Effect of 2-amino-4-phosphonobutyrate on the OFF responses of frog retinal ganglion cells and local ERG after glycinergic blockade

Perfusion with the ON channel blocker 2-amino-4-phosphonobutyrate (APB) of dark adapted frog eyecups not only abolished the ganglion cells' (GC) ON responses and the ERG b-wave, but markedly potentiated the OFF responses of ON-OFF and phasic OFF-GCs and the d-wave amplitude of simultaneously recorded local ERG. Glycinergic blockade by strychnine prevented this potentiating effect in 31 out of 69 GCs, but did not change it at all in the other cells. At the same time the d-wave potentiation was preserved during the glycinergic blockade in all eyecups. The results indicate that glycinergic transmission is involved in the inhibition exerted from ON upon OFF channel in some but not all frog retinal GCs.     

The oscillation-like activity in bullfrog ON–OFF retinal ganglion cell

Oscillatory activity of retinal ganglion cell (RGC) has been observed in various species. It was reported such oscillatory activity is raised within large neural network and involved in retinal information coding. In the present research, we found an oscillation-like activity in ON–OFF RGC of bullfrog retina, and studied the mechanisms underlying the ON and OFF activities respectively. Pharmacological experiments revealed that the oscillation-like activity patterns in both ON and OFF pathways were abolished by GABA receptor antagonists, indicating GABAergic inhibition is essential for generating them. At the meantime, such activities in the ON and OFF pathways showed different responses to several other applied drugs. The oscillation-like pattern in the OFF pathway was abolished by glycine receptor antagonist or gap junction blocker, whereas that in the ON pathway was not affected. Furthermore, the blockade of the ON pathway by metabotropic glutamate receptor agonist led to suppression of the oscillation-like pattern in the OFF pathway. These results suggest that the ON pathway has modulatory effect on the oscillation-like activity in the OFF pathway. Therefore, the mechanisms underlying the oscillation-like activities in the ON and OFF pathways are different: the oscillation-like activity in the ON pathway is likely caused by GABAergic amacrine cell network, while that in the OFF pathway needs the contributions of GABAergic and glycinergic amacrine cell network, as well as gap junction connections.     

Crizotinib-Induced Abnormal Signal Processing in the Retina

Molecular target therapy for cancer is characterized by unique adverse effects that are not usually observed with cytotoxic chemotherapy. For example, the anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor crizotinib causes characteristic visual disturbances, whereas such effects are rare when another ALK-tyrosine kinase inhibitor, alectinib, is used. To elucidate the mechanism responsible for these visual disturbances, the responses to light exhibited by retinal ganglion cells treated with these agents were evaluated using a C57BL6 mouse ex vivo model. Both crizotinib and alectinib changed the firing rate of ON and OFF type retinal ganglion cells. However, the ratio of alectinib-affected cells (15.7%) was significantly lower than that of crizotinib-affected cells (38.6%). Furthermore, these drugs changed the response properties to light stimuli of retinal ganglion cells in some of the affected cells, i.e., OFF cells responded to both ON and OFF stimuli, etc. Finally, the expressions of ALK (a target receptor of both crizotinib and alectinib) and of MET and ROS1 (additional target receptors of crizotinib) were observed at the mRNA level in the retina. Our findings suggest that these drugs might target retinal ganglion cells and that the potency of the drug actions on the light responses of retinal ganglion cells might be responsible for the difference in the frequencies of visual disturbances observed between patients treated with crizotinib and those treated with alectinib. The present experimental system might be useful for screening new molecular target agents prior to their use in clinical trials.     

Effect of GABAergic inhibition on odorant concentration coding in mushroom body intrinsic neurons of the honeybee

Kenyon cells, the intrinsic neurons of the insect mushroom body, have the intriguing property of responding in a sparse way to odorants. Sparse neuronal codes are often invariant to changes in stimulus intensity and duration, and sparse coding often depends on global inhibition. We tested if this is the case for honeybees’ Kenyon cells, too, and used in vivo Ca²⁺ imaging to record their responses to different odorant concentrations. Kenyon cells responded not only to the onset of odorant stimuli (ON responses), but also to their termination (OFF responses). Both, ON and OFF responses increased with increasing odorant concentration. ON responses were phasic and invariant to the duration of odorant stimuli, while OFF responses increased with increasing odorant duration. Pharmacological blocking of GABA receptors in the brain revealed that ionotropic GABA_A and metabotropic GABA_B receptors attenuate Kenyon cells’ ON responses without changing their OFF responses. Ionotropic GABA_A receptors attenuated Kenyon cell ON responses more strongly than metabotropic GABA_B receptors. However, the response dynamic, temporal resolution and paired-pulse depression did not depend on GABA_A transmission. These data are discussed in the context of mechanisms leading to sparse coding in Kenyon cells.     

Response dynamics of bullfrog ON-OFF RGCs to different stimulus durations

Stimulus duration is an important feature of visual stimulation. In the present study, response properties of bullfrog ON-OFF retinal ganglion cells (RGCs) in exposure to different visual stimulus durations were studied. By using a multi-electrode recording system, spike discharges from ON-OFF RGCs were simultaneously recorded, and the cells’ ON and OFF responses were analyzed. It was found that the ON response characteristics, including response latency, spike count, as well as correlated activity and relative latency between pair-wise cells, were modulated by different light OFF intervals, while the OFF response characteristics were modulated by different light ON durations. Stimulus information carried by the ON and OFF responses was then analyzed, and it was found that information about different light ON durations was more carried by transient OFF response, whereas information about different light OFF intervals were more carried by transient ON response. Meanwhile, more than 80 % information about stimulus durations was carried by firing rate. These results suggest that ON-OFF RGCs are sensitive to different stimulus durations, and they can efficiently encode the information about visual stimulus duration by firing rate.     

Non-peptidergic small diameter primary afferents expressing VGluT2 project to lamina I of mouse spinal dorsal horn

The aim of this study was to investigate the expression of prostaglandin EP1 receptor within the ventrolateral periaqueductal grey (VL PAG). The role of VL PAG EP1 receptor in controlling thermonociception and rostral ventromedial medulla (RVM) activity in healthy and neuropathic rats was also examined. EP1 receptor was indeed found to be expressed within the VL PAG and co-localized with vesicular GABA transporter. Intra-VL PAG microinjection of ONO-DI-004, a selective EP1 receptor agonist, dose-dependently reduced tail flick latency as well as respectively increasing and decreasing the spontaneous activity of ON and OFF cells. Furthermore, it increased the ON cell burst and OFF cell pause. Intra-VL PAG prostaglandin E2 (PGE2) behaved similarly to ONO-DI-004. The effects of ONO-DI-004 and PGE2 were antagonized by intra-VL PAG L335677, a selective EP1 receptor antagonist. L335677 dose-dependently increased the tail flick latency and ongoing activity of the OFF cells, while reducing the ongoing ON cell activity. It also decreased the ON cell burst and OFF cell pause. In neuropathic rats using spare nerve injury (SNI) of the sciatic nerve model, EP1 receptor expression decreased in the VL PAG. However, ONO-DI-004 and L335677 were able to alter pain responses and ON and OFF cell activity, as they did in healthy animals. Collectively, these data show that within the VL PAG, EP1 receptor has a facilitatory effect on the nociceptive response and consistently affects RVM neuron activity. Thus, the blockade of EP1 receptor in the VL PAG leads to antinociception in neuropathic pain conditions, despite its down-regulation. The expression of EP1 receptor on GABAergic neurons is consistent with an EP1 receptor blockade-induced disinhibition of the antinociceptive descending pathway at VL PAG level.     

Analysis of synaptic inputs to on-off amacrine cells of the carp retina

To elucidate the synaptic transmission between bipolar cells and amacrine cells, the effect of polarization of a bipolar cell on an amacrine cell was examined by simultaneous intracellular recordings from both cells in the isolated carp retina. When either an ON or OFF bipolar cell was depolarized by an extrinsic current step, an ON-OFF amacrine cell was transiently depolarized at the onset of the current but no sustained polarization during the current was detected. The current hyperpolarizing the OFF bipolar cell also produced the transient depolarization of the amacrine cell at the termination of the current. These responses had a latency of approximately 10 ms. The amplitude of the current-evoked responses changed gradually with current intensity within the range used in these experiments. They were affected by polarization of the amacrine cell membrane; the amplitude of the current-evoked responses as well as the light-evoked responses was increased when the amacrine cell membrane was hyperpolarized, while the amplitude was decreased when the cell was depolarized. These results confirm directly that ON-OFF amacrine cells receive excitatory inputs from both ON and OFF bipolar cells: the ON transient is due to inputs from ON bipolar cells, and the OFF transient to inputs from OFF bipolar cells. The steady polarization of bipolar cells is converted into transient signals during the synaptic process.     

Two-photon imaging reveals somatodendritic chloride gradient in retinal ON-type bipolar cells expressing the biosensor Clomeleon

A somatodendritic gradient of Cl(-) concentration ([Cl(-)](i)) has been postulated to generate GABA-evoked responses of different polarity in retinal bipolar cells, hyperpolarizing in OFF cells with low dendritic [Cl(-)](i), and depolarizing in ON cells with high dendritic [Cl(-)](i). As glutamate released by the photoreceptors depolarizes OFF cells and hyperpolarizes ON cells, the bipolars' antagonistic receptive field (RF) could be computed by simply integrating glutamatergic inputs from the RF center and GABAergic inputs from horizontal cells in the RF surround. Using ratiometric two-photon imaging of Clomeleon, a Cl(-) indicator transgenically expressed in ON bipolar cells, we found that dendritic [Cl(-)](i) exceeds somatic [Cl(-)](i) by up to 20 mM and that GABA application can lead to Cl(-) efflux (depolarization) in these dendrites. Blockers of Cl(-) transporters reduced the somatodendritic [Cl(-)](i) gradient. Hence, our results support the idea that ON bipolar cells employ a somatodendritic [Cl(-)](i) gradient to invert GABAergic horizontal cell input.     

Kinetics of intramembrane charge movement and conductance activation of batrachotoxin-modified sodium channels in frog node of Ranvier

Sodium current and intramembrane gating charge movement (Q) were monitored in voltage-clamped frog node of Ranvier after modification of all sodium channels by batrachotoxin (BTX). Sodium current activation followed a single-exponential time course, provided a delay was interposed between the onset of the step ON depolarization and that of the current change. The delay decreased with increased ON depolarization and, for a constant ON depolarization, increased with prehyperpolarization. ON charge movement followed a single-exponential time course with time constants tau Q,ON slightly larger than tau Na, ON. For pulses between -70 and -50 mV, tau Q,ON/tau Na,ON = 1.14 +/- 0.08. The OFF charge movement and OFF sodium current tails after a depolarizing pulse followed single-exponential time courses, with tau Q, OFF larger than tau Na, OFF. tau Q,OFF/tau Na,OFF increased with OFF voltage from 1 near -100 mV to 2 near -160 mV. At a set OFF potential (-120 mV), both tau Q,OFF and tau Na,OFF increased with ON pulse duration. The delay in INa activation and the effect of ON pulse duration on tau Q,OFF and tau Na,OFF are inconsistent with a simple two-state, single-transition model for the gating of batrachotoxin-modified sodium channels.     

Non-centered spike-triggered covariance analysis reveals neurotrophin-3 as a developmental regulator of receptive field properties of ON-OFF retinal ganglion cells

The functional separation of ON and OFF pathways, one of the fundamental features of the visual system, starts in the retina. During postnatal development, some retinal ganglion cells (RGCs) whose dendrites arborize in both ON and OFF sublaminae of the inner plexiform layer transform into RGCs with dendrites that monostratify in either the ON or OFF sublamina, acquiring final dendritic morphology in a subtype-dependent manner. Little is known about how the receptive field (RF) properties of ON, OFF, and ON-OFF RGCs mature during this time because of the lack of a reliable and efficient method to classify RGCs into these subtypes. To address this deficiency, we developed an innovative variant of Spike Triggered Covariance (STC) analysis, which we term Spike Triggered Covariance - Non-Centered (STC-NC) analysis. Using a multi-electrode array (MEA), we recorded the responses of a large population of mouse RGCs to a Gaussian white noise stimulus. As expected, the Spike-Triggered Average (STA) fails to identify responses driven by symmetric static nonlinearities such as those that underlie ON-OFF center RGC behavior. The STC-NC technique, in contrast, provides an efficient means to identify ON-OFF responses and quantify their RF center sizes accurately. Using this new tool, we find that RGCs gradually develop sensitivity to focal stimulation after eye opening, that the percentage of ON-OFF center cells decreases with age, and that RF centers of ON and ON-OFF cells become smaller. Importantly, we demonstrate for the first time that neurotrophin-3 (NT-3) regulates the development of physiological properties of ON-OFF center RGCs. Overexpression of NT-3 leads to the precocious maturation of RGC responsiveness and accelerates the developmental decrease of RF center size in ON-OFF cells. In summary, our study introduces STC-NC analysis which successfully identifies subtype RGCs and demonstrates how RF development relates to a neurotrophic driver in the retina.     

Presynaptic inhibition modulates spillover, creating distinct dynamic response ranges of sensory output

Sensory information is thought to be modulated by presynaptic inhibition. Although this form of inhibition is a well-studied phenomenon, it is still unclear what role it plays in shaping sensory signals in intact circuits. By visually stimulating the retinas of transgenic mice lacking GABAc receptor-mediated presynaptic inhibition, we found that this inhibition regulated the dynamic range of ganglion cell (GC) output to the brain. Presynaptic inhibition acted differentially upon two major retinal pathways; its elimination affected GC responses to increments, but not decrements, in light intensity across the visual scene. The GC dynamic response ranges were different because presynaptic inhibition limited glutamate release from ON, but not OFF, bipolar cells, which modulate the extent of glutamate spillover and activation of perisynaptic NMDA receptors at ON GCs. Our results establish a role for presynaptic inhibitory control of spillover in determining sensory output in the CNS.     

Responses of recurrent nets of asymmetric ON and OFF cells

A neural field model of ON and OFF cells with all-to-all inhibitory feedback is investigated. External spatiotemporal stimuli drive the ON and OFF cells with, respectively, direct and inverted polarity. The dynamic differences between networks built of ON and OFF cells (“ON/OFF”) and those having only ON cells (“ON/ON”) are described for the general case where ON and OFF cells can have different spontaneous firing rates; this asymmetric case is generic. Neural responses to nonhomogeneous static and time-periodic inputs are analyzed in regimes close to and away from self-oscillation. Static stimuli can cause oscillatory behavior for certain asymmetry levels. Time-periodic stimuli expose dynamical differences between ON/OFF and ON/ON nets. Outside the stimulated region, we show that ON/OFF nets exhibit frequency doubling, while ON/ON nets cannot. On the other hand, ON/ON networks show antiphase responses between stimulated and unstimulated regions, an effect that does not rely on specific receptive field circuitry. An analysis of the resonance properties of both net types reveals that ON/OFF nets exhibit larger response amplitude. Numerical simulations of the neural field models agree with theoretical predictions for localized static and time-periodic forcing. This is also the case for simulations of a network of noisy integrate-and-fire neurons. We finally discuss the application of the model to the electrosensory system and to frequency-doubling effects in retina.     

Directional selectivity is formed at multiple levels by laterally offset inhibition in the rabbit retina

The excitatory and inhibitory inputs to directionally selective (DS) ganglion cells are themselves directionally selective. Directionality is achieved because excitation is reduced during null-direction movement along a GABAergic pathway. Inhibition is reduced during preferred-direction movement along a pathway that includes cholinergic synapses. Both excitation and inhibition are made directional by laterally offset inhibitory signals similar to the spatial offset of the direct inhibitory input to the DS cell dendrites. Thus, spatially offset lateral inhibition generates directionality at three different levels in the DS circuitry. We also found that for stimuli falling within the dendritic field, cholinergic input is delivered to the OFF but not the ON dendrites. Cholinergic pathways from outside the dendritic field reach both ON and OFF dendrites, but both of these pathways are normally inactivated by GABAergic synapses.     

Cell-type specific dendritic contacts between retinal ganglion cells during development.

The extent of a neuron's dendritic field defines the region within which information is processed. The dendritic fields of functionally distinct ON and OFF center retinal ganglion cells (RGCs) form separate mosaics across the retina. Within each mosaic, neighboring dendritic fields overlap by a constant amount, sampling the visual field with the appropriate coverage. Contact-mediated lateral inhibition between neighboring RGCs has long been thought to regulate both the extent and overlap of dendritic fields during development. Here we show that dendro-dendritic contact exists between developing RGCs and occurs in a manner that would regulate the formation of ON and OFF mosaics separately. Dye-filled neighboring ON and OFF ferret alpha RGCs were reconstructed using multiphoton microscopy. At all neonatal ages examined, we observed dendro-dendritic contacts between RGCs of the same sign (ON/ON; OFF/OFF), but never between cells of opposite signs (ON/OFF). Terminal dendrites of one cell often touched a dendrite of its neighbor as they intersected. In some instances, the distal dendrite of one cell formed a fascicle with the proximal process of its neighbor. Alpha cells did not form contacts with neighboring beta cells of the same sign. Together, these observations suggest that dendro-dendritic contact between RGCs is cell-type specific. Dendritic contacts were observed even before the alpha cell arbors were completely stratified, suggesting that cell-cell recognition may take place early in their development. For each cell type, the relative overlap of dendritic fields was constant with age, despite a two-fold increase in field area. We suggest that dendro-dendritic contacts may be sites of intercellular signaling that could regulate local extension of dendrites to maintain the relative overlap of RGCs within a mosaic during development.     

The development of intrinsic excitability in mouse retinal ganglion cells

Activity-dependent refinement of synaptic connections occurs throughout the developing nervous system, including the visual system. Retinal ganglion cells (RGCs) overproduce synapses then refine them in an activity-dependent manner that segregates RGC connections into multicellular patterns, such as eye-specific regions and retinotopic maps. Ferrets additionally segregate ON and OFF retinogeniculate pathways in an activity-dependent manner. It was unknown whether differences in ON versus OFF intrinsic and spontaneous activity occur in postnatal mouse. The work reported here measured the intrinsic properties and spontaneous activity of morphologically identified postnatal mouse RGCs, and tested the hypothesis that mouse ON and OFF RGCs develop differences in spontaneous activity. We found developmental changes in resting potential, action potential threshold, depolarization to threshold, action potential width, action potential patterns, and maximal firing rates. These results are consistent with the maturation of the intrinsic properties of RGCs extending through the first three postnatal weeks. However, there were no differences among mouse ON, OFF, and multistratified RGCs in intrinsic excitability, spontaneous synaptic drive or spontaneous action potential patterns. The absence of differences between ON and OFF activity patterns is unlike the differences that arise in ferrets. In contrast to the ferret, the ON and OFF target neurons in the mouse are organized in a random pattern, not layers. This supports the hypothesis that the absence of systematic differences in activity results in the nonlayered distribution of retinogeniculate connections.     

Morphological and functional organization of ON and OFF pathways in the adult newt retina

Morphological and functional organization of ON and OFF pathways in the adult newt retina were examined by intracellular recording and staining techniques and immunohistochemistry. Synaptotagmin immunoreactivity discriminated three broad bands within the IPL: the distal band (sublamina I), the middle band (sublamina II) consisting of two dense punctate bands (sublaminae II(a) and II(b)), and proximal band (sublamina III). The Lucifer-yellow labeled OFF amacrine and ganglion cells send their processes mainly in sublamina I and/or II(a) where OFF bipolar cells extend their axon terminals, while ON amacrine and ganglion cells send their processes in sublamina III and/or II(b) where ON bipolar cells extend their axon terminals. Processes of ON-OFF amacrine and ganglion cells ramify broadly in the whole thickness of the IPL. Many bipolar cells responded to light spot with a transient hyperpolarization at both light onset and offset. They are probably subtypes of ON bipolar cells, because their axon terminals branch mainly in sublaminae III and/or II(b), although a few cells ramified the axon at both sublaminae II(a) and III. Two immunohistochemical markers for bipolar cells, PKC and RB-1, identified axon terminals in sublaminae III and/or II(b). From the ramification pattern of axon terminal, they are probably subtypes of ON bipolar cells. ChAT-ir amacrine cells ramified their dendrites in either sublamina I or II(b). Altogether, present studies support the general idea of segregation of ON and OFF pathways in sublaminae a and b of the IPL.     

Visual stimulation is required for refinement of ON and OFF pathways in postnatal retina

ON and OFF pathways separately relay increment and decrement luminance signals from retinal bipolar cells to cortex. ON-OFF retinal ganglion cells (RGCs) are activated via synaptic inputs onto bistratified dendrites localized in the ON and OFF regions of the inner plexiform layer. Postnatal maturational processes convert bistratifying ON-OFF RGCs to monostratifying ON and OFF RGCs. Although visual deprivation influences refinement of higher visual centers, no previous studies suggest that light regulates either the development of the visual-evoked signaling in retinal ON and OFF pathways, nor pruning of bistratified RGC dendrites. We find that dark rearing blocks both the maturational loss of ON-OFF responsive RGCs and the pruning of dendrites. Thus, in retina, there is a previously unrecognized, pathway-specific maturation that is profoundly affected by visual deprivation.     

Axonal Synapses Utilize Multiple Synaptic Ribbons in the Mammalian Retina

In the mammalian retina, bipolar cells and ganglion cells which stratify in sublamina a of the inner plexiform layer (IPL) show OFF responses to light stimuli while those that stratify in sublamina b show ON responses. This functional relationship between anatomy and physiology is a key principle of retinal organization. However, there are at least three types of retinal neurons, including intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells, which violate this principle. These cell types have light-driven ON responses, but their dendrites mainly stratify in sublamina a of the IPL, the OFF sublayer. Recent anatomical studies suggested that certain ON cone bipolar cells make axonal or ectopic synapses as they descend through sublamina a, thus providing ON input to cells which stratify in the OFF sublayer. Using immunoelectron microscopy with 3-dimensional reconstruction, we have identified axonal synapses of ON cone bipolar cells in the rabbit retina. Ten calbindin ON cone bipolar axons made en passant ribbon synapses onto amacrine or ganglion dendrites in sublamina a of the IPL. Compared to the ribbon synapses made by bipolar terminals, these axonal ribbon synapses were characterized by a broad postsynaptic element that appeared as a monad and by the presence of multiple short synaptic ribbons. These findings confirm that certain ON cone bipolar cells can provide ON input to amacrine and ganglion cells whose dendrites stratify in the OFF sublayer via axonal synapses. The monadic synapse with multiple ribbons may be a diagnostic feature of the ON cone bipolar axonal synapse in sublamina a. The presence of multiple ribbons and a broad postsynaptic density suggest these structures may be very efficient synapses. We also identified axonal inputs to ipRGCs with the architecture described above.     

A damped oscillation in the intramembranous charge movement and calcium release flux of frog skeletal muscle fibers

Asymmetric membrane currents and calcium transients were recorded simultaneously from cut segments of frog skeletal muscle fibers voltage clamped in a double Vaseline-gap chamber in the presence of high concentration of EGTA intracellularly. An inward phase of asymmetric currents following the hump component was observed in all fibers during the depolarization pulse to selected voltages (congruent to -45 mV). The average value of the peak inward current was 0.1 A/F (SEM = 0.01, n = 18), and the time at which it occurred was 34 ms (SEM = 1.8, n = 18). A second delayed outward phase of asymmetric current was observed after the inward phase, in those experiments in which hump component and inward phase were large. It peaked at more variable time (between 60 and 130 ms) with amplitude 0.02 A/F (SEM = 0.003, n = 11). The transmembrane voltage during a pulse, measured with a glass microelectrode, reached its steady value in less than 10 ms and showed no oscillations. The potential was steady at the time when the delayed component of asymmetric current occurred. ON and OFF charge transfers were equal for all pulse durations. The inward phase moved 1.4 nC/microF charge (SEM = 0.8, n = 6), or about one third of the final value of charge mobilized by these small pulses, and the second outward phase moved 0.7 nC/microF (SEM = 0.8, n = 6), bringing back about half of the charge moved during the inward phase. When repolarization intersected the peak of the inward phase, the OFF charge transfer was independent of the repolarization voltage in the range -60 to -90 mV. When both pre- and post-pulse voltages were changed between -120 mV and -60 mV, the equality of ON and OFF transfers of charge persisted, although they changed from 113 to 81% of their value at -90 mV. The three delayed phases in asymmetric current were also observed in experiments in which the extracellular solution contained Cd2+, La3+ and no Ca2+. Large increases in intracellular [Cl-] were imposed, and had no major effect on the delayed components of the asymmetric current. The Ca2+ transients measured optically and the calculated Ca2+ release fluxes had three phases whenever a visible outward phase followed the inward phase in the asymmetric current. Several interventions intended to interfere with Ca release, reduced or eliminated the three delayed phases of the asymmetric current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Removal of extracellular chloride suppresses transmitter release from photoreceptor terminals in the mudpuppy retina

Removal of extracellular Cl- has been shown to suppress light-evoked voltage responses of ON bipolar and horizontal cells, but not photoreceptors or OFF bipolar cells, in the amphibian retina. A substantial amount of experimental evidence has demonstrated that the photoreceptor transmitter, L-glutamate, activates cation, not Cl-, channels in these cells. The mechanism for Cl-free effects was therefore reexamined in a superfused retinal slice preparation from the mudpuppy (Necturus maculosus) using whole-cell voltage and current clamp techniques. In a Cl-free medium, light-evoked currents were maintained in rod and cone photoreceptors but suppressed in horizontal, ON bipolar, and OFF bipolar cells. Changes in input resistance and dark current in bipolar and horizontal cells were consistent with the hypothesis that removal of Cl- suppresses tonic glutamate release from photoreceptors. The persistence of light-evoked voltage responses in OFF bipolar cells, despite the suppression of light-evoked currents, is due to a compensatory increase in input resistance. Focal application of hyperosmotic sucrose to photoreceptor terminals produced currents in bipolar and horizontal cells arising from two sources: (a) evoked glutamate release and (b) direct actions of the hyperosmotic solution on postsynaptic neurons. The inward currents resulting from osmotically evoked release of glutamate in OFF bipolar and horizontal cells were suppressed in a Cl-free medium. For ON bipolar cells, both the direct and evoked components of the hyperosmotic response resulted in outward currents and were thus difficult to separate. However, in some cells, removal of extracellular Cl- suppressed the outward current consistent with a suppression of presynaptic glutamate release. The results of this study suggest that removal of extracellular Cl- suppresses glutamate release from photoreceptor terminals. Thus, it is possible that control of [Cl-] in and around photoreceptors may regulate glutamate release from these cells.