Color-opponent responses of small and giant bipolar cells in the carp retina.

The physiological and morphological properties of color-opponent bipolar cells in the carp retina were studied. Fifty nine OFF-center bipolar cells and 63 ON-center bipolar cells out of about 500 total bipolar cells recorded showed color-opponent responses. The OFF-center color-opponent bipolar cells were classified into three subgroups according to their spectral and spatial responses. Fifty OFF-center color-opponent cells responded with depolarization to a blue light spot and with hyperpolarization to a red spot in the receptive-field center. The polarity of the surround response was opposite to that of center response at each wavelength. Therefore these cells were classified as OFF double-opponent cells (OFF-DO). Eight cells responded with hyperpolarization to a blue and green spot and with depolarization to a red spot. The surround responses of those cells were depolarizing at any wavelength (R+G- cell). One responded with hyperpolarization to a blue and red spot and with depolarization to a green spot. The surround response showed a different spectral characteristic from that of the center response. It responded with depolarization to a blue and green annulus and with hyperpolarization to a red annulus (R-G+B- cell). The ON-center color-opponent bipolar cells were similarly classified into three subgroups. Sixty of ON-center color-opponent cells were the double color-opponent type (ON-DO cell), showing the responses of opposite polarity to the OFF-DO cells. Two cells were classified as R- G+ cell, and one cell as R+G-B+ cell. Both OFF- and ON-DO cells were identified by their morphology as Cajal's giant bipolar cells, and R+G-, R-G+, R-G+B-, and R+G-B+ cells as Cajal's small bipolar cells. The analysis of the latency and the ionic mechanisms of their responses suggest that DO cells under light-adapted conditions receive direct inputs from long-wavelength (red) cones, RG cells from middle-wavelength (green) cones, and RGB cells from short-wavelength (blue) cones. Possible mechanisms of the opponent inputs to these bipolar cells are discussed.

Intraventricular injection of melatonin inhibits naloxone-induced, but not NMDA- or LHRH-induced LH release in ovariectomized estrogen-primed rats.

The present study was aimed to examine the possible functional relationship between melatonin and hypothalamic transmitters, endogenous opioids and excitatory amino acids in controlling gonadotropin secretion in ovariectomized estrogen-primed rats. An intravenous injection of naloxone (mu opioid receptor antagonist), N-methyl-D-aspartate (NMDA; NMDA receptor agonist) or luteinizing hormone-releasing hormone (LHRH) significantly elevated serum luteinizing hormone (LH) concentrations within 10 min. An intraventricular treatment with melatonin, which did not affect the basal LH concentration by itself, significantly suppressed the effect of naloxone. However, the same melatonin treatment did not inhibit the NMDA-induced or LHRH-induced LH secretion. These results support the hypothesis that melatonin has a suprapituitary site of action to inhibit LHRH release, and suggest that the site of its action may be located downstream to that of naloxone action and upstream to that of NMDA in the hypothalamic LHRH neuronal pathway.

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.

Differential inhibition of NMDA- and naloxone-induced LH release by NMDA receptor antagonist and CRH in ovariectomized estrogen-primed rats.

A possible functional relationship between endogenous opioid peptides (EOPs), corticotropin-releasing hormone (CRH) and excitatory amino acids (EAAs) in the control of LH secretion was investigated in ovariectomized estrogen-primed rats. An intraventricular (icv) injection of an EAA agonist, N-methyl-D-aspartate (NMDA), or an EOP antagonist, naloxone, produced an abrupt increase in the serum LH level. While icv pretreatment of the animals with 2-amino-5-phosphonovaleric acid, a specific NMDA receptor antagonist, did not affect by itself basal LH levels, it significantly suppressed the NMDA-induced and also the naloxone-induced LH release. An icv injection of CRH also interfered with the naloxone-induced LH release. However, the NMDA-induced LH release was not affected by an icv injection of CRH or of beta-endorphin. These results suggest that the sites of EOP and CRH inhibition may be located upstream of the site of NMDA stimulation on the GnRH neuronal pathway, and that CRH can inhibit LH secretion without mediation by EOP neurons.

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.

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).

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.

Acute stress suppresses the N-methyl-D-aspartate-induced luteinizing hormone release in the ovariectomized estrogen-primed rat.

The effects of N-methyl-D-aspartate (NMDA) and luteinizing hormone releasing hormone (LH-RH) on luteinizing hormone (LH) secretion were examined in ovariectomized estrogen-primed rats under nonstressed and acutely stressed conditions. The basal LH levels were significantly elevated 15 min after the onset of acute immobilization stress, but were not altered in emotionally stressed or nonstressed rats. Intravenous injections of 10 and 40 mg/kg NMDA significantly elevated serum LH levels by 161 and 212%, respectively, from baseline within 10 min in nonstressed animals. However, the NMDA-induced LH release was significantly reduced when tested 30 min after the onset of acute immobilization stress. Acute emotional stress, which did not affect the baseline LH, also suppressed the LH release response to NMDA, suggesting that the reduced LH responses to NMDA in stressed animals was not due to the elevated baseline level. Pituitary LH release responses to LH-RH were not affected by acute immobilization. We conclude from these results: (1) acute immobilization stress exerts both stimulatory and inhibitory effects on LH release, while acute emotional stress has only an inhibitory effect in estrogen-primed ovariectomized rats; (2) this inhibition occurs at the suprapituitary level, and (3) it involves a suppression of the responsiveness of the hypothalamic LH-RH neuronal system to the excitatory amino acid input.

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.

Chondroitin sulfate immobilized onto culture substrates modulates DNA synthesis, tyrosine aminotransferase induction, and intercellular communication in primary rat hepatocytes.

Newly prepared phosphatidylethanolamine (PE) conjugates of glycosaminoglycans (GAGs) enabled us to immobilize GAGs to solid phase through hydrophobic interaction. Using these compounds, called GAG-PEs, we studied the effects of GAGs immobilized to culture plates coated with various extracellular matrix (ECM) proteins in terms of cell-substrate adhesion, DNA synthesis, tyrosine aminotransferase (TAT) induction, and intercellular communication in primary rat hepatocytes. Treatment with chondroitin sulfate (CS)-PE at 10 micrograms/ml made laminin, fibronectin, vitronectin, and collagens type I-V less adhesive as substrates for cell attachment and inhibited cell spreading on these substrates. The effect on cell attachment was lost after long incubations over 2 h. Dermatan sulfate (DS)-PE was also inhibitory, but less effective. The conjugates of heparan sulfate (HS), heparin, and hyaluronan were much less effective. DNA synthesis initiated by EGF in the culture on laminin substrates was inhibited most strongly by CS-PE, as well as by DS-PE and hyaluronan-PE, but not by either HS-PE or heparin-PE. With type I collagen substrates, GAG-PEs had similar effects but to lesser extent. TAT induction in the culture on laminin substrates but not on type I collagen substrates was significantly enhanced by CS-PE. In terms of DNA synthesis and TAT induction, the culture on laminin substrates treated with CS-PE was comparable to that at higher cell density on the non-treated laminin substrates. Intercellular communication assessed by dye coupling was maintained longer on the substrates treated with CS-PE. Taken together, our results demonstrate that CS immobilized especially onto laminin substrates inhibits the growth of hepatocytes and enhances their differentiated functions by modulating cell-ECM protein interactions.

Positron emission tomography and plasma biochemistry findings in schizophrenic patients before and after electroconvulsive therapy.

The clinical effects of electroconvulsive therapy (ECT) on the morbidity of paranoid schizophrenic patients were assessed by positron emission tomography (PET) and plasma biochemistry studies before and after ECT. The present study included five patients whose average age was 41.4 years. The average duration of illness was 23.0 years. To avoid any effect of changes in drugs on PET, no changes were made in the medication of any of the five patients during the study period. ECT improved the clinical symptoms in every patient. Regional cerebral blood flow (rCBF) on PET in both temporal lobes and the left cerebellum was higher in paranoid schizophrenia before ECT than in normal subjects, and rCBF after ECT in both frontal lobes, the right temporal lobe and the right putamen was lower than before ECT as mental symptoms improved. These findings suggest high cerebral blood flow volume in paranoid schizophrenia. Plasma biochemistry studies revealed a lower level of 3-methoxy-4-hydroxyphenylglycol (MHPG) after ECT than before ECT, but a higher level of prolactin existed.

Effects of pinealectomy and melatonin on the timing of the proestrous luteinizing hormone surge in the rat.

Effects of pinealectomy and melatonin replacement on the timing of the preovulatory luteinizing hormone (LH) surge were examined in female rats housed under a light-dark cycle (lights on 06:30-18:30 h). Animals were pinealectomized or sham-operated 21-28 days before bleeding. They were sequentially bled in the afternoon of proestrus through the indwelling cardiac cannula without anesthesia. Proestrous LH surges were observed in all pinealectomized rats, but the onset times of the LH surge in these animals showed a significantly greater variance than those in sham-operated controls. A single melatonin injection at 18:00 or 21:00, shortly before or after the light-dark transition, on the day before proestrus (diestrus II) reduced the variance in the LH surge onset in pinealectomized rats in a dose-dependent manner. In contrast, melatonin injected at 14:00 or 23:00 on diestrus II or at 04:00 on proestrus was ineffective. These results showed that the pineal gland is involved in controlling the timing of the proestrous LH surge in the rat. The pineal signal may be transmitted by melatonin, a major product of the gland.

Brain morphogenesis of the red sea bream, Pagrus major (teleostei).

Post-embryonic morphogenesis and regional volumetric growth of the brain in larval and juvenile red sea bream, Pagrus major, were analyzed by employing a computer-aided three-dimensional reconstruction of serial sections of the head. The relative volume of the optic tectum rapidly increases after hatching until day 8, implying the importance of the visual sense for the first feeding, which begins on day 4. The differentiation and outgrowth of the primary gustatory center in the medulla after day 43 may reflect the increasing number of taste buds, suggesting a relationship to the onset of benthic feeding as juveniles. The rather rapid increases in the relative volumes of the corpus and valvula cerebelli after day 28 and their progressive morphogenesis may be correlated with improved motor performance. The relative volumes of the octaval-lateral line centers, including the eminentia granularis and the crista cerebellaris, and the relative volume of the torus longitudinalis, increase in parallel with those of the corpus and valvula cerebelli.

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.

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.

Intracellular and extracellular chloride ions in the horizontal cells of the stingray retina.

Intracellular and extracellular concentrations of chloride ([Cl-]i, [Cl-]o) ions in the horizontal cells of the stingray retina were studied by means of ion-selective microelectrodes. The electrodes used were of the double-barreled type and Corning's #477315 resin was used as the exchanger. The average [Cl-]o and [Cl-]i in the L-type cells were 320 and 130 mM, respectively (n = 37), and the equilibrium potential (ECl) was calculated as -23.3 mV. In some cases, dark membrane potentials were more positive than the ECl. With white light stimulus, the membrane hyperpolarized to a more negative level than ECl. Based on the experiments described above, the hypothesis that the hyperpolarizing response of horizontal cells is due to the permeability change of the membrane to chloride ion was excluded in the stingray retina.

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.

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.

Effects of nitro compounds, isosorbide dinitrate, 5-isosorbide mononitrate and glyceryl trinitrate on Ca-uptake into Ca-stores and Ca-release from Ca-stores in rabbit isolated femoral veins and femoral arteries.

In organ bath studies, effects of isosorbide dinitrate (ISDN), 5-isosorbide mononitrate (ISMN), a major metabolite of ISDN, and glyceryl trinitrate (GTN) on Ca-uptake into Ca-stores and Ca-release from Ca-stores were tested in the rabbit isolated femoral veins and femoral arteries. ISDN (10(-4) M) and GTN (10(-4) M) inhibited Ca-uptake in the femoral veins but not in the femoral arteries. The selectivity to the femoral veins was not observed in ISMN (10(-3) M) and GTN (3 X 10(-6) M). All the nitro compounds inhibited Ca-release from Ca-stores more effectively in the femoral veins than in the femoral arteries. The present results may explain the selectivity of the nitro compounds to the femoral veins.