Voltage noise in Limulus visual cells.

Intracellular recordings from Limulus eccentric cells suggest that the generator potential arises from the superposition of numerous discrete fluctuations in membrane conductance. If this is so, a relation between frequency response to flickering light and noise characteristics under steady light may be predicted. This prediction is verified experimentally. If a discrete fluctuation model is assumed, the data indicate that increased light has two major effects: (i) the discrete events are strongly light-adapted to smaller size, and (ii) the time course of each event becomes briefer.

Enhancement of flicker by lateral inhibition.

Sinusoidal modulation of illumination on the compound eye of the horseshoe crab, Limulus, produces a corresponding variation in the rate of discharge of optic nerve impulses. Increasing the area of illumination decreases the variation at low frequencies of modulation, but unexpectedly enhances-or "amplifies"-the variation at the intermediate frequencies to which the eye is most sensitive. Both effects must result from inhibition since it is the only significant lateral influence in this eye.

Frequency characteristics of retinal neurons in the carp.

Frequency characteristics of various retinal neurons in the carp were studied using sinusoidally modulated light as an input. They were affected by both intensity and pattern of illumination. In the horizontal cells, in which the effect of light intensity was studied most extensively, an increase in the light intensity brought about a decrease of the gain, which was more marked at lower frequencies, resulting in a shift of cutoff frequency towards higher frequencies and in a slight low frequency attenuation. A decrease in the area illuminated had an effect similar to a decrease in the light intensity. In the receptor, the low frequency attenuation was not apparent even at high light intensities. The adaptation process in receptors was not sufficient to explain the low frequency attenuation in the horizontal cells, and a possible contribution of negative feedback from horizontal cells to receptors was suggested. In the bipolar cell, the lateral interaction played an important role. An increase in an area resulted in the suppression of the response at low frequencies where the phases of the center and the surround responses were opposed, but in the augmentation near 5 Hz where the two responses were in phase. In amacrine cells, a low frequency attenuation and a phase advance at low frequencies were very prominent, and were considered to be due mainly to a process designated here as the neural adaptation.

Analyses of bipolar cell responses elicited by polarization of horizontal cells.

Simultaneous intracellular recordings were made from a bipolar cell and a horizontal cell in the carp retina. The properties of the bipolar cell were studied while injecting current into the horizontal cell. Hyperpolarization of horizontal cells, irrespective of their type, elicited a hyperpolarizing response in on-center bipolar cells and a depolarizing response in off-center bipolar cells. Analyses of the ionic mechanisms of bipolar cell responses revealed that depolarization of horizontal cells simulated and hyperpolarization opposed the effect of central illumination. The effect of polarization was exerted in such a manner that each type of horizontal cells modified the transmission from those photoreceptors from which they receive main inputs. In on-center bipolar cells, for example, the L-type horizontal cells receiving inputs mainly from red cones modified the cone-bipolar transmission accompanied by a conductance change of K+ and/or Cl- channels, and the intermediate horizontal cells receiving inputs from rods modified the rod-bipolar transmission accompanied by a conductance change of Na+ channels. In off-center bipolar cells, the effect of polarization of any type of horizontal cells was mediated mainly by conductance changes of Na+ channels. Feedback mechanisms from horizontal cells to photoreceptors could explain these results reasonably well.

Application of transretinal current stimulation for the study of bipolar-amacrine transmission.

Transretinal current flowing from the receptor side to the vitreous side depolarizes the axon terminals of retinal cells and facilitates the release of transmitter. Such current elicited a depolarizing response in off-center bipolar cells and a hyperpolarizing response in on-center bipolar cells. It also elicited a response of relatively complex waveform in amacrine cells. The responses elicited in bipolar cells were suppressed in the presence of 5-10 mM glutamate in the perfusing Ringer solution, while the responses of amacrine cells persisted, although their waveform changed to a simple one that showed monotonic depolarization irrespective of the type of amacrine cell and were accompanied by a decrease in the membrane resistance. The results indicate excitatory synaptic transmission from bipolar cells to amacrine cells. Since the response elicited by current in ON-OFF cells was almost identical to those elicited in ON or OFF amacrine cells, the transient nature of their light response cannot be due to their membrane properties. ON-OFF cells responded to transretinal current flowing in the opposite direction with a small hyperpolarization accompanied by a resistance increase. The hyperpolarizing response was suppressed by the addition of GABA in glutamate Ringer solution. The results suggest an activation by the current of GABA-ergic feedback pathways from amacrine cells to bipolar cells.

Ionic mechanisms of two types of on-center bipolar cells in the carp retina. II. The responses to annular illumination.

On-center bipolar cells in the dark-adapted carp retina were divided into four types (A, B, C, and D) on the basis of response wave forms, spectral response properties, and electrical membrane properties. Type A and B cells responded to a spot of light with a transient depolarization followed by a plateau, whereas the response of type C and D cells were approximately rectangular in shape. The center and surround responses of type A cells had maximum spectral response of approximately 525 nm in the lower mesopic range; the polarity of both responses was reversed at positive membrane potentials as the membrane was depolarized by extrinsic current. The center and surround responses of type D cells had a maximum spectral response of approximately 625 nm in the mesopic or photopic range; the polarity of both responses was reversed at membrane potentials that were more negative than those at the dark level. The results suggest that the center and surround responses mediated by rods are generated by changes in sodium conductance, but in opposite ways; whereas those mediated by red cones are generated by changes in potassium and/or chloride conductances. In type B and C cells, which probably receive inputs from both rods and/or green cones as well as red cones, the center responses were composed of the two ionic mechanisms described above. The surround responses of many type B and C cells were dominated by only one ionic mechanism with a negative reversal potential, but in some type B cells the surround responses were resulted from two ionic mechanisms similar to those of the center responses.

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.

Ionic mechanisms of two types of on-center bipolar cells in the carp retina. I. The responses to central illumination.

Properties of the depolarizing response of on-center bipolar cells to a light spot stimulus were studied in the carp retina. On-center bipolar cells were classified into two types, cone-dominant and rod-dominant, according to their major input from cones and rods. Cone-dominant bipolar cells responded to spectral light with the maximum amplitude near 625 nm, suggesting major input from red cones. The response was accompanied by a resistance increase and showed a reversal potential at -63 +/- 21 mV when the membrane was hyperpolarized by current. The results suggest that the photoresponse of cone-dominant cells is due to a decrease of gK and/or gCl, membrane conductances to potassium and chloride, respectively. Rod-dominant bipolar cells responded to spectral light with the maximum amplitude near 525 nm under scotopic conditions and near 625 nm under photopic conditions, providing evidence that they receive input from rods and red cones. In the scoptopic condition their response was accompanied by a resistance decrease and showed a reversal potential at 29 +/- 13 mV, whereas in the photopic condition the response in most of them was accompanied by a resistance increase, at least in their part and showed a reversal at -53 +/- 11 mV. The results suggest that the photoresponse activated by rod input is due to an increase in gNa. In the mesopic condition rod-dominant cells showed complex electrical membrane properties as the result of electric interaction between the above two differnt ionic mechanisms activated by rod and cone inputs.

A quantitative description of the dynamics of excitation and inhibition in the eye of Limulus.

By means of intracellular microelectrode techniques, we have measured the dynamics of the several processes which translate light stimulation into spike activity in the Limulus eye. The transductions from light to voltage and from voltage to spike rate, and the lateral inhibitory transduction from spike rate to voltage, we have characterized by transfer functions. We have checked the appropriateness of treating the eye as a system of linear transducers under our experimental conditions. The response of the eye to a large spot of light undergoing sine flicker has been correctly predicted.

Electrical and morphological properties of off-center bipolar cells in the carp retina.

Electrical membrane properties of carp off-center bipolar cells were studied by injecting a ramp of current (changing at a rate of about 0.3 nA/s) through one barrel of a double-barreled electrode and recording the voltage drop through the other barrel. The light-elicited response reversed the polarity at a positive membrane potential. The current-voltage curve showed inward and outward rectifications which modified the amplitude of the light-elicited response. The degree of membrane rectifications varied considerably among the cells sampled. Off-center bipolar cells were stained by iontophoretic injections of Lucifer-yellow dye after studying their electrical properties. There was a significant correlation between the difference in cell morphology and the degree of membrane rectification. The cells, which were characterized by small cell bodies lying close to the proximal part of the inner nuclear layer and by thin primary dendrites, tended to show rectifying membrane properties, compared to those characterized by large cell bodies lying close to the distal part of the inner nuclear layer and by thick primary dendrites. A site showing rectifying membrane properties and the origins of a large variability in membrane rectifications are discussed in detail.

Three types of horizontal cells in the stingray retina: their morphology and physiology.

Histological and electrophysiological features of horizontal cells were studied in the stingray retina in which the ratio of rods to cones was about five to one. There were three distinct sublayers of horizontal cells; external, middle and internal. The type of horizontal cells recorded was identified by dye injection after recording. Relatively thick external horizontal cells sent their short dendrites into both rod and cone terminals to make synaptic contacts. The response recorded from them was not color-coded but showed a Purkinje shift depending on the adaptational state. Middle horizontal cells were flat and usually two or three of them overlying each other constituted the middle sublayer. Their response, which was difficult to record in the light-adapted retina, was characterized by a slow time course, a prolongation of the response to bright flashes and a maximum sensitivity in the green region of the spectrum without any sign of Purkinje shift. Internal horizontal cells were color-coded. They responded with hyperpolarization at shorter wavelengths and with depolarization at longer wavelengths. It was concluded that the internal horizontal cells receive inputs from cones and the middle horizontal cells from rods, whereas the external horizontal cells receive inputs from both rods and cones.

Melatonin inhibits naloxone-induced luteinizing hormone release in ovariectomized estrogen-primed rats.

The present study aimed to examine the effect of melatonin on naloxone-induced luteinizing hormone (LH) secretion in ovariectomized estrogen-primed rats. A single intracerebroventricular (i.c.v.) injection of naloxone (mu opioid receptor blocker, 15 micrograms) or an intravenous (i.v.) injection of LH-releasing hormone (LHRH, 50 ng/kg) elicited a transient and significant increase in the serum LH concentration within 10 min. While an i.c.v. injection of 100 ng melatonin by itself did not change the basal LH release, it almost completely inhibited the naloxone-induced LH release. Melatonin (10 ng) also significantly reduced the effect of naloxone. However, an i.c.v. injection of 100 ng melatonin did not affect the LHRH-induced LH release. In separate experiments, the effect of melatonin on naloxone-induced pulsatile LH secretion was studied in estrogen-treated rats. A continuous i.v. infusion of naloxone (20 mg/kg/h) induced LH pulses in rats treated i.c.v. with saline. An i.c.v. administration of 100 ng melatonin, which by itself did not affect basal LH secretion, significantly reduced the frequency, but not the amplitude, of LH pulses induced by the naloxone infusion. These results show that melatonin has a suprapituitary site of action to inhibit naloxone-induced LH release, and suggest that melatonin has an effect in inhibiting the activity of the hypothalamic LHRH pulse generator, either directly or indirectly, in female rats.

A possible role of the pineal gland in acute immobilization-related suppression of naloxone-induced LH release in ovariectomized estrogen-primed rats.

It has been recently reported that acute immobilization stress almost completely suppresses the luteinizing hormone (LH) release induced by naloxone, a mu-opioid antagonist, in ovariectomized estrogen-primed rats. The present study examined the possible involvement of the pineal gland in the acute immobilization-related suppression of the naloxone-induced LH release. An intraventricular (ICV) injection of 15 microg naloxone produced an abrupt increase in circulating LH concentrations in non-stressed rats. The naloxone-induced LH release was completely eliminated when tested 60 min after the end of a 30 min session of acute immobilization. The same stress conditions did not affect LH-releasing hormone (LHRH)-induced LH release, suggesting that the stress-related suppression of the naloxone-induced LH release was a suprapituitary event. In chronically-pinealectomized rats, but not in sham-pinealectomized rats, naloxone injected 60 min after the end of the stress session evoked a significant increase in serum LH concentrations. However, naloxone injected ICV during the acute immobilization did not elicit LH release in either pinealectomized or sham-operated rats. Under non-stressed conditions, the LH secretory response to naloxone was similar in pinealectomized and sham-operated animals. The same stress (30 min immobilization) significantly increased pineal melatonin content as well as plasma melatonin concentrations in rats bearing intact pineal glands, indicating that stress actually affected the pineal function. These results provide evidence for a role of the pineal in the suppression of the LH response to naloxone after stress, but not during stress.

Acute immobilization stress and intraventricular injection of CRF suppress naloxone-induced LH release in ovariectomized estrogen-primed rats.

The present study was undertaken to evaluate the role and possible interaction of the endogenous opioid peptide (EOP) and corticotropin-releasing factor (CRF) in the acute stress-induced suppression of gonadotropin secretion in ovariectomized estrogen-primed rats. An intravenous (i.v.) injection of naloxone (10 or 20 mg/kg), an EOP antagonist, significantly elevated serum luteinizing hormone (LH) levels within 10 min in non-stressed animals. The naloxone-induced LH release was completely eliminated when tested 30 min after the onset of acute immobilization. In a subsequent study, it was found that suppression of the naloxone-induced LH release occurred as early as 5 min after the stress onset, and was still evident 60 min after the end of a 30-min period of immobilization. The effect of naloxone was restored 3 h after liberation of the animal from the 30-min immobilization. An intraventricular (i.c.v.) injection of CRF (1 or 5 micrograms) also significantly suppressed, in a dose-related manner, the effect of a subsequent i.v. injection of naloxone. However, an i.c.v. injection of alpha-helical CRF(9-41) (25 or 50 micrograms), a CRF antagonist, prior to immobilization, could not interfere with the suppressive effect of stress on naloxone-induced LH release. These results suggest that both acute immobilization stress and CRF can inhibit the LH secretory activity without mediation by EOP neurons. However, the stress-related suppression may involve non-CRF mechanism(s).

An analysis of inputs to ON-OFF amacrine cells in the carp retina with kynurenic acid.

Amacrine cell inputs were studied in the carp retina. Responses to light of bipolar and amacrine cells in off-pathways were suppressed by 0.5 mM kynurenic acid (Kyn), while those in on-pathways were not. In ON-OFF amacrine cells, the sustained potential level during illumination shifted in the depolarizing direction. The depolarizing response elicited in off-center bipolar cells by transretinal current was also suppressed, but those elicited in amacrine cells were not. The results indicated that off-pathways are selectively suppressed by 0.5 mM Kyn at the level of receptor-bipolar synapses and demonstrated that sustained levels of ON-OFF amacrine responses are determined by a balance of inputs from on- and off-center bipolar cells.

The expression of rat GAP-43 cDNA in transgenic carp.

To understand more about growth-associated protein-43 (GAP-43), we produced transgenic carp by introduction of a rat GAP-43 cDNA linked to the Rous sarcoma virus-long terminal repeat into fertilized eggs. Of 180 eggs microinjected with exogenous gene, 59 embryos hatched and 4 fish were found to contain the exogenous gene sequences in the genomic DNA. From a mature female transgenic carp, parthenogenetically, 126 progeny were derived and 52 of them survived for more than 90 days. The exogenous gene sequences were detected in 22 F1 progeny, and its messenger RNA was detected in all of 10 transgenic F1 carp examined. In serum-free medium, cultured retinal ganglion cells isolated from transgenic carp elongated their axons, while non-transgenic cells did not elongate axons.

Dual effect of glutamate and aspartate on the on-center bipolar cell in the carp retina.

In on-center bipolar cells of the carp retina, glutamate and aspartate applied extracellularly by pressure injection produced hyperpolarizing potential changes. These responses were not blocked by Co2+. Examination of reversal potentials revealed two different conductance changes. One of them appears to be an increase in K+ and/or Cl- conductance and the other a decrease in Na+ conductance. The results are discussed with relation to the difference in ionic mechanisms of cone and rod inputs.

Push-pull modulation of ganglion cell responses of carp retina by amacrine cells.

The responses of a ganglion and an amacrine cell were recorded simultaneously in the carp retina. Sinusoidal current injected into amacrine cells modulated ganglion cell discharges either in phase (excitation) or in opposite phase (inhibition). ON-center ganglion cells received excitatory inputs and OFF-center ganglion cells received inhibitory inputs from ON-center amacrine cells. They received inputs of opposite polarity from OFF-center amacrine cells. Namely, inputs from ON-center and OFF-center amacrine cells augment the responses of ON-center and OFF-center ganglion cells in a push-pull manner.

Permissive role of corticotropin-releasing factor in the acute stress-induced prolactin release in female rats.

The present study examined the role of corticotropin-releasing factor (CRF) in the acute stress-induced release of prolactin (PRL) in ovariectomized estrogen-primed female rats. Acute immobilization stress induced a marked increase in serum PRL levels in animals treated with saline intraventricularly (i.c.v.). However, a prior icv injection of alpha-helical CRF(9-41), a CRF antagonist, completely eliminated the immobilization-induced PRL release in the majority of animals, providing evidence for involvement of CRF in the acute stress-induced PRL release. On the other hand, an i.c.v. injection of CRF did not affect basal PRL release at any dose in non-stressed animals, suggesting that the peptide plays a permissive role which enables other undefined stress mediator(s) to stimulate PRL release.

Voltage dependency of light-evoked on-off transient amacrine cell responses in carp retina.

The voltage dependency of the ON and the OFF components of transient amacrine cell responses was studied using two-electrode voltage-clamp and current-clamp techniques in the isolated retina of the carp. The two independent approaches gave similar data. When cells were voltage clamped near their resting potentials, both response components were associated with transient inward currents. Hyperpolarization increased response size (current or voltage) whilst depolarization decreased it. Response reversal, or a tendency for it, occurred at membrane potentials significantly more positive than the resting level with some quantitative variability. These data support the view that the ON-OFF depolarizations represent basically excitatory postsynaptic potentials and that the transience of the responses cannot mainly be due to any voltage-dependent conductance.