had an effect on larval growth and metamorphosis of Hermissenda crassicornis in the laboratory.

Induction of metamorphosis inHermissenda crassicornislarvae (Molluscs: Nudibranchia) by GABA, choline and serotonin

Summary The nudibranch mollusc Hermissenda crassicornis is currently used as a biomedical model in neurobiological studies. It possesses planktotrophic larvae which metamorphose in the laboratory in the presence of the hydroid Tubularia crocea in yields of about 2–5% (Tamse et al., 1990). This study presents evidence that artificial metamorphic inducers such as GABA (γ-aminobutyric acid), choline, and serotonin (alone or combined with the natural inducer T. crocea), can be used to improve the metamorphic success in H. crassicornis larvae. GABA at 10−5M and 10−4M, choline at 10−3M and 10−4M, and serotonin at 10−5 M and 10−4M, were most effective, while serotonin at 10−3 M was toxic. Larvae 47 and 63 days old metamorphosed at different rates when exposed to the same concentrations of chemicals, but these had no positive effect on young larvae, i.e., 28 and 39 days old. On the other hand, when young larvae were exposed to the natural inducer, an habituation phenomenon was observed, i.e., the larvae did not m...

Food Detection and Preferences of the Nudibranch Mollusc Hermissenda crassicornis: Experiments in a Y-Maze

Previous articleNext article No AccessBEHAVIORFood Detection and Preferences of the Nudibranch Mollusc Hermissenda crassicornis: Experiments in a Y-MazeE. Tyndale, C. Avila, and A. M. KuzirianE. Tyndale Search for more articles by this author , C. Avila Search for more articles by this author , and A. M. Kuzirian Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Biological Bulletin Volume 187, Number 2October 1994 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.1086/BBLv187n2p274 Views: 93Total views on this site Copyright © 1994 by Marine Biological LaboratoryPDF download Crossref reports no articles citing this article.

Sequential modification of membrane currents with classical conditioning

Pavlovian conditioning of the nudibranch mollusc Hermissenda crassicornis was previously shown to produce long-lasting reduction of two K+ currents measured across the Type B photoreceptor soma membrane (Alkon et al., 1982a; Alkon et al., 1985). Pavlovian conditioning of the rabbit was also shown to be followed by persistent K+ current reduction (Disterhoft et al., 1986). Here we report the first evidence that Ca2+ currents can also be modified by conditioning. The amplitude of the currents rather than their voltage-dependence remains reduced at least 1-2 d after conditioning (but not control procedures). Conditioning-induced changes of both K+ and Ca2+ currents increased as a function of training, the Ca2+ currents only changing substantially with greater than or equal to 250 trials. The later changes of the Ca2+ current may function to limit the magnitude of excitability increases due to associative learning.

Effects of alpha 2-adrenergic agonists and antagonists on photoreceptor membrane currents.

Type B photoreceptors of the nudibranch mollusc Hermissenda crassicornis receive excitatory synaptic potentials (EPSPs) whose frequency is controlled by potential changes of a neighboring cell known as the S optic ganglion cell which is thought to be electrically coupled to the presynaptic source of these EPSPs, the E optic ganglion cell. The frequency of the EPSPs increases when a conditioned stimulus (light) is paired with an unconditioned stimulus (rotation) during acquisition of a Pavlovian conditioned response. The results of the present study are consistent with an adrenergic origin for these EPSPs. Noradrenergic agonists (greater than 100 microM), norepinephrine and clonidine, only slightly depolarize the type B cell but clearly prolong its depolarizing response to light. Serotonin, by contrast, causes hyperpolarization of the type B cell's resting potential as well as after a light step. Clonidine reduces voltage-dependent outward K+ currents (IA, an early current, ICa2+-K+, a late Ca2+-dependent current) that control the type B cell's excitability (and thus its light response and membrane potential). These effects of clonidine are reduced or blocked by the alpha 2-receptor antagonist, yohimbine (0.5 microM), but not the alpha 1-blocker, prazosin. The same yohimbine concentration also blocked depolarizing synaptic excitation of the type B cell in response to depolarization of a simultaneously impaled S optic ganglion cell. Histochemical techniques (both the glyoxylic acid method of de la Torre and Surgeon and the formaldehyde-induced fluorescence or Falck-Hillarp method) demonstrated the presence of a biogenic amine(s) within a single neuron in each optic ganglion as well as three or four cells within the vicinity of previously identified visual interneurons. No serotonergic neurons were found within the optic ganglion or in proximity to visual interneurons. A clonidine-like synaptic effect on type B cells, therefore, could amplify conditioning-specific changes of membrane currents by increasing type B depolarization and possibly, as well, by elevating intracellular second messengers.

Autoradiographic measurement of tritiated agmatine as an indicator of physiologic activity inHermissenda visual and vestibular neurons

[3H]Agmatine (amino-4-guanidobutane) has been shown to be potentially useful for identifying and assessing the ACh sensitivity of specific neurons. Small cationic amines are able to permeate ACh-activated ion channels in sympathetic neurons and vertebrate endplates. Sensory neurons of the photic pathway in the nudibranch mollusc Hermissenda crassicornis are cholinergic and the synaptic interactions between the photic and vestibular systems have been well characterized electrophysiologically. We have therefore tested the feasibility of using autoradiography with [3H]agmatine, (a) to identify known ACh-responsive postsynaptic cells and (b) to examine its ability to serve as an indicator of physiologic activity within the photic and vestibular pathways under conditions of darkness and light stimulation. Scintillation counting revealed that approximately 70% of the radioactivity was associated with the CNS while approximately 30% was found in the processing fluids, indicating that routine glutaraldehyde-osmium fixation and subsequent processing for epoxy embedding allows retention of substantial amounts of the radiolabel. The autoradiographic results consistently demonstrated that the uptake patterns for [3H]agmatine did reflect some of the known neuronal interactions under the experimental conditions of light and dark. The accuracy extended to the second order cells of the optic ganglion and to putative interneurons along the photic tract in the cerebropleural ganglion. Since all the neurons in these pathways are unipolar with their synaptic interactions occurring only at the terminal endings, the radiolabel accumulated in the somata resulted from retrograde axonal transport. In the photic-vestibular pathways, the highest silver grain densities were found over structures (cell bodies or axon tracts) with increased synaptic activity coupled with higher levels of cellular activity (i.e. increased excitatory postsynaptic potentials or increased spontaneous impulse activity). Slightly less label was found in cells which received increased numbers of inhibitory postsynaptic potentials that produced hyperpolarization and a transient cessation of impulse activity under conditions of illumination. Therefore, the uptake levels of [3H]agmatine as revealed by autoradiography appear to reflect not only changes in sensitivity or density of ACh-activated channels but also changes in cellular activity as indicated by increased amounts of retrograde transport. These results represent the first example of the effective use of this radiolabel as an indicator of synaptic activity in invertebrates and in sensory systems.

INDIVIDUAL VARIATION IN ASSOCIATIVE LEARNING OF THE NUDIBRANCH MOLLUSCHERMISSENDA CRASSICORNIS

Retention of learned suppression of positive phototaxis in the nudibranch mollusc Hermissenda crassicornis, induced by exposure to trials of paired light and rotation, was determined for individuals within groups trained in two, three, four, and six daily sessions of 100 trials each. Significant increases in latency to light (acquisition) were measured within all paired treatment groups when these were tested before treatment and 24 hours after the last session. No significant differences in latency were found within four unpaired and one random control group. Next, retention of phototactic suppression (increased latency to respond to light) for each individual was assessed by comparing its post-treatment suppression ratio (SR) scores to a population median score derived from the frequency distribution of scores from a naive group of animals repeatedly tested over a 31-day period. Retention, defined as the consecutive number of days post-treatment on which an animal's SR scores were suppressed below the population median score, was significantly longer in groups trained four and six days than in the two- and three-day paired treatment groups. When retention day score distributions from paired groups were compared to those from the unpaired and random control groups, a significant increase in phototactic suppression was found only for groups trained four and six days. Maximum retention, or resistance to extinction, was measured at 17-18 days (one animal) after 6 sessions. All paired treatments contained animals which did not acquire the association. Retention increased with experience (number of sessions) and the number of animals per group which showed no acquisition decreased.Investigations on the neural correlates of this behavioral change in Hermissenda are currently in progress; an understanding of the relationship between the degree of phototactic suppression in a sample of animals and the number of training sessions will aid in design and interpretation of experiments in which biophysical and biochemical data are correlated with behavioral measures.

Extinction of associative learning in Hermissenda: Behavior and neural correlates

The nudibranch mollusk Hermissenda crassicornis' normal attraction to light can be suppressed by repeated pairings of light with rotation. The present study examines the effects of extinction procedures on this simple form of associative learning. Presentation of non-reinforced light steps following associative conditioning resulted in an attenuation of phototaxic suppression, evident at both short- and long-term retention intervals. The absence of habituation of phototaxic behavior, coupled with the failure to demonstrate spontaneous recovery of extinguished conditioned suppression, indicates that extinction of associative conditioning in Hermissenda depends little upon non-associative learning processes. Electrophysiological evidence indicates that the Type B photoreceptors, which have been causally implicated in the acquisition and retention of associative learning, play an important role in mediating extinction as well. Enhanced input resistances and light responses of B cells, which are produced by associative training, are absent for animals subsequently exposed to light unaccompanied by rotation. In terms of both behavioral and electrophysiological measures, extinction appears to result primarily from a reversal of the original acquisition process.

Protein phosphorylation/dephosphorylation and the transient, voltage-dependent potassium conductance in Hermissenda crassicornis.

Recent interest in the modulation of ionic conductances in excitable membranes, particularly K+ conductances, has focused on biochemical mechanisms such as protein phosphorylation. Several distinct types of voltage-dependent K+ currents (e.g. transient, delayed, and Ca2+-dependent) can be separated by electrophysiological and pharmacological means. We have found that two treatments, 4-aminopyridine and high external K+, which preferentially reduce the transient K+ current also reduce the level of 32P incorporation in a 25,000 molecular weight phosphoprotein band in eyes and ganglia of the nudibranch mollusc Hermissenda crassicornis. The effect of agents which reduce the transient K+ current is relatively specific because 32P incorporation in this band is not significantly affected by Ba2+ which preferentially blocks the delayed K+ current, or by Ni2+, which blocks the Ca2+ channel and in turn inhibits the Ca2+-dependent K+ current. Additional studies indicate that a change in endogenous impulse activity is not required for the expression of the 4-aminopyridine effect on protein phosphorylation. The concentration dependence and time course of the 4-aminopyridine effect on 32P incorporation are in reasonable agreement with those reported for the blockade of the transient K+ current by 4-aminopyridine. Following removal of 4-aminopyridine, 32P incorporation increases in the 25,000 molecular weight phosphoprotein band and in a 23,000 molecular weight Ca2+-stimulated phosphoprotein band. The data presented here suggest that there is a relationship between the transient K+ current and the state of phosphorylation of specific neuronal proteins in Hermissenda and point to further studies to identify the phosphoproteins and the mechanism which leads to the observed changes in protein phosphorylation.

Primary changes of membrane currents during retention of associative learning.

A single identified neuron was repeatedly isolated by axotomy from the central nervous system of the nudibranch mollusk Hermissenda crassicornis. An early voltage-dependent outward K+ current of this neuron was reduced and more rapidly inactivated for animals previously trained with paired but not randomized light and rotation. Since this current change can affect interneuron and motorneuron output via known synaptic pathways, it helps explain a long-lasting behavioral change that shows the defining features of vertebrate associative learning.

Change in a specific phosphoprotein band following associative learning in Hermissenda.

Although cellular neurophysiological studies of invertebrates have provided some understanding of the cellular basis of modifiable behaviour (for reviews, see refs 1–5), little is known about biochemical changes in the specific neural pathways accompanying alterations in behaviour. The nervous systems and sensory structures of invertebrates are especially suitable to an analysis of the biochemical mechanisms involved in behavioural modification because they typically contain a relatively small number of neurones, some of which have large, identifiable somata, and because associative learning has been studied in several invertebrate preparations6–10. Studies with the nudibranch mollusc Hermissenda crassicornis have demonstrated a short-term non-associative change in phototactic behaviour after training with light and rotation11. More recently8,12, an example of associative learning in Hermissenda was produced by the temporal association of light and a presumed aversive stimulus. This associative learning is accompanied by neurophysiological changes in the B-type photoreceptors12. We now report that there is also a change in the level of incorporation of 32P in a 20,000-molecular weight (MW) phosphoprotein; as the eye of Hermissenda consists of only five photoreceptors, a lens and a few pigment and epithelial cells, a biochemical change specific to associative learning has thus been localized to a few cells within a nervous system.

An infraciliary network in statocyst hair cells.

Ultrastructural analysis of the statocyst, a primitive vestibular organ, of the nudibranch mollusc Hermissenda crassicornis, indicates that in addition to the basal foot, there is an infraciliary rootlet system between basal bodies of adjacent sensory cilia. These rootlets project perpendicularly from the basal bodies and parallel to the cell surface in an astral array. A polarity within the network also appears to exist; the array is longest and most extensive on the side of the basal body directed away from the cell centre, but the overall arrangement of the basal bodies indicates a multidirectional sensitivity for each of the 13 sensory cells. This rootlet system, in conjunction with the attachment system of the basal bodies to the cell membrane (button anchors), may serve an integrative function for the mechanical stimuli experienced by sensory cells and/or be involved with their transductive processes by maximizing the stress to, and membrane distortion of, the transductive site caused by weighting of the cilia. Evidence was also obtained for the intracellular synthesis of statoconia by the nonsensory supporting cells.

CELLULAR ANALYSIS OF A GASTROPOD (HERMISSENDA CRASSICORNIS) MODEL OF ASSOCIATIVE LEARNING

Intuitive formulations concerning the nature of cellular substrates for associative learning are considered. Invertebrate models of vertebrate learning are discussed. A cellular analysis of a long-term associative behavioral change is reviewed for the nudibranch mollusc Hermissenda crassicornis. Convergence of neuronal pathways which mediate natural sensory stimuli used for associative training have been determined. Primary biophysical changes within single identified neurons are analyzed for their causal role in learning behavior. A sequence of neural processes is proposed to underlie acquisition and retention of an associative behavioral change of Hermissenda. Paired sensory stimuli enhance voltage-dependent currents by means of converging sensory pathways. Repeated pairings cause progressive membrane depolarization, which in turn causes long-lasting conductance changes. Biochemical correlates of these phenomena are presented.

Motile statocyst cilia transmit rather than directly transduce mechanical stimuli.

We have investigated the role of motile cilia in mechanotransduction by statocysts of the nudibranch mollusk Hermissenda crassicornis. Movement of the cilia that experience the weight of statoconia causes increased variance of voltage noise and membrane depolarization of the statocyst hair cell. Two complementary approaches were used to immobilize the cilia. Vanadate anion was iontophoretically injected into hair cells. This reversible inhibitor of vibratile form and to assume a more classic, pliable beat pattern. Voltage noise decreased as the cilia slowed and bent more extremely, nearly disappearing as motility was lost. When the intracellular vanadate concentration approached 10(-5) M, the cilia were arrested in an effective stroke against the cell membrane. The cell no longer depolarized upon gravitational or local mechanical stimulation. Rapid reversal of ciliary inhibition by norepinephrine or slow reversal with time restored both the voltage noise and depolarization response. Cilia were rendered rigid and upright by covalent cross-linkage of their membrane "sleeve" to the 9 + 2 axoneme, using the photoactivated, lipophilic, bifunctional agent 4,4'-dithiobisphenyl azide. In the initial stages of cross-linkage, the cilia remained vibratile but slowed and moved through wider excursions. Voltage noise decreased in frequency but increased in amplitude. When the cilia were fully arrested, voltage noise was minimized while the resting potential and membrane resistance remained essentially constant. Mechanical stimulation of the rigid cilia, normal to the cell membrane, elicited a generator potential of the same amplitude but of greater duration than before treatment. Because cilia that are partially arrested by vanadate undergo increased bending, although the hair cell shows decreased noise, neither the axoneme nor the ciliary membrane proper would appear to be sites of direct transduction. In cells with beating but stiffened cilia, however, the voltage noise becomes amplified, implying an increased efficiency of transduction. We suggest that active but rigid flexure of the axoneme is involved in amplification and continuous signal detection. The basal insertion area is the most likely transduction site, being the terminal leverage point through which force is applied to the plasma membrane via the flexing ciliary shaft.

Ultrastructure of photoreceptors in the eye of Hermissenda labelled with intracellular injections of horseradish peroxidase.

The terminal processes of single and of pairs of identified photoreceptors in the eyes of the nudibranch mollusc Hermissenda crassicornis were studied by light and transmission electron microscopy after their somata were labelled by intracellular iontophoresis of horseradish peroxidase (HRP). The HRP spread from the somata into the axons and fine terminal processes within the neuropil of the cerebropleural ganglia. The photoreceptors ended in extensive secondary branches in the neuropil where previous electrophysiological studies had indicated the probable site of synaptic interactions between photoreceptors. Clear round vesicles (54-126 nm diameter) within labelled processes were similar to the vesicles found in the somata and axon hillocks. The terminal processes of pairs of type B photoreceptors contained different intensities of the HRP label. Uniform intensities of the HRP label were found in the terminal processes of single type B photoreceptors. These differences in intensity suggested that the terminal processes were from different type B photoreceptors. This finding suggests that the connections between type B photoreceptors are probably monosynaptic.

Cholinergic features of photoreceptor synapses in Hermissenda.

1. A number of observations, as listed below, suggested a cholinergic basis for inhibitory interactions between photoreceptors of the eye in the nudibranch mollusk Hermissenda crassicornis. 2. The isolated eyes synthesized and accumulated acetylcholine but not other putative neurotransmitter substances. Synthesis and accumulation were determined by electrophoretic separation of products that incorporated radioactive label. Electron microscopic visualization of clear round vesicles within the photoreceptors' somata and axon hillocks was consistent with synthesis and storage of acetylcholine within these cells. 3. Pharmacologic experiments indicated the presence of cholinergic receptors on the terminal branches of the photoreceptors, which are pre- and postsynaptic to each other. Carbachol or nicotine produced hyperpolarization of the photoreceptors' membrane accompanied by a reduction of the input resistance. The reversal potential of carbachol-induced hyperpolarization coincided with the reversal potentials of the IPSPs that followed, one for one, impulses of neighboring photoreceptors. Eserine often caused blockade of the IPSPs. This blockade was associated with substantial membrane hyperpolarization and reduction of membrane resistance. 4. Neuronal endings within the optic tract in the area of the photoreceptor's terminal branches stained for acetylcholinesterase. 5. The results of these different experiments, especially when considered together, strongly suggest, although by no means unequivocally demonstrate, that the neurotransmitter of the photoreceptors is acetylcholine.