Stretch-induced Ca2+ independent ATP release in hippocampal astrocytes.

KEY POINTS: Similar to neurons, astrocytes actively participate in synaptic transmission via releasing gliotransmitters. The Ca2+ -dependent release of gliotransmitters includes glutamate and ATP. Following an 'on-cell-like' mechanical stimulus to a single astrocyte, Ca2+ independent single, large, non-quantal, ATP release occurs. Astrocytic ATP release is inhibited by either selective antagonist treatment or genetic knockdown of P2X7 receptor channels. Our work suggests that ATP can be released from astrocytes via two independent pathways in hippocampal astrocytes; in addition to the known Ca2+ -dependent vesicular release, larger non-quantal ATP release depends on P2X7 channels following mechanical stretch. ABSTRACT: Astrocytic ATP release is essential for brain functions such as synaptic long-term potentiation for learning and memory. However, whether and how ATP is released via exocytosis remains hotly debated. All previous studies of non-vesicular ATP release have used indirect assays. By contrast, two recent studies report vesicular ATP release using more direct assays. In the present study, using patch clamped 'ATP-sniffer cells', we re-investigated astrocytic ATP release at single-vesicle resolution in hippocampal astrocytes. Following an 'on-cell-like' mechanical stimulus of a single astrocyte, a Ca2+ independent single large non-quantal ATP release occurred, in contrast to the Ca2+ -dependent multiple small quantal ATP release in a chromaffin cell. The mechanical stimulation-induced ATP release from an astrocyte was inhibited by either exposure to a selective antagonist or genetic knockdown of P2X7 receptor channels. Functional P2X7 channels were expressed in astrocytes in hippocampal brain slices. Thus, in addition to small quantal ATP release, larger non-quantal ATP release depends on P2X7 channels in astrocytes.

Ginsenoside Rd protects neurons against glutamate-induced excitotoxicity by inhibiting ca(2+) influx.

Our previous studies have demonstrated that ginsenoside Rd (GSRd), one of the principal ingredients of Pana notoginseng, has neuroprotective effects against ischemic stroke. However, the possible mechanism(s) underlying the neuroprotection of GSRd is/are still largely unknown. In this study, we treated glutamate-injured cultured rat hippocampal neurons with different concentrations of GSRd, and then examined the changes in neuronal apoptosis and intracellular free Ca(2+) concentration. Our MTT assay showed that GSRd significantly increased the survival of neurons injured by glutamate in a dose-dependent manner. Consistently, TUNEL and Caspase-3 staining showed that GSRd attenuated glutamate-induced cell death. Furthermore, calcium imaging assay revealed that GSRd significantly attenuated the glutamate-induced increase of intracellular free Ca(2+) and also inhibited NMDA-triggered Ca(2+) influx. Thus, the present study demonstrates that GSRd protects the cultured hippocampal neurons against glutamate-induced excitotoxicity, and that this neuroprotective effect may result from the inhibitory effects of GSRd on Ca(2+) influx.

Effects of vagotomy, splanchnic nerve lesion, and fluorocitrate on the transmission of acute hyperosmotic stress signals to the supraoptic nucleus.

The response to hyperosmotic stresses in the abdominal cavity is regulated, in part, by vasopressin (VP)-secreting neurons in the supraoptic nucleus (SON). How osmotic stress signals are transmitted to the brain is incompletely understood, and whether the transmission routes for osmotic stress signals differ between acute and chronic stresses is unknown. Here we investigated the role of the vagus, splanchnic nerves, and astrocytes in the SON in transducing acute hyperosmotic-stress signals from the abdominal cavity. We found that acute administration of hyperosmotic saline triggered the activation of neurons as well as astrocytes in the SON and the adjoining ventral glia limitans (SON-VGL). Severing the subdiaphragmatic vagal nerve (SDV) prevented the normal response of cells in the SON to HS treatment and attenuated the release of VP into the bloodstream. Lesioning the splanchnic nerves (SNL) diminished HS-induced release of VP, but to a much lesser extent than SDV. Furthermore, SNL did not significantly affect the up-regulation of Fos in SON neurons or the up-regulation of Fos and GFAP in SON and SON-VGL astrocytes that normally occurred in response to HS and did not affect HS-induced expansion of the SON-VGL. Inhibiting astrocytes with fluorocitrate (FCA) prevented the response of the SON to HS and attenuated the release of VP, similarly to SDV surgery. These results suggest that the vagus is the principle route for the transmission of hyperosmotic signals to the brain and that astrocytes in the SON region are necessary for the activation of SON neurons and the release of VP into the bloodstream.

Activity-induced rapid synaptic maturation mediated by presynaptic cdc42 signaling.

Maturation of presynaptic transmitter secretion machinery is a critical step in synaptogenesis. Here we report that a brief train of presynaptic action potentials rapidly converts early nonfunctional contacts between cultured hippocampal neurons into functional synapses by enhancing presynaptic glutamate release. The enhanced release was confirmed by a marked increase in the number of depolarization-induced FM4-64 puncta in the presynaptic axon. This rapid presynaptic maturation can be abolished by treatments that interfered with presynaptic BDNF and Cdc42 signaling or actin polymerization. Activation of Cdc42 by applying BDNF or bradykinin mimicked the effect of electrical activity in promoting synaptic maturation. Furthermore, activity-induced increase in presynaptic actin polymerization, as revealed by increased concentration of actin-YFP at axon boutons, was abolished by inhibiting BDNF and Cdc42 signaling. Thus, rapid presynaptic maturation induced by neuronal activity is mediated by presynaptic activation of the Cdc42 signaling pathway.

Acute hyperosmotic stimulus-induced Fos expression in neurons depends on activation of astrocytes in the supraoptic nucleus of rats.

Acute hyperosmolarity induced a time-dependent expression of Fos protein in both neurons and astrocytes of the rat supraoptic nucleus, with peak Fos expression occurring at 45 min in astrocytes and at 90 min in neurons after hypertonic stimulation in vivo. To determine whether the two cell types were activated separately or in an integrated manner, animals were pretreated with fluorocitrate, a glial metabolic blocker or carbenoxolone, a gap junction blocker followed by an acute hypertonic stimulation similar to that of the controls. Antibodies against glial fibrillary acidic protein, connexin 43, vasopressin, and oxytocin were used in serial sections to identify the cellular elements of the supraoptic nucleus. It was found that interruption of astrocyte metabolism with fluorocitrate significantly reduced Fos protein expression in both astrocytes and neurons, whereas blockage of gap junctions with carbenoxolone clearly reduced Fos protein expression in neurons, but not in astrocytes. These results indicate that both neurons and astrocytes in the rat supraoptic nucleus are involved in regulating osmolarity. Astrocytes are activated first, whereas connexin 43 functional hemichannels in SON astrocytes are required for the subsequent activation of the neurons.

Reciprocal pathway between medullary visceral zone and hypothalamic supraoptic nucleus or paraventricular nucleus involved in hyperosmotic regulation.

In this study we try to simultaneously investigate the response of neurons and astrocytes of rats following hyperosmotic stimulation and test the possibility that the reciprocal pathways between medullary visceral zone (MVZ) and hypothalamic paraventricular nucleus (PVN) or supraoptic nucleus (SON). Hyperosmotic pressure animal model was established by administering 3% sodium chloride as drinking water to rats. The distribution and expression of the HRP retrogradely labeled neurons, Fos, tyrosine hydroxylase (TH) or vasopressin (VP) positive neuron and glial fibrillary acidic protein (GFAP) positive astrocytes in the MVZ, SON and PVN were observed by quadruplicate-labeling methods of WGA-HRP retrograde tracing combined with anti-Fos, TH (or VP) and GFAP immunohistochemical technique. Fos positive neurons within the MVZ, PVN and SON increased markedly. There were also a large number of GFAP positive structures in the brain and their distribution pattern was fundamentally similar or analogous to Fos positive neurons in the above-mentioned areas. The augmented GFAP reactivities took on hypertrophic cell bodies, thicker and longer processes. Quadruplicate immunohistochemical staining showed that a neuron could be closely surrounded by many astrocytes and they formed neuron-astrocytic complex (N-ASC). Fos+/TH+/HRP+/GFAP+ and Fos+/VP+/HRP+/GFAP+ quadruplicate labeled N-ASC could be found in the MVZ, PVN and SON, respectively. The present results indicated that the neurons and astrocytes might be very active following hyperosmotic pressure and N-ASC as a functional unit might serve to modulate osmotic pressure. There were reciprocal osmoregulation pathways between the MVZ and SON or PVN in the brain.

Involvement of activated astrocyte and microglia of locus coeruleus in cardiac pain processing after acute cardiac injury.

OBJECTIVE: It is still not known whether the glial cell activation of locus coeruleus (LC) is involved in the neurophysiologic mechanism of the acute phase of heart disease. The aim of this study was to investigate whether the glial cell activation of LC responds to acute cardiac injury (ACI). METHODS: In this study, ACI was established by intramyocardial injection of formalin. Afterward, we analysed c-Fos, OX42, GFAP and P2X(4)R expression levels in the LC of the rats by immunofluorescence staining or Western blot analysis. RESULTS: There was no significant difference in the levels of these markers in the LC between the normal control and the sham-operated groups. Following ACI, up-regulation of GFAP, OX42 and P2X(4)R expression levels were observed in locus coeruleus of the rats. The peak expression time was at hour 24. P2X(4)R was colocalized with OX42 in activated microglias, but not with GFAP in activated astrocytes. Compared with the control group, the ACI group showed a high expression level of c-Fos at hour 1 with a peak expression level at hour 2. CONCLUSION: The results showed that LC glia cells, like neurons, could sensitively respond to cardiovascular nociception induced by ACI at different time points. Results of this study may provide insights into the role of glial activation in response to ACI and may represent a potential strategy for investigation of neurophysiologic mechanism of cardiac pain.

Blocking the glial function suppresses subcutaneous formalin-induced nociceptive behavior in the rat.

This study examined whether glial cells in the trigeminal nucleus caudalis (Sp5C) were necessary for orofacial nociception and nociceptive processing induced by subcutaneously (s.c.) injection of 5% formalin into left mystacial vibrissae. The immunohistochemical, immunoelectron microscopical methods and behavior assessment were used in this study. Two hours after administration of carbenoxolone (CBX, a gap junction blocker) or fluorocistrate (FCA, a glail metabolic inhibitor) into the cerebellomedullary cistern, the nociceptive behavior and scratching-cumulative time reduced significantly (P<0.01). FCA attenuated obviously the expression of Fos/NeuN-immunoreactive (-IR) neurons (mean+/-S.E.M.=29+/-2.5) and Fos/glial fibrillary acidic protein (GFAP)-IR astrocytes (7.2+/-2.2) in Sp5C. CBX decreased the number of Fos/NeuN-IR neurons (25+/-1.7), but did not affect Fos/GFAP-IR astrocytes (16.2+/-5.4), compared with vehicle-preadministered rats (Fos/NeuN-IR neurons 135+/-4.2, and Fos/GFAP-IR astrocytes 25.8+/-4). Immunoelectron microscopy established that Cx32/Cx43 heterotypic gap junctions (HGJs) were present on junction areas between astrocytes and neurons within Sp5C. The number of HGJs increased significantly following formalin s.c. injection. It suggests that the Sp5C astrocytes may play an active regulating role in orofacial nociception via Cx32/Cx43 HGJs between astrocytes and neurons of Sp5C.

Topiramate reduced sweat secretion and aquaporin-5 expression in sweat glands of mice.

Decreased sweat secretion is a primary side effect of topiramate in pediatric patients, but the mechanism underlying this effect remains unclear. This study aimed to better understand how topiramate decreases sweat secretion by examining its effect on the expression of carbonic anhydrase (CA) II and aquaporin-5 (AQP5), total CA activity, as well as on tissue morphology of sweat glands in mice. Both developing and mature mice were treated with a low (20 mg/kg/day) and high dose (80 mg/kg/day) of topiramate for 4 weeks. Sweat secretion was investigated by an established technique of examining mold impressions of hind paws. CA II and AQP5 expression levels were determined by immunofluorescence and immunoblotting and CA activity by a colorimetric assay. In mature mice, topiramate treatment decreased the number of pilocarpine reactive sweat glands from baseline in both the low and high dose groups by 83% and 75%, respectively. A similar decrease was seen in developing mice. Mature mice with reactive sweat glands that declined more than 25% compared to baseline were defined as anhidrotic mice. These mice did not differ from controls in average secretory coil diameter, CA II expression and CA activity. In contrast, anhidrotic mice did show a reduction in membrane AQP5 expression in sweat glands after topiramate delivery. Thus, sweat secretion and membrane AQP5 expression in mouse sweat glands decreased following topiramate administration. These results suggest dysregulation of AQP5 may be involved in topiramate-induced hypohidrosis and topiramate may serve as a novel therapy for hyperhidrosis.

Electro-acupuncture of Tsusanli and Shangchuhsu regulates gastric activity possibly through mediation of the vagus-solotary complex.

BACKGROUND/AIMS: Acupuncture has been reportedly used to treat gastrointestinal diseases, however, its precise mechanism remains unknown. METHODOLOGY: In our study, the effects and mechanism of electro-acupuncture (EA) at Tsusanli (ST 36), Shangchuhsu (ST 37) on regulation of gastric activity were observed. RESULTS: EA at Tsusanli showed that gastric electric change was the most obvious, with significantly higher frequency and wave amplitude compared with that of the Shangchuhsu group and other groups. EA at Shangchuhsu demonstrated that the change of gastric electric level was much higher than that of the non-acupoint group and control group. After bilateral vagotomy, Tsusanlis was electro-acupunctured, the changes of electro-gastric graph (EGG) weren't significant with the control group. The frequency of electro-physiological activity in nucleus of solitary tract (NTS) and dorsal motor nucleus of the vagus nerve (DMV) in the Tsusanli group was markedly increased compared with that in the other group. Fos and GFAP expression in NTS and DMV in the Tsusanli group was significantly higher than that in other groups and control group. The results have indicated that EA at Tsusanli and Shangchuhsu cannot only regulate gastric activity, but also activate neurons and astrocytes in NTS and DMV. The effects on regulation and activation of EA at Tsusanli were very obvious. CONCLUSIONS: Our study suggests that this electroacupuncture regulation of gastric activity may partially depend upon integrated nerve pathway and related central neurons and astrocytes in the vagus-solitary complex.

Electro-acupuncture of Foot YangMing regulates gastric activity possibly through mediation of the dorsal vagal complex.

Acupuncture at some specific acupoints of Foot Yangming can regulate gastric activity. However, its precise mechanism remains unknown. In our study, the effects and mechanism of electro-acupuncture (EA) at Tsusanli (ST 36), Shangchuhsu (ST 37) on the regulation of gastric activity were observed. EA at Tsusanli showed that gastric electric change had a significantly higher frequency and wave amplitude as compared to that of the Shangchuhsu group and other groups. EA at Shangchuhsu demonstrated the change of gastric electric was greater than that of the non-acupoint group and the control group. After bilateral vagotomy, the change of electro gastric graph (EGG) of EA at Tsusanlis was not significant compared to the control group. In the mean time, we have observed the electric discharge of the neurons in NTS and DMV. The frequency of electro-physiological activity in nucleus of solitary tract (NTS) and dorsal motor nucleus of the vagus nerve (DMV) in Tsusanli group and Shangchuhsu group were markedly increased compared with that in other groups. The results have indicated that EA at Tsusanli and Shangchuhsu not only regulate gastric activity, but also activate neurons in NTS and DMV significantly. Our study suggests that the effect of EA at Tsusanli and Shangchuhsu on the gastric activity may partially depend upon integrated nerve pathway and related central neurons in dorsal vagal complex.

[Effect of carbenoxolone on expression of Fos, NMDAR2 and GFAP in the hippocampus of pentylenetetrazo-kindled epileptic rats].

OBJECTIVE: Gap junctions, the clusters of intercellular channels, play an important role in synchronizing electrical activity. This study investigated the effect of gap junction blocker carbenoxolone (CBX) on epileptic activity in pentylenetetrazo (PTZ)-kindled rats. METHODS: Thirty adult male SD rats were randomly divided into three groups: control, PTZ-kindled and CBX-treated groups (n=10 each). The rats from the PTZ-kindled and the CBX-treated groups were intraperitoneally injected with PTZ (35 mg/kg x d) to induce epilepsy. After epilepsy kindling, they were intraperitoneally injected for 3 days with CBX (10 mg/kg) (CBX-treated group) or with normal saline (PTZ-kindled group). The control group received intraperitoneal injections of normal saline. Anti-GFAP, anti-Fos, and anti-NMDARZ immunohistochemical ABC methods were used to detect the expression of GFAP-Li, Fos-Li and NMDAR2-Li in the hippocampus respectively. RESULTS: Spontaneous seizures occurred in PTZ-kindled epileptic rats. CBX administration reduced spontaneous seizures. The NMDAR2-Li and Fos-Li neurons as well as GFAP-Li astrocytes in hippocampi increased in PTZ-kindled epileptic rats compared with controls. The numbers of Fos-Li (93.75 +/-7.94 vs 165.25 +/-15.87, P < 0.05) and NMDAR2-Li neurons (61.47 +/-3.62 vs 148.72 +/-14.53, P < 0.01) in the CBX-treated group were significantly less than in the PTZ-kindled group. There were no significant differences in the GFAP-Li expression between the CBX-treated and the PTZ-kindled groups. CONCLUSIONS: CBX may inhibit spontaneous seizures and decrease the numbers of Fos-Li and NMDARZ-Li neurons, thus providing anti-epileptic effects.

The lumbar spinal cord glial cells actively modulate subcutaneous formalin induced hyperalgesia in the rat.

We investigated the response and relationship of glial cells and neurons in lumbar spinal cord to hyperalgesia induced by the unilateral subcutaneous formalin injection into the hindpaw of rats. It was demonstrated that Fos/NeuN immunoreactive (-IR) neurons, glial fibrillary acidic protein (GFAP)-IR astrocytes and OX42-IR microglia were distributed in dorsal horn of lumbar spinal cord, predominantly in the superficial layer. In the time-course studies, GFAP-IR astrocytes were firstly detected, OX42-IR microglia were sequentially observed, Fos/NeuN-IR neurons were found slightly late. Immunoelectron microscopy studies established that many heterotypic gap junctions (HGJs), which consisting of Cx43-IR astrocytic process on one side and Cx32-IR dendrite on the other side, were present in superficial layer of dorsal horn. Ninety-one HGJs were found in 100 areas of experimental rats and occupied 91%, while only 39% HGJs were found in control rats. In experimental rats pretreated with intrathecal (i.t.) application of the carbenoxolone (a gap junction blocker) or fluorocitrate (a glial metabolic inhibitor), the paw withdrawal thermal latency was prolonged than those application of the sterile saline (i.t.). It suggests that spinal cord glial cells may play an important role for modulation of hyperalgesia induced by noxious stimuli through HGJs which located between astrocytes and neurons.

GFAP and Fos immunoreactivity in lumbo-sacral spinal cord and medulla oblongata after chronic colonic inflammation in rats.

AIM: To investigate the response of astrocytes and neurons in rat lumbo-sacral spinal cord and medulla oblongata induced by chronic colonic inflammation, and the relationship between them. METHODS: Thirty-three male Sprague-Dawley rats were randomly divided into two groups: experimental group (n = 17), colonic inflammation was induced by intra-luminal administration of trinitrobenzenesulfonic acid (TNBS); control group (n = 16), saline was administered intra-luminally. After 3, 7, 14, and 28 d of administration, the lumbo-sacral spinal cord and medulla oblongata were removed and processed for anti-glial fibrillary acidic protein (GFAP), Fos and GFAP/Fos immunohistochemistry. RESULTS: Activated astrocytes positive for GFAP were mainly distributed in the superficial laminae (laminae I-II) of dorsal horn, intermediolateral nucleus (laminae V), posterior commissural nucleus (laminae X) and anterolateral nucleus (laminae IX). Fos-IR (Fos-immunoreactive) neurons were mainly distributed in the deeper laminae of the spinal cord (laminae III-IV, V-VI). In the medulla oblongata, both GFAP-IR astrocytes and Fos-IR neurons were mainly distributed in the medullary visceral zone (MVZ). The density of GFAP in the spinal cord of experimental rats was significantly higher after 3, 7, and 14 d of TNBS administration compared with the controls (50.4+/-16.8, 29.2+/-6.5, 24.1+/-5.6, P<0.05). The density of GFAP in MVZ was significantly higher after 3 d of TNBS administration (34.3+/-2.5, P<0.05). After 28 d of TNBS administration, the density of GFAP in the spinal cord and MVZ decreased and became comparable to that of the controls (18.0+/-4.9, 14.6+/-6.4, P>0.05). CONCLUSION: Astrocytes in spinal cord and medulla oblongata can be activated by colonic inflammation. The activated astrocytes are closely related to Fos-IR neurons. With the recovery of colonic inflammation, the activity of astrocytes in the spinal cord and medulla oblongata is reduced.

[Effects of N-methyl-D-aspartate receptor in visceral, hypersensitivity in rats with colonic inflammation].

OBJECTIVE: To investigate the effects of N-methyl-D-aspartate receptor (NMDAR) in the spinal dorsal horn in visceral hypersensitivity in rats with colonic inflammation. METHODS: Seventy adult male Sprague-Dawley (SD) rats were randomly divided into the experimental group and the control group. Colonic inflammation was induced in the experimental rats by intraluminal administration of trinitrobenzenesulfonic acid (TNBS). Saline was administered intraluminally in the control rats. After 3, 7, 14, and 28 days of administration, abdominal contractions induced by inflation of a balloon colonically inserted were recorded in rats by implanting electrodes in the abdominal striated muscles. Immunohistochemistry method was used to study the expression of NMDAR1 and NMDAR2A/B in lumbarsacral spinal cord after inflammation. RESULTS: Colonic distension evoked a significant increase of abdominal contractions after 3, 7 and 14 days of TNBS administration. After 28 days of TNBS administration, abdominal contractions were still significantly increased in 2 TNBS-treated rats compared with the control rats. After 7 and 14 days of TNBS administration, NMDAR1 and NMDAR2A/B-immunoreactive cells were significantly increased compared with the control group (P <0.05). Twenty-eight days after TNBS administration, the number of NMDAR1-IR and NMDAR2A/B-IR neurons was still significantly increased in 4 TNBS-treated rats compared with the saline-treated rats (P < 0.05). CONCLUSION: NMDAR was involved in the transmission of visceral nociceptive stimuli. After the remission of colonic inflammation, increased expression of NMDAR1 and NMDAR2A/B in the spinal dorsal horn may induce persistent neuronal hyperactivity, which results in visceral hypersensitivity.

Dentate granule cell neurogenesis after seizures induced by pentylenetrazol in rats.

Epileptic seizures originating from the limbic system have been shown to stimulate the proliferation rate of granule cell precursors in the adult brain, but it is not clear if other type(s) of seizures have the similar effects. This study examined the effects of pentylenetrazol (PTZ)-induced generalized clonic seizures on dentate granule cell neurogenesis in adult rats. Using systemic bromodeoxyuridine (BrdU) to label dividing cells, we studied the proliferation rate of neural precursor cells in the dentate gyrus at various time points after PTZ-induced seizures. The double-label immunofluorescence with confocal microscopy was used to determine the newborn cell phenotypes. Quantitative analysis of BrdU labeling revealed a significant increase in the proliferation rate of neural precursor cells in the dentate gyrus 3, 7, and 14 days after seizures. The number of BrdU-labeled cells in the dentate gyrus returned to baseline levels by 28 days after the initial seizures. Most of newborn cells migrated into the granule cell layer from the subgranular zone, displayed the neuronal phenotype, and developed morphological characteristics of differentiated dentate granule cells. These results indicated that neuron proliferation in the dentate gyrus was enhanced during a time window (3-14 days) after PTZ-induced seizures. Its underlying mechanism is discussed.

Ultrastructure of junction areas between neurons and astrocytes in rat supraoptic nuclei.

AIM: To determine the ultrastructure of junction areas between neurons and astrocytes of supraoptic nuclei in rats orally administered 30 g/L NaCl solution for 5 days. METHODS: The anti-connexin (CX) 43 and anti-CX32 double immunoelectromicroscopic labeled method, and anti-Fos or anti-glial fibrillary acidic protein (GFAP) immunohistochemistry were used to detect changes in the junctional area between neurons and astrocytes in supraoptic nuclei of 5 rats after 30 g/L NaCL solution was given for 5days. RESULTS: A heterotypic connexin32/connexin43 gap junction (HGJ) between neurons and astrocytes (AS) in rat supraoptic nuclei was observed, which was characterized by the thickening and dark staining of cytomembranes with a narrow cleft between them. The number of HGJs and Fos like immunoreactive (-LI) cells was significantly increased following hyperosmotic stimuli, that is, the rats were administered 30 g/L NaCl solution orally or 90 g/L NaCl solution intravenously. HGJs could be blocked with carbenoxolone (CBX), a gap junction blocker, and the number of Fos-LI neurons was significantly decreased compared with that in rats without CBX injection, while Fos-LI ASs were not affected. CONCLUSION: HGJ may be a rapid adaptive signal structure between neurons and ASs in response to stimulation.

[Three-dimensional structural reconstruction of the neurons and astrocytes in rat hippocampal CA3 area].

OBJECTIVE: To observe the distribution of the pyramidal neurons and the surrounding astrocytes in rat hippocampal CA3 area and reconstruct their three-dimension structure. METHODS: Whole-cell patch-clamp recording of the brain slices, intracellular staining with Lucifer yellow, glial fibrillary acidic protein (GFAP) immunofluorescence, and confocal laser scanning microscopy were utilized in this study. RESULTS: According to the firing patterns, the hippocampal pyramidal neurons were classified into phasic or non-phasic firing neurons. Monolayer scanning and three-dimensional reconstruction showed that many astrocytes aggregated in tight clusters around the pyramidal neurons positive for Lucifer yellow staining, forming close contact. The two types of neurons contacted the astrocytes in different manners. Surrounding the cell body and dendrites of the non-phasic firing neurons, many astrocytes were seen to form contact with the neurons, whereas the phasic firing neurons formed contact with the astrocytes through the dendrites. CONCLUSION: The spatial distribution of the astrocytes surrounding different types of the hippocampal neurons may differ.

[Spinal cord neuronal apoptosis induced by triorthocresyl phosphate poisoning in hens].

OBJECTIVE: To investigate the role of neuronal apoptosis in organophosphorus poisoning-induced delayed neuropathy (OPIDN) and its dynamic pathological changes. METHODS: To establish OPIDN animal model, triorthocresyl phosphate (TOCP)was given to hens with a single dose (1 000 mg/kg, im). Changes of neuropathology, number of neurons and apoptotic cells in the third lumbar spinal cord were observed by HE, Nissl and TUNEL methods 3, 5, 7, 10, 14, 18 days after injection. RESULTS: The hens showed OPIDN typical signs (progressive ataxia and hypotonia) about 9 days after TOCP exposure. HE staining revealed dark red nucleus in neurons of anterior horn of lumbar spinal cord 5 days after exposure, but this phenomenon disappeared 18 days later. Nissl method showed that the number of neurons in anterior horn of spinal cord decreased [from (82 +/- 4) cell/mm(2) to (66 +/- 6) cell/mm(2)]. TUNEL positive cells began to appear [(22 +/- 2) cell/mm(2)] 5 days after TOCP exposure, and reached the peak [(27 +/- 3) cell/mm(2)] 7 days later, and disappeared 18 days later. CONCLUSION: Neuronal apoptosis in anterior horn of spinal cord of hens appeared in OPIDN, suggesting that cellular apoptosis may play an important role in the pathogenesis of OPIDN.

[Influences of repeated lower +Gz exposures on high +Gz exposure induced brain injury in rats].

Objective. To explore the influence of repeated lower +Gz exposures on high +Gz exposure induced brain injury in rats. Method. Forty SD male rats were randomly divided into control group (5 rats), +10 Gz/5 min group (5 rats) +4 Gz exposure one time, three times and five times group (10 rats each group). After 1 d or 6 d of +4 Gz exposure each group were exposed to +10 Gz again. Three days after +10 Gz exposure the neuron damage was observed by light microscope in HE stained section. Result. There was no brain damage after repeated +4 Gz/3 min exposure 5 times so it was reasonable to use this exposure intensity as ischemia stimulation. +10 Gz/5 min exposure could result in irreversible neuron damage such as neuron degeneration and coagulation necrosis. The experiment results suggested that after +10 Gz/5 min exposure there were degenerated neurons in cortex, hippocampus and thalamus. The number of degenerated neurons were obviously decreased in cortex, hippocampus and thalamus when exposed to +10 Gz/5 min again after repeated +4 Gz/3 min 3-5 times. Conclusion. The degree of neuron damage was obviously slight at the time of exposure to +10 Gz/5 min again after repeated +4 Gz/3 min 3-5 times. The ischemia tolerance at the time of exposure to +Gz was similar to other brain ischemia.