Depolarization waves in the embryonic CNS triggered by multiple sensory inputs and spontaneous activity: optical imaging with a voltage-sensitive dye.

Previously, we discovered a novel type of depolarization wave in the embryonic chick brain by using a multiple-site optical recording technique with a fast voltage-sensitive dye. This depolarization wave traveled widely over almost all the region of the CNS. This profile has raised the possibility that the depolarization wave plays some global roles in development of the CNS, rather than contributing to a specific neuronal circuit formation. To obtain more information concerning this issue, in the present study, we examined whether the depolarization wave was triggered by various types of peripheral nerve inputs. Stimulation applied to the vagus, glossopharyngeal, cochlear and trigeminal nerves evoked widely spreading depolarization waves with similar spatiotemporal distribution patterns. The developmental sequence of wave expression was parallel to the development of the excitatory postsynaptic potentials in each sensory nucleus. The depolarization wave was accompanied by a Ca(2+)-wave, suggesting that not only electrical synchrony, but also large-scale Ca(2+)-transients may affect developmental processes in the embryonic brain. Furthermore, we found that the depolarization wave also occurred spontaneously. The waveform and distribution patterns of the spontaneous optical signals were similar to those of the cranial nerve-evoked depolarization wave. These results demonstrated that the depolarization wave in the embryonic chick brain is triggered by multiple sources of external and endogenous activity. This profile supports the idea that this depolarization wave may not serve as a simple regulator of specific neuronal circuit formation, but might play more global roles in CNS development.

Role of adenosine and P2 receptors in the penile tumescence in anesthetized dogs.

We studied the role of adenosine and P2 receptors in the pelvic nerve stimulation-induced penile tumescence in anesthetized dogs. A local intracavernous injection of adenosine induced the tumescence, which was abolished by intracavernous 8-(p-sulfophenyl)theophylline (8-SPT), an unspecific adenosine receptor antagonist, and by 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol (ZM241385), an adenosine A(2A) receptor antagonist. ATP also induced the tumescence, which was diminished by 8-SPT, but not by reactive blue-2, a P2 receptor antagonist. Neither intracavernous beta, gamma-meATP nor ADP(beta)S, P2X and P2Y receptor agonists, induced tumescence. N(G)-nitro-L-arginine (L-NAME), a nitric oxide synthase inhibitor, and T-1032, a phosphodiesterase type V inhibitor, had no effects on the tumescence induced by adenosine. 8-SPT and reactive blue-2 had no effects on the tumescence induced by pelvic nerve stimulation. These results show that although exogenous adenosine and ATP induce tumescence, neither the adenosine nor the P2 receptor is involved in the tumescence induced by pelvic nerve stimulation in anesthetized dogs.

Optical imaging of spreading depolarization waves triggered by spinal nerve stimulation in the chick embryo: possible mechanisms for large-scale coactivation of the central nervous system.

Using a multiple-site optical recording technique with a voltage-sensitive dye, we found that widely spreading depolarization waves were evoked by dorsal root stimulation in embryonic chick spinal cords. Spatiotemporal maps of the depolarization waves showed that the signals were mainly distributed in the ventral half of the slice, with the highest activity in the ventrolateral area. The propagation velocity of the waves was estimated to be in the order of mm/s. Depolarization waves were evoked in the ventral root-cut preparation, but not in the dorsal root-cut preparation, suggesting that the wave was triggered by synaptic inputs from the primary afferents, and that activation of the motoneurons was not essential for wave generation. In intact spinal cord-brain preparations, the depolarization wave propagated rostrally and caudally for a distance of several spinal segments in normal Ringer's solution. In a Mg(2+)-free solution, the amplitude and extent of the signals were markedly enhanced, and the depolarization wave triggered in the cervical spinal cord propagated to the brainstem and the cerebellum. The depolarization wave demonstrated here had many similarities with the vagus nerve-evoked depolarization wave reported previously. The results suggest that functional cell-to-cell communication systems mediated by the depolarization wave are widely generated in the embryonic central nervous system, and could play a role in large-scale coactivation of the neurons in the spinal cord and brain.

Developmental changes in trial-to-trial variations in whisker barrel responses studied using intrinsic optical imaging: comparison between normal and de-whiskered rats.

We used an intrinsic optical imaging technique to examine postnatal developmental changes in the rat barrel response to a single whisker movement. We compared the optical response patterns between control and de-whiskered rats, from which whiskers were removed except for the D1 whisker just after birth. Barrel responses were evoked by D1-whisker movement stimulation, and the intrinsic optical signals were detected from the somatosensory cortex through the dura mater. In the control rats, the area of the barrel response decreased gradually as postnatal development proceeded from 2 to 7 wk, until reaching the adult pattern. On the other hand, in the de-whiskered rats, the barrel response area did not change during development and showed a larger size than in the control rats. We also compared the trial-to-trial variations in the barrel responses between the two groups. In the control rats, trial-to-trial variations in the optical responses were observed under the same conditions of whisker stimulation, and the extent of the variations decreased with postnatal development up to 7 wk. In the de-whiskered rats, trial-to-trial variations were also observed, but the extent was larger and unchanged during development. In both groups, the positions of the response area were the same with respect to the bregma. These results suggest that the decrease in the area and variations in the optical responses are caused by interactions of the corresponding whisker barrel with neighboring barrels and that these interactions are necessary for the developmental stabilization of the intracortical horizontal connections, which are widespread and have high plasticity in early postnatal periods.

Multiple-site optical recording reveals embryonic organization of synaptic networks in the chick spinal cord.

We examined embryonic expression of postsynaptic potentials in stages 26-31 (E5 to E7) chick spinal cord slices. Slow optical signals related to the postsynaptic potentials which were evoked by electrical stimulation of afferent fibers were identified in the dorsal grey matter and the ventral motoneuronal area. In cervical spinal cord (C13) preparations, the dorsal slow signal appeared from stage 28 (E6), whilst the ventral slow signal was recognized from stage 29. At stages 26 and 27 (E5), no slow signal was observed in either the dorsal or ventral regions. On the other hand, in lumbosacral spinal cord (LS5) preparations, the dorsal, as well as ventral, slow signals appeared from stage 29; at stage 28 no slow signal was detected in the dorsal or ventral regions. These results suggest that there are differences in the ontogenetic expression of synaptic functions between the dorsal and ventral regions, and between the cervical and lumbosacral spinal cords. In embryos older than stage 29, removal of Mg2+ from the bathing solution markedly enhanced the amplitude and incidence of the ventral slow signal. In addition, in C13 preparations at stage 28, removal of Mg2+ elicited small slow signals in the ventral region in which no synaptic response was evoked in normal Ringer's solution. The slow signals induced in the Mg2+-free solution were blocked by 2-amino-5-phosphonovaleric acid (APV), showing that they are attributable to N-methyl- D-aspartate (NMDA) receptors. These results suggest that functional synaptic connections via polysynaptic pathways are already generated on motoneurons, but are suppressed by a Mg2+ block on the NMDA receptors at developmental stages when synaptic transmission from the primary afferents to the dorsal interneurons is initially expressed in the dorsal region.

Effects of anisomycin on LTP in the hippocampal CA1: long-term analysis using optical recording.

Long-term potentiation (LTP) in the hippocampal CA1 region and in the dentate gyrus consists of different stages: early LTP lasting minutes or several hours, and late LTP lasting longer than 4 h. It has been suggested that the late phase of LTP is dependent on protein synthesis. However, the experimental results of the effects of protein synthesis inhibitors are still confusing. We applied optical recording techniques to rat hippocampal slices, and re-evaluated the effects of a protein synthesis inhibitor, anisomycin, on LTP. Using a voltage-sensitive oxonol dye, NK3630 (RH482), LTP in the CA1 region could be monitored optically for a long-term period (7-8 h). In the presence of anisomycin, the potentiation of the EPSP (excitatory postsynaptic potential) lasted about 2-3 h, followed by a gradual decline in the signal amplitude. Statistically, significant effects of anisomycin were observed 6 h after LTP induction for 100 Hz tetanus and 8 h after LTP induction for 400 Hz tetanus. These results suggest that the early phase of LTP is independent of protein synthesis, while the late phase of potentiation (> 3-5 h) depends on protein synthesis.

Optical responses to micro-application of GABA agonists in the embryonic chick brain stem.

We investigated optical responses induced by micro-application of muscimol and baclofen in the embryonic chick brain stem. Muscimol evoked biphasic optical signals which were similar to those induced by GABA. The first component was a dye-dependent absorption change that reflected membrane depolarization, and the second component was an intrinsic optical change coupled with changes in the membrane potential. On the other hand, baclofen did not elicit any optical change. The optical responses induced by muscimol persisted in the presence of picrotoxin and 2-hydroxysaclofen, suggesting that they contain a component which is not mediated by classical GABA receptors.

Spreading depolarization waves triggered by vagal stimulation in the embryonic chick brain: optical evidence for intercellular communication in the developing central nervous system.

Throughout experiments on multiple-site voltage-sensitive dye recordings of neural activity in embryonic chick brain preparations, we have found a novel type of depolarization waves which spread widely from the brainstem to the whole brain region at a rapid rate (mm/s). This depolarization wave was triggered by glutamate-mediated postsynaptic potentials and was especially correlated to N-methyl-D-aspartate receptor function. Evidence that the spreading depolarization wave is eliminated by octanol or 18beta-glycyrrhetinic acid suggests that the depolarization wave depends on functions of gap junctions. The profile obtained with Ca(2+)-imaging experiments also suggests that the propagation of the depolarization wave is accompanied by a calcium wave. These results provide new evidence for intercellular functional communication between neural cells in the vertebrate central nervous system during embryonic development.

Pharmacological profile of T-1032, a novel specific phosphodiesterase type 5 inhibitor, in isolated rat aorta and rabbit corpus cavernosum.

This study was designed to examine the pharmacological properties of T-1032 (methyl-2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate), a novel phosphodiesterase type 5 inhibitor, in isolated rat aorta and rabbit corpus cavernosum. T-1032 (3x10(-11) to 3x10(-7) M) caused an endothelium-dependent relaxation in the isolated rat aorta precontracted with phenylephrine, and the relaxation was accompanied by an increase in cGMP but not cAMP levels. The T-1032-induced relaxation was attenuated by N(G)-nitro-L-arginine methyl ester (L-NAME) (10(-3) M), a nitric oxide (NO) synthase inhibitor, or 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) (10(-5) M), a guanylyl cyclase inhibitor. T-1032 (10(-9), 10(-8) M) produced a potentiation of the relaxation induced by sodium nitroprusside, but not of the relaxation induced by isoproterenol. In the isolated rabbit corpus cavernosum precontracted with phenylephrine, the electrical field stimulation-induced relaxation was attenuated by treatment with tetrodotoxin (10(-6) M) as well as L-NAME (10(-4) M). The L-NAME-inhibited relaxation was restored by treatment with L-arginine (5x10(-4) M). T-1032 (10(-9) to 10(-6) M) and sildenafil (10(-9) to 10(-6) M) produced a potentiation of the electrical field stimulation-induced relaxation as well as a decrease in basal tension in a concentration-dependent manner. It was concluded that T-1032 had potentiating effects on the NO/cGMP signaling pathway in isolated tissues, probably through specific blockade of phosphodiesterase type 5. T-1032 would be a useful compound to examine the physiologic functions of phosphodiesterase type 5 in mammalian tissues.

Optical detection of embryogenetic expression of vagal excitability in the rat brain stem.

We traced and identified the ontogenetic expression of neural excitability related to the vagus nerve in the embryonic rat brain stem. Multiple-site optical recordings of neural activities revealed two response areas in the E12 rat brain stem: one corresponding to the dorsal motor nucleus of the vagus nerve, and the other reflecting the activities of sensory nerve fibers. In embryos younger than E11, no optical response was identified, suggesting that excitability of the motoneurons and/or sensory nerve fibers is first generated no later than E12. A contour line map of the neural responses suggested that, in contrast to older embryos, the functional organization of the vagal nucleus is not orderly at the time of the initial expression of neural excitability.

Evaluation of voltage-sensitive dyes for long-term recording of neural activity in the hippocampus.

We searched for an optimal voltage-sensitive dye for optical measurements of neural activity in the hippocampal slice by evaluating several merocyanine-rhodanine and oxonol dyes. The wavelength dependence (action spectra), pharmacological effects of staining, signal size, signal-to-noise ratio, and the utility of the dyes for long-term continuous recording were examined for four merocyanine-rhodanine dyes (NK2761, NK2776, NK3224 and NK3225), which had been reported to be optimal in embryonic nervous systems, and for two oxonol dyes (NK3630 (RH482) and NK3041 (RH155)), which have been among the most popular potentiometric probes for the hippocampal slice preparation. NK2761, NK3224 and NK3225 provided large signal-to-noise ratios, and proved to be useful for optical recordings lasting several hours. NK3630 was most suitable for long-term recording, although the signal-to-noise ratio was slightly inferior to that of the merocyanine-rhodanines. Using NK3630 (RH482) on the hippocampal slice preparation, we demonstrate here that long-term potentiation can be monitored stably for more than 8 hr.

Optical mapping reveals the functional organization of the trigeminal nuclei in the chick embryo.

The functional organization of the trigeminal nuclei during embryogenesis was investigated using multiple-site optical recording with a fast voltage-sensitive dye. Brainstem preparations with three classified trigeminal nerve afferents, the ophthalmic, maxillary and mandibular nerves, together with motor nerve fibers, were dissected from five- to eight-day-old chick embryos. Electrical responses evoked by trigeminal nerve stimulations were optically recorded simultaneously from many loci of the stained preparations. We identified three response areas related to the trigeminal nerve: area I, located cephalic to the level of the trigeminal ganglion; area II, located caudal to the level of the trigeminal ganglion; and area III, located at the level of the trigeminal root. The neural responses in areas I and II were evoked by ophthalmic, maxillary or mandibular nerve stimulation, while the responses in area III were detected when the stimulation was applied to the trigeminal motor nerve. In comparison with the morphology indicated by DiI labeling, the results suggest that areas I, II and III correspond to the principal sensory nucleus of the trigeminal nerve, the spinal sensory nucleus of the trigeminal nerve and the trigeminal motor nucleus, respectively. We identified two components of the optical response: a fast and a slow signal. In five-day-old preparations, fast spike-like signals related to action potentials were recorded from the three response areas. In six-day-old preparations, slow optical signals which reflect glutamate-mediated excitatory postsynaptic potentials were detected from area II only when the ophthalmic nerve was stimulated: no slow signal was evoked by maxillary or mandibular nerve stimulation. In seven- and eight-day-old preparations, slow signals were detected from both areas I and II with every nerve stimulation. These results suggest that synaptic function is first generated in the spinal trigeminal nucleus by the six-day embryonic stage, and the developmental organization of synaptic function is not the same in the three trigeminal nerves or in the two sensory nuclei. Contour line maps of the signal amplitude revealed that the size and the area of the neural responses within the trigeminal nuclei changed dramatically with development. We compared the spatial distribution and temporal dynamics of the optical signals between the ophthalmic, maxillary and mandibular nerve stimulations, and we found that somatotopic organization is less clear in a rostrocaudal/mediolateral X-Y plane, although the areas of the maxillary and mandibular nerves appeared to separate in the lateral direction.

Production and characterization of specific anti-peptide antiserum against free alpha-subunit of rat pituitary glycoprotein hormones.

To obtain an antibody specific for the alpha-subunit of rat pituitary glycoprotein hormones, we synthesized a peptide corresponding to the sequence 37-53 (ST-7: Phe-Ser-Arg-Ala-Tyr-Pro-Thr-Pro-Ala-Arg-Ser-Lys-Lys-Thr-Met-Leu-Val) of the rat alpha-subunit. The polyclonal antiserum against this peptide was generated in rabbits. This region is hydrophilic and highly conserved among several mammalian species. Noncompetitive binding tests showed that the ST-7 antiserum had specific affinity for the rat free alpha-subunit, but not for rat intact LH, FSH, and TSH. The ST-7 antiserum immunostained two types of cells in the rat anterior pituitary, i.e., gonadotrophs and thyrotrophs. This was also the case in mouse, cattle, sheep, and pig, which have an identical sequence of ST-7 in their alpha-subunit. The pituitary cells of horse (Arg substituted for Lys as residue 48 of the rat alpha-subunit), human, and eel (Leu for Ala at residue 45), chicken (Met for Ala at residue 45), and bullfrog (Tyr for Phe at residue 37 and Met for Ala at residue 45) were not stained with the ST-7 antiserum. This study indicated that the ST-7 antiserum is sequence-specific for the alpha-subunit and is therefore useful for immunohistochemical studies on the secretory pathway of the free alpha-subunit.

Mutagenicity of dihydroxybenzenes and dihydroxynaphthalenes for Ames Salmonella tester strains.

The mutagenicity of 3 dihydroxybenzene (DHB) and 9 dihydroxynaphthalene (DHN) isomers was examined by using 5 different Ames Salmonella mutagenicity tester strains in the presence and absence of phenobarbital and 5,6-benzoflavone-treated rat liver S9-mix. Of the 3 DHB isomers, 1,4-DHB (hydroquinone) was mutagenic, and of the 9 DHN isomers, 1,3-DHN (naphthoresorcinol), 1,4-DHN (hydronaphthoquinone), 1,6-DHN and 1,7-DHN were mutagenic. Mutagenicity of all the compounds tested was observed in the absence of S9-mix, while 1,4-DHN and 1,6-DHN were also mutagenic in the presence of S9-mix. The mutagenicity of 1,4-DHB and 1,4-DHN for TA104, which is a strain sensitive to oxidative mutagens, was almost completely or partially inhibited by superoxide dismutase (SOD) and/or catalase, indicating the involvement of activated oxygen species in mutagenesis. Furthermore, from the finding that the 4 DHNs were mutagenic for TA2637, the strain sensitive to frameshift mutagens, it is possible that the mutagenicity of DHNs for S. typhimurium was also attributable to DNA adducts that form with quinones and/or semiquinones through oxidation of DHNs. The mutagenicity of 1,3-DHN, which showed the largest number of revertants in strains TA100, TA98, TA2637 and TA104, was greatly decreased, when their pKM101 plasmid-deficient strains, TA1535, TA1538, TA1537 and TA2659 were used. This observation suggests that an SOS repair system was involved in the mutagenesis of 1,3-DHN for S. typhimurium.

Immunocytochemical localization of prohormone convertases PC1/PC3 and PC2 in rat pancreatic islets.

The prohormone convertases PC1/PC3 and PC2 are endoproteases involved in prohormone cleavage at pairs of basic amino acids. To determine the cellular and subcellular distribution of PC1/PC3 and PC2 in the rat pancreas, we generated their polyclonal antisera in rabbits, using as immunogens two synthetic peptide antigens corresponding to amino acids 442-459 (ST-28) of PC1/PC3 and 613-629 (ST-29) of PC2 and two bacterially expressed antigens covering amino acids 145-414 (KN-1) of PC1/PC3 and 385-637 (KN-2) of PC2. Western blot analysis revealed the presence of PC1/PC3 (87 and 68 kDa) and PC2 (75 and 70 kDa) in rat pancreatic islets, indicating that the antisera are specific for the corresponding antigens. Immunocytochemical staining of serial sections demonstrated that the antibody against PC1/PC3 immunostained only insulin-producing cells, whereas the PC2 antibody stained insulin, glucagon-, somatostatin-, and pancreatic polypeptide-producing cells. Double-immunolabeling of the prohormone convertases and pancreatic hormones with gold particles of different sizes revealed that insulin-positive secretory granules were also immunolabeled with PC1/PC3 and PC2 antibodies, whereas glucagon-, somatostatin-, or pancreatic polypeptide-positive granules were labeled only with the PC2 antibody. This differential localization of PC1/PC3 and PC2 provides a further problem on the substrate-specificity of these enzymes in the processing of pancreatic prohormones.

Injury to infrapatellar branch of saphenous nerve in arthroscopic knee surgery.

Injury to the infrapatellar branch of the saphenous nerve has been reported as a complication of arthroscopic examination and surgery of the knee. The authors studied the anatomic distribution of this branch in cadavers, and investigated the incidence of this complication in 68 patients. The results of anatomic study showed that blind puncture is safe within an approximate 30-mm area from the medial margin of the patella at the level of midpatella, and within an approximate 10-mm area from the medial margin of the patellar ligament at the level of the distal pole of the patella. In 30% of examined cadavers, the infrapatellar branch of the saphenous nerve transverses and runs laterally before it crosses the proximal edge of the tibia. Anatomic findings indicated that blind puncture to the knee in a 90 degrees flexion position should be done horizontally and parallel to the articular surface to reduce the incidence of nerve injury. The results of this study of patients who had arthroscopy from 1990 to 1991 revealed a 22.2% incidence rate of sensory disturbances in the area where the infrapatellar branch is distributed. The incidence can be minimized by clarifying the distribution of the infrapatellar nerve branch in relation to palpable landmarks.

Mutagenicity of benzoquinones for Ames Salmonella tester strains.

The mutagenicity of 12 simple benzoquinone (BQ) derivatives was studied using five different Ames Salmonella mutagenicity tester strains in the presence and absence of S9 mix. Seven of the BQs used displayed mutagenicity with and/or without S9 mix, and most of them produced a marginal increase in revertants. p-Benzoquinone (p-BQ) showed the most potent mutagenic activity (17 induced revertants/nmol/plate for strain TA104 without S9 mix) among the BQs tested. TA104, which is sensitive to oxidative mutagens, was the most sensitive to the mutagenicity of the BQs of the five strains used, while the second most sensitive strain was TA2637, which detects bulky DNA adducts. Significant reductions in the mutagenicity of p-BQ, and 2,3-diCl-5,6-diCN-BQ without S9 mix were observed in the presence of catalase. These findings suggest that the mutagenicity of BQs for S. typhimurium is attributable to oxidative injury after BQ reduction and to DNA adducts that form with BQs that have electrophilic substituents.

[New sex pheromone isolated from the abdominal glands of male newt, Cynops pyrrhogaster]. / Une nouvelle phéromone sexuelle isolée à partir des glandes abdominales du Triton mâle, Cynops pyrrhogaster.

Sodefrin, a novel decapeptide which attracts female newts, Cynops pyrrhogaster was isolated from the abdominal gland of the male of the same species. Synthetic sodefrin was tested for its activity with sexually active females and males and sexually inert females. It attracted only sexually active females. The effect of sodefrin was blocked by a bilateral nostril plugging with cotton balls soaked in melted vaseline. Immunoelectronmicroscopic study using antiserum against sodefrin revealed that sodefrin was located mainly in the secretory granules in the epithelial cells of the abdominal gland of the cloaca.

Mutagenicity and cytotoxicity of naphthoquinones for Ames Salmonella tester strains.

The molecular mechanisms involved in quinone cytotoxicity, especially mutagenicity, are still largely unknown. In order to better understand the molecular aspects of the mechanisms of quinone mutagenicity and cytotoxicity, we examined them by using a series of 13 simple structural naphthoquinone (NQ) derivatives for 9 Ames Salmonella mutagenicity tester strains in the presence of absence of liver homogenate S9 mix from rats induced with phenobarbital and 5,6-benzoflavone. Most NQs used in this study showed mutagenicity with and/or without S9 mix. The most potent mutagenic NQ was 2,3-dichloro-1,4-NQ, with mutagenicity of 18 induced revertants/nmol/plate for strain TA104 without S9 mix. Among the strains used, TA104, which is sensitive to oxidative mutagens, was the most sensitive to the NQs, and the second most sensitive strain was TA2637, which detects bulky DNA adducts. The relationship of mutagenic potency to the one-electron reduction potential with TA104 suggested that the higher redox potential NQs were more mutagenic than the lower redox potential NQs. Significant reduction of the mutagenicity of 1,4-naphthoquinone without S9 mix was observed in the presence of catalase. Enhancement of the mutagenic potential of the NQs by the pKM101 plasmid implicated in error-prone repair was also observed. The most cytotoxic NQ was 2,3-dichloro-5,8-dihydroxy-1,4-NQ, and the least cytotoxic NQ was beta-NQ-4-sulfonic acid potassium salt, a 700-fold range in potency. The cytotoxic effect of the NQs was largely dependent on the structures of their substituents.(ABSTRACT TRUNCATED AT 250 WORDS)