Salamander Retina Research Articles

A slowly inactivating potassium current truncates spike activity in ganglion cells of the tiger salamander retina

Voltage-gated ganglion cell membrane currents were studied under whole-cell patch clamp in isolation and in retinal slices. The cells were identified by (1) backfilling their axons with rhodamine and later identifying them by their fluorescence in the slice or the mix of isolated cells or (2) by filling them with Lucifer yellow during recording in retinal slices. Both methods yielded cells with similar currents. In some cases, isolated cells lacked processes yet showed currents similar to other cells, suggesting that voltage-gated currents in all cells were located primarily at the soma. Both a conventional inactivating sodium current and a sustained calcium current were found. We describe 3 inactivating outward currents, ordered in their rate of inactivation. The fastest current resembled IA reported by Connor and Stevens (1971a, b). A slower current labeled IB inactivated with a time constant of 339 msec at 0 mV. The current with slowest inactivation is labeled IC here, inactivating with a time constant of 4.03 sec at 0 mV. An additional outward current was sustained and calcium dependent labeled IK(Ca). IB was the largest of these currents. It was slower than IA, was not blocked by 4AP, and inactivated over a much more positive potential range. IB appears to play an important role in spike generation, different from that of IA: Its inactivation leads to a slow depolarizing shift of the membrane during a current step, truncating spike activity after about 300-700 msec as the membrane potential enters the region of sodium inactivation. We analyze how the inactivating outward current acts to ensure a graded spiking response and to truncate the spiking output in the presence of large excitatory inputs.

Colocalization of GABA and glycine immunoreactivities in a subset of retinal neurons in tiger salamander

Alternate serial 1 μm Durcupan resin sections of tiger salamander retina were stained with antisera against GABA and glycine using postembed immunocytochemical techniques. Although the vast majority of neurons were labeled by either GABA or glycine antiserum, a small percentage of presumed amacrine cells in the inner nuclear layer and cells in the ganglion cell layer were clearly labeled by both antisera, indicative of colocalization of endogenous GABA and glycine. Although there is a greater than 90% chance that a labeled cell will be clearly labeled for either GABA or glycine immunoreactivity, the possibility for cotransmission of two inhibitory transmitters must be considered for a small percentage of these retinal neurons.

Localization of putative GABAergic neurons in the larval tiger salamander retina by immunocytochemical and autoradiographic methods.

Putative GABAergic neurons in the larval tiger salamander retina were localized by a comparative analysis of glutamate decarboxylase immunoreactivity (GAD-IR), GABA-like immunoreactivity (GABA-IR), and high-affinity 3H-GABA uptake at the light microscopical level. Preliminary data showed that all GAD-IR neurons were double labeled for GABA-IR. However, because the weak somatic labeling with GAD-IR, we could not determine if the converse were true. Neurons commonly labeled with GABA-IR and 3H-GABA uptake include horizontal cells, type I (outer) and type II (inner) bipolar cells, type I (inner) and type II (outer) amacrine cells, and cell bodies in the ganglion cell layer (GCL). In addition, interplexiform cells were identified with GABA-IR. The presence of GABA-IR ganglion cells was indicated by GABA-IR fibers in the optic fiber layer and optic nerve as well as by a GABA-IR cell in the GCL that included a labeled axon. The percentage of labeled somas in the inner nuclear layer (INL) compared to all cells in each layer was similar for the two methods: 30% in INL 1 (outer layer of somas), 15% in INL 2 (middle layer), 43-52% in INL 3 (inner layer), and about 21-26% in the GCL. Labeled processes were found in three bands in the inner plexiform layer, with the densest band located in the most proximal part. Postembedding labeling of 1-micron Durcupan resin sections for GABA-IR showed the same general pattern as obtained with 10-microns cryostat sections, with additional staining, however, of type II (inner) bipolar cell Landolt's clubs. Extensive colocalization of labeling was indicated, and we conclude that GABA-IR can serve as a valid and reliable marker for GABA-containing neurons in this retina and suggest that GABA serves as a transmitter for horizontal cells, several types of amacrine cell, a type of interplexiform cell, and perhaps a small percentage of type I and type II bipolar cells and ganglion cells.

Growth and synapse formation among major classes of adult salamander retinal neurons in vitro

Adult neurons, isolated from the salamander retina, were maintained in low-density cell culture and examined for synapse formation by electrophysiological and electron microscopic techniques. Morphologically identifiable rod, cone, horizontal, bipolar, and amacrine/ganglion cells survived for many months, grew processes, and formed numerous cell contacts. Intracellular recordings showed the presence of a variety of voltage- and time-dependent conductances and both electrical and chemical transmission among these cells. At the ultrastructural level, gap junctions, monad ribbon synapses, and conventional synapses, like those present in the intact retina, were observed in sibling cultures. Thus, all major classes of adult retinal neurons, in addition to ganglion cells, are able to regenerate processes and reform synapses. The regenerated synaptic contacts are functional and structurally diverse.

The off-overshoot responses of photoreceptors and horizontal cells in the light-adapted retinas of the tiger salamander

Depolarizing overshoot responses at the cessation of a test light step were observed in horizontal cells (HCs) and in a population of photoreceptors (rod cs) in light-adapted retinas of the tiger salamander. An anode break regenerative conductance may contribute to the overshoot responses in rod cs (o-wave). The overshoot responses in HCs consist of two components: a fast α-wave whose amplitude and time course follow those of the o-wave; and a slow β-wave whose amplitude and time course vary with the HC membrane voltage. These results are consistent with the notion that the α-wave is a postsynaptic response to the voltage overshoots of the o-waves in rod cs and the β-wave is mediated by voltage-dependent conductances in the HC membrane. A possible function of the HC overshoot responses is to reset the amplitude of the light-adapted HC responses during repetitive or rapidly changing light stimulation.

Light microscopic localization of putative glycinergic neurons in the larval tiger salamander retina by immunocytochemical and autoradiographical methods.

Putative glycinergic neurons in the larval tiger salamander retina were localized by a comparative analysis of high affinity 3H-glycine uptake and glycine-like immunoreactivity (Gly-IR) at the light microscopic level. Commonly labeled neurons include at least three types of amacrine cell (Type IAd, Type IAb, Type IIAd; distinguished by soma location and dendritic ramification), cell bodies in the ganglion cell layer (GCL), and rarely observed Type II (inner) bipolar cells. With the increased resolution provided by Gly-IR, we identified a Type IAa amacrine cell, two types of Type IIAd amacrine cells, and Gly-IR interplexiform cells. Gly-IR axons in longitudinal sections of the optic nerve indicate the presence of Gly-IR ganglion cells. The percentage of labeled somas in the inner nuclear layer (INL) compared to all cells in each layer was similar for the two methods: 30-40% in INL 2 (middle layer of somas), 30-40% in INL 3 (inner layer of somas), and about 5% in the GCL. Labeled processes were found throughout the full thickness of the inner plexiform layer (IPL), but with a much denser band in the proximal half (sublamina b). The only major difference between the two methods (3H-glycine uptake vs. Gly-IR) was that Type I (outer) bipolar cells were labeled only by 3H-glycine uptake; these cells were more lightly labeled with silver grains than cell bodies in either INL 2 or INL 3. Postembed labeling of 1 micron Durcupan plastic sections for Gly-IR showed the same pattern, but with much higher resolution, as obtained with 10 micron cryostat sections. This study indicates extensive colocalization of labeling by both probes in INL 2, INL 3, the IPL, and the GCL. We conclude that Gly-IR can serve as a valid and reliable marker for glycine-containing neurons in this retina and suggest that glycine serves as a transmitter for several morphologically distinct types of amacrine cell, an interplexiform cell, and perhaps a small percentage of Type II bipolar cells and ganglion cells.

Melatonin enhances horizontal cell sensitivity in salamander retina

Intracellular electrophysiological recording techniques were utilized to investigate the possible function of retinal melatonin in the larval tiger salamander. Endogenous retinal melatonin was present and appeared to bind to a membrane-enriched fraction of the salamander retina, as determined by radioimmunoassay and receptor binding studies. Melatonin added through the purfusion bath to flat-mounted retinas resulted in a horizontal cell(HC) hyperpolarization of 10–20 mV. Additionally, the amplitude of HC responses to short test flashes increased in the presence of melatonin. Voltage-intensity plots revealed that application of 500 μM of melatonin caused an increase of the HC light sensitivity and this effect was reversible. These results suggest that melatonin synthesized and released during the dark period of the diurnal cycle may alter the sensitivity of second-order neurons at a time of day when photopic input is at its lowest level.

Localization of tyrosine-hydroxylase-like-immunoreactive amacrine cells in the larval tiger salamander retina.

Immunocytochemistry was used to localize the populations of tyrosine-hydroxylase-like (TH)-immunoreactive cells in the tiger salamander retina. Ninety percent of these cells possessed somas that were situated in the innermost cell row of the inner nuclear layer and were classified as amacrine cells. Ten percent of TH-immunoreactive somas were located in the ganglion cell layer and were tentatively designated as those of displaced amacrine cells. The processes of TH-immunoreactive cells ramified most heavily in sublayer 1 of the inner plexiform layer, while a relatively small number of TH-labelled processes distributed in sublayers 3 and 5. Less than 1% of TH-immunoreactive cells in the amacrine cell layer exhibited a short process of somal origin that extended distally toward the outer plexiform layer. However, these processes did not cross the whole of the inner nuclear layer, and no immunolabelling was observed in the outer plexiform layer. An examination of retinal whole-mounts revealed that TH-immunoreactive amacrine and displaced amacrine cells were distributed throughout the center and periphery of the retina. The density of TH-immunolabelled amacrine cells was calculated to be 49 +/- 13 (mean +/- standard error) cells per mm2. The vast majority of TH-immunoreactive amacrine and displaced amacrine cells exhibited a stellate appearance and gave rise to three or more primary dendrites. A few TH-amacrine and displaced amacrine cells possessed two primary dendrites that emerged from opposite sides of their somas. The processes of TH-immunoreactive cells were generally poorly branched and varicose with terminal branches sometimes appearing thin and beaded. Because some TH-immunolabelled processes were very long, there was considerable overlap between the dendritic fields of neighboring TH-cells. Lastly, individual TH-immunoreactive amacrine and displaced amacrine cells were often observed in whole-mounts to provide processes that ramified at more than one level of the inner plexiform layer.

A presynaptic action of glutamate at the cone output synapse

Neurotransmitter release from many central nervous system synapses is regulated by 'autoreceptors' at the synaptic terminal, which bind the released transmitter and alter release accordingly. The photoreceptors of lower vertebrates are thought to use glutamate as a neurotransmitter. Glutamate conveys the visual signal to postsynaptic bipolar and horizontal cells, but has been reported not to act on the photoreceptors themselves. We show here that glutamate evokes a current, carried largely by chloride ions, in cones isolated from the tiger salamander retina. This response is localized to the synaptic terminal of the cone. Removing external sodium blocks this action of glutamate. These results suggest the existence of a positive feedback loop at the cone output synapse: over most of the light-response range, glutamate released by depolarization of the cone will cause further depolarization, increasing the gain of phototransduction. Glutamate released from rods may also polarize cones, modulating the gain of the cone output synapse. This system is surprisingly different from the autoreceptor systems for most other transmitters, which act in a negative feedback way.

Localization of neurotensin-like immunoreactive amacrine cells in the larval tiger salamander retina.

Light microscopic immunocytochemistry was used to localize the populations of NT-like immunoreactive amacrine cells in the larval tiger salamander retina. Seventy-nine percent of NT-immunostained cells observed in transverse cryo-prepared sections were classified as Type 1 amacrine cells. Another 6% were classified as Type 2 amacrine cells, while 15% of the NT-cells had their cell bodies situated in the ganglion cell layer and were tentatively designated as displaced amacrine cells. Each type of NT-like immunoreactive cell was observed in the central and peripheral retina. NT-immunostained processes were observed to ramify in sublayers 3 and 5 of the inner plexiform layer. An examination of retinal whole mounts revealed that NT-amacrine cells were distributed throughout the center and periphery of the retina at a density of 82 +/- 24 cells/mm2. The dendritic fields of NT-immunostained amacrine and displayed amacrine cells were observed to be either symmetrically or asymmetrically distributed about their somas. Symmetrical dendritic fields were generally oval-shaped and ranged in diameter from 250 to 500 micron (major axis) by 150 to 250 micron (minor axis). Asymmetrical dendritic fields were observed to encompass one-half or less of an imaginary circle surrounding their soma of origin and were orientated in all directions. The processes forming asymmetrical dendritic fields ranged from 75 to 260 micron in length. Furthermore, partial overlap was often observed between the dendritic fields of adjacent NT-amacrine cells.

Electrical coupling between rods and cones in the tiger salamander retina.

Electrical coupling between rods and cones was studied in the salamander (Ambystoma tigrinum) retina by measuring the light responses and spectral sensitivities of rods and cones and by measuring the voltage responses from a rod to current pulses injected into a cone. A population of 10-20% of the photoreceptors exhibited a mixed-response waveform of the rod and the cone under dark-adapted conditions, and a response waveform closely resembled that of a cone in the presence of background illumination. Lucifer yellow injection revealed that these cells are morphologically identical to rods, and thus they are named rodcs. Dark-adapted rodcs exhibited a rod-like spectral sensitivity with a peak at approximately 520 nm that shifted to a cone-like spectral sensitivity with a peak at approximately 620 nm in response to background light (Purkinje shift). The voltage response of a rodc to a -1-nA current step injected into an adjacent cone is approximately 3.6 times larger than that of a rod to the same current step. These results indicate that there is a population of rods (rodcs) in the tiger salamander retina that is strongly coupled to the cones and that these cells allow significant mixture of rod and cone signals at the photoreceptor level.

Synaptic transmission from rods to horizontal cells in dark-adapted tiger salamander retina

Synaptic transmission from rods to horizontal cells in dark-adapted tiger salamander retina

Synaptic transmission from rods to horizontal cells in dark-adapted tiger salamander retina

Synaptic transmission between rods and horizontal cells (HCs) was studied by simultaneously recording from the pre- and postsynaptic cells in the living retinal slices of the larval tiger salamander. Voltage tails after the termination of a flash were observed in dark-adapted rods and HC somata, but not in cones. These simultaneously recorded voltage tails were used to isolate the rod input from the cone input to the HCs. The rod voltage versus the HC voltage in the response tails yielded the input-output relation of the rod-HC synapse. Two factors, a luminance-dependent signal delay between the rod and HC responses and a voltage-dependent depolarizing current in the HCs, complicate the analysis of the rod-HC synapse. A single input-output relation of the rod-HC synapse was obtained after these two factors had been corrected. The rod-HC synapse was approximately linear near the rod dark potential (−40 mV) with a synaptic gain around 12, but it became progressively nonlinear at more hyperpolarized potentials. The synaptic gain at V rod=−50 mV was about 0.65.

Structure of the receptive fields of bipolar cells in the salamander retina.

1. The receptive-field structure of bipolar cells in the salamander retina has been examined using isolated retinae from dark-adapted eyes. 2. Receptive-field mapping was carried out with a 25-microns diam spot of light whose wavelength and intensity was intended to stimulate rods rather than cones. 3. Both hyperpolarizing and depolarizing bipolar cells showed receptive fields having a single central point of maximum sensitivity from which sensitivity declined radially. Antagonistic surrounds could not be demonstrated using a small spot of light. 4. The diameter of receptive fields was found to vary between 374 and 662 micron, consistent with a single bipolar cell being effectively connected to 323-1,275 rods. 5. Lucifer yellow injections of bipolar cells revealed dendritic arbors whose greatest dimensions varied between 43 and 70 microns, consistent with a direct synaptic connection of between 10 and 24 rods to each bipolar cell. 6. We rule out signal spread within the rod network, extensive lateral ramification of rod process, nonlinearity of synaptic transmission, and light scatter, as possible explanations of large bipolar cell receptive fields. It seems likely, instead, that signals are extensively shared between bipolar cells.

Synaptic connections between neurons in living slices of the larval tiger salamander retina

Synaptic connections between retinal cells were studied by recording simultaneously from pre- and postsynaptic cells in the retinal slice preparation. The time course and waveform of the pre- and postsynaptic light responses were monitored, and the postsynaptic voltage responses to presynaptic current injection were measured. Results obtained provide direct evidence showing that the rod-HBC and rod-HC synapses are sign-preserving, and the rod-DBC. HC-HBC and amacrine-DBC synapses are sign-inverting. Moreover, the synaptic delays between rods and bipolar cells are shorter than that between rods and horizontal cells. The methodology of making retinal slices and the subsequent recording and stimulation procedures are described. The advantages of the retinal slice preparation and its potential in retinal research are discussed.

Interactions between enkephalin and γ-aminobutyric acid in the larval tiger salamander retina

Both double-label and intracellular electrophysiological recording techniques were utilized to investigate the interactions between enkephalin and γ-aminobutyric acid in the larval tiger salamander retina.Double-label studies revealed that the vast majority ( 96%) 96%) of enkephalin-immunostained amacrine cells also exhibit high affinity uptake of [ 3H]γ-aminobutyric acid. Electrophysiological evidence demonstrated that morphine and γ-aminobutyric acid exert opposite effects on a population of On-Off ganglion cells. γ-Aminobutyric acid decreased the activity of these cells, while enkephalin increased their activity. These findings support the idea that opiate-mediated pathways inhibit GABAergic pathways in the vertebrate retina.

Direct excitatory and lateral inhibitory synaptic inputs to amacrine cells in the tiger salamander retina

Two distinct synaptic currents in amacrine cells were measured using whole cell patch clamp in retinal slices. Synaptic activity was elicited with transretinal current passed from photoreceptors to ganglion cells to depolarize bipolar cell terminals. Recordings made in line with the stimulating electrodes revealed inward synaptic currents that reversed near 0 mV, positive to spike threshold. Recording 400 μm lateral to the stimulating electrodes revealed synaptic currents that reversed near-60 mV, negative to spike threshold. The lateral signal was blocked by tetrodotoxin and strychnine. A current with revesal potential similar to the lateral input was elicited by direct application of glycine to the amacrine cells. The results suggest that bipolar, but not amacrine input can elicit spikes in neighboring amacrine cells, that amacrine cells are mutually inhibitory and the inhibitory lateral transmission is limited to the extent of processes of the excited, spiking cell.

Changes in response waveform of retinal horizontal cells during dark and light adaptation.

Changes in response waveform of retinal horizontal cells during dark and light adaptation were examined in the tiger salamander retina. In addition to sensitivity changes, the response waveform of HC was altered during visual adaptation: following bleaching or in the presence of background illumination, a transient depolarizing overshoot was observed at the cessation of the test flash. In contrast to the hyperpolarizing light responses, the depolarizing off-overshoots do not follow the standard threshold recovery time course during dark adaptation, neither do they obey Weber's law for increment thresholds during light adaptation. Simultaneous intracellular recordings from photoreceptors and HC revealed that the off-overshoot responses in light-adapted HC are probably originated from a population of cells in the photoreceptor layer which are strongly coupled with the cones.

Light-dependent synaptic delay between photoreceptors and horizontal cells in the tiger salamander retina

A long delay between the time to peak of the photoreceptor and horizontal cell light responses was observed in dark-adapted tiger salamander retinas. The rise of the HC light responses follows a biphasic time-course which is probably mediated by the removal of the rod and cone transmitters, but not by voltage-dependent mechanisms in the HC membrane. A bleaching white light shortens the signal delay and results in a faster and monophasic rise of the HC light responses mediated predominately by the cones.

Synaptic transmission in amphibian retinae during conditions unfavourable for calcium entry into presynaptic terminals.

Toad (Bufo marinus) retinae were peeled from the pigment epithelium and superfused over the photoreceptor surface with a calcium-poor, cobalt-rich medium. The shape of the electroretinogram indicated that post-synaptic neurones received synaptic input. Adding the putative transmitters glutamate and N-acetylhistidine changed the shape of the electroretinogram. The change suggests that an excess of the putative transmitters blocked a component of synaptic transmission that persisted when a retina was bathed in cobalt. Salamander (Ambystomatigrinum) retinae in hemisected eye cups were superfused over their vitreal surface. Intracellular responses were recorded from photoreceptors. Reducing the calcium concentration in the superfusing medium from 1 mM to less than 10 microM slowly changed responses produced by light. The change indicates that the calcium concentration in the extracellular space surrounding photoreceptors fell to less than 100 microM. When retinae were superfused with a medium containing 1 mM-calcium, 3 mM-barium, and 10 mM-tetraethylammonium (TEA), rods produced action potentials that were later blocked by adding 1 mM-cobalt. Blocking calcium channels with cobalt and lowering the extracellular calcium concentration should together block calcium-dependent synaptic transmission. Intracellular responses were recorded from horizontal cells. After replacing external calcium with cobalt the membrane potential hyperpolarized and responses produced by light became smaller but did not entirely disappear. The responses that remained were less sensitive to light and had an altered shape. The change was reversible. Similar responses could be recorded after prolonged (30-120 min) exposure to cobalt. Electrical synapses between horizontal cells were uncoupled by adding 10 microM-forskolin to the cobalt medium. The polarity of a response could then be reversed if a cell was depolarized by injecting current. The observation of a reversal potential demonstrates that the response was produced by a conductance change. Intracellular responses were recorded from depolarizing and hyperpolarizing bipolar cells while retinae were superfused with cobalt-rich medium. After changing to a cobalt-free medium containing 1 mM-calcium, responses produced by light were slightly smaller. Large responses were recorded after superfusing with cobalt-rich, calcium-poor medium for 30-120 min. The results indicate that synaptic transmission by photoreceptors continues during conditions expected to block the entry of calcium into their presynaptic terminals.