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Previous studies suggested that oxidative stress is related to the onset and development of osteoporosis. Moreover, it was demonstrated that berberine has a protective effect against oxidative stress-induced injuries. In this study, we aimed to investigate the effect and mechanism of action of berberine on rats with induced osteoporosis. Sixty 8-week-old female Wistar rats were randomly divided into the following 6 groups: control saline-treated, osteoporosis saline-treated, 3 osteoporosis berberine-treated groups (Ber 5, 10, and 20 mg/kg/body weight, respectively), and osteoporosis alendronate-treated (ALD) group. Osteoporosis was induced by bilateral ovariectomy. All treatments were performed for 8 weeks. The bone mineral density (BMD), serum alkaline phosphatase (ALP), osteocalcin, calcium, phosphorus, superoxide dismutase (SOD), methylenedioxyamphetamine (MDA), and glutathione peroxidase (GSH-Px) level was determined in the rat femur tissue. The gene and protein expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) was analyzed by quantitative reverse transcription PCR and Western blot, respectively. The BMD, SOD and GSH-Px levels, and the expression of OPG were significantly lower in osteoporosis compared to control group (all P < 0.05). The serum levels of osteocalcin, ALP, and MDA, and the expression of RANKL were significantly higher in osteoporosis compared to control group (all P < 0.05). Berberine, especially the high doses of berberine, effectively increased SOD, GSH-Px, and OPG levels as well as decreased serum osteocalcin, ALP, MDA and RANKL levels in berberine-treated osteoporosis groups (all P < 0.05). To conclude, oxidative stress may promote the development of osteoporosis in rats through the RANK/RANKL/OPG pathway. The antioxidative effect of berberine reduces the development of osteoporosis in rats to some extent.

Previous studies suggested that oxidative stress is related to the onset and development of osteoporosis. Moreover, it was demonstrated that berberine has a protective effect against oxidative stress-induced injuries. In this study, we aimed to investigate the effect and mechanism of action of berberine on rats with induced osteoporosis. Sixty 8-week-old female Wistar rats were randomly divided into the following 6 groups: control saline-treated, osteoporosis saline-treated, 3 osteoporosis berberine-treated groups (Ber 5, 10, and 20 mg/kg/body weight, respectively), and osteoporosis alendronate-treated (ALD) group. Osteoporosis was induced by bilateral ovariectomy. All treatments were performed for 8 weeks. The bone mineral density (BMD), serum alkaline phosphatase (ALP), osteocalcin, calcium, phosphorus, superoxide dismutase (SOD), methylenedioxyamphetamine (MDA), and glutathione peroxidase (GSH-Px) level was determined in the rat femur tissue. The gene and protein expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) was analyzed by quantitative reverse transcription PCR and Western blot, respectively. The BMD, SOD and GSH⁃Px levels, and the expression of OPG were significantly lower in osteoporosis compared to control group (all p < 0.05). The serum levels of osteocalcin, ALP, and MDA, and the expression of RANKL were significantly higher in osteoporosis compared to control group (all p < 0.05). Berberine, especially the high doses of berberine, effectively increased SOD, GSH⁃Px, and OPG levels as well as decreased serum osteocalcin, ALP, MDA and RANKL levels in berberine-treated osteoporosis groups (all p < 0.05). To conclude, oxidative stress may promote the development of osteoporosis in rats through the RANK/RANKL/OPG pathway. The antioxidative effect of berberine reduces the development of osteoporosis in rats to some extent.

TACC3, a member of the transforming acidic coiled-coil protein (TACC) family, is a multifunctional protein that is involved in various biological functions, including proliferation and differentiation of tumor cells, cancer progression and metastasis. The aims of the present study were to examine whether TACC3 expression is associated with the proliferation and migration of osteosarcoma (OS) cells and to investigate the potential underlying molecular mechanisms of TACC3 in OS. First, the levels of mRNA and protein expression in OS cell lines by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively were examined. Second, the effects of TACC3 knockdown and overexpression on the proliferative, migratory and invasive capacities of OS cells were investigated. Finally, western blot analysis was employed to detect the potential mechanism of TACC3 in osteosarcoma. TACC3 expression was significantly increased in osteosarcoma tissues and cell lines, compared to matched controls. The knockdown of TACC3 was able to significantly inhibit the proliferation, migration and invasion of osteosarcoma cells, whereas the overexpression of TACC3 was able to promote cell proliferation and migration. Mechanistically, TACC3 may promote the migration and invasion of osteosarcoma cells via through nuclear factor-κB signaling. These data suggest that TACC3 has an important part in the progression of osteosarcoma and may serve as a potential target for gene therapy.

Deep brain stimulation (DBS) is a widely used neurosurgical approach to treating tremor and other movement disorders1,2,3. In addition, the use of DBS in a number of psychiatric diseases, including obsessive-compulsive disorders and depression, is currently being tested4,5,6. Despite the rapid increase in the number of individuals with surgically implanted stimulation electrodes, the cellular pathways involved in mediating the effects of DBS remain unknown1. Here we show that DBS is associated with a marked increase in the release of ATP, resulting in accumulation of its catabolic product, adenosine. Adenosine A1 receptor activation depresses excitatory transmission in the thalamus and reduces both tremor- and DBS-induced side effects. Intrathalamic infusion of A1 receptor agonists directly reduces tremor, whereas adenosine A1 receptor–null mice show involuntary movements and seizure at stimulation intensities below the therapeutic level. Furthermore, our data indicate that endogenous adenosine mechanisms are active in tremor, thus supporting the clinical notion that caffeine, a nonselective adenosine receptor antagonist, can trigger or exacerbate essential tremor7. Our findings suggest that nonsynaptic mechanisms involving the activation of A1 receptors suppress tremor activity and limit stimulation-induced side effects, thereby providing a new pharmacological target to replace or improve the efficacy of DBS.

Cortical spreading depression (CSD) is a self-propagating wave of cellular depolarization that has been implicated in migraine and in progressive neuronal injury after stroke and head trauma. Using two-photon microscopic NADH imaging and oxygen sensor microelectrodes in live mouse cortex, we find that CSD is linked to severe hypoxia and marked neuronal swelling that can last up to several minutes. Changes in dendritic structures and loss of spines during CSD are comparable to those during anoxic depolarization. Increasing O2 availability shortens the duration of CSD and improves local redox state. Our results indicate that tissue hypoxia associated with CSD is caused by a transient increase in O2 demand exceeding vascular O2 supply.

Previously, we were able to demonstrate the neuroprotective effect of quercetin in an animal model of acute traumatic spinal cord injury. The objective of the present study was to determine whether any neuroprotective effect is seen when quercetin is administered in an animal model of traumatic brain injury. Twenty-six adult male Sprague-Dawley rats were submitted to moderate fluid percussion injury in the anterior midline position. Animals were divided into two experimental groups: one group received 25 mumol/kg quercetin starting 1 h after injury, while animals in the second group received saline vehicle (n = 13 per group). Eight animals were used as uninjured healthy controls. Eight animals in each experimental group were sacrificed at 24 h, while five animals per group were allowed to recover for 72 h following injury. Compound action potential amplitudes (CAPAs) were recorded on 400-microm vibrotome sections of the corpus callosum superfused with oxygenated artificial CSF (n = 3 per animal) in 20 experimental animals and five healthy controls. Three brains from animals in each experimental group and healthy controls were used for histological, immunocytochemical and biochemical analysis after sacrifice at 24 h. CAPAs in uninjured animals had a mean of 1.12 mV. This decreased to 0.55 mV in saline vehicle-treated injured animals by 24 h and changed little over the next 3 days. CAPAs were significantly better at 0.82 mV at 24 h and 0.76 mV at 3 days in quercetin-treated injured animals when compared to injured saline vehicle controls. Quercetin significantly prevented decrease of glutathione levels and decreased myeloperoxidase activity. We conclude that this dietary flavonoid has therapeutic potential following brain trauma.

Hypersynchronous neuronal firing is a hallmark of epilepsy, but the mechanisms underlying simultaneous activation of multiple neurons remains unknown. Epileptic discharges are in part initiated by a local depolarization shift that drives groups of neurons into synchronous bursting. In an attempt to define the cellular basis for hypersynchronous bursting activity, we studied the occurrence of paroxysmal depolarization shifts after suppressing synaptic activity using tetrodotoxin (TTX) and voltage-gated Ca(2+) channel blockers. Here we report that paroxysmal depolarization shifts can be initiated by release of glutamate from extrasynaptic sources or by photolysis of caged Ca(2+) in astrocytes. Two-photon imaging of live exposed cortex showed that several antiepileptic agents, including valproate, gabapentin and phenytoin, reduced the ability of astrocytes to transmit Ca(2+) signaling. Our results show an unanticipated key role for astrocytes in seizure activity. As such, these findings identify astrocytes as a proximal target for the treatment of epileptic disorders.

This study describes a new method used to evaluate axonal physiological dysfunction following fluid percussion induced traumatic brain injury (TBI) that may facilitate the study of the mechanisms and novel therapeutic strategies of posttraumatic diffuse axonal injury (DAI). Stimulated compound action potentials (CAP) were recorded extracellularly in the corpus callosum of superfused brain slices at 3 h, and 1, 3, and 7 days following central fluid percussion injury and demonstrated a temporal pattern of functional deterioration. The maximal CAP amplitude (CAPA) covaried with the intensity of impact 1 day following sham, mild (1.0-1.2 atm), and moderate (1.8-2.0 atm) injury (p0.05; 1.11 +/- 0.10, 0.82 +/- 0.11, and 0.49 +/- 0.08 mV, respectively). The CAPA in sham animals were approximately 1.1 mV and did not vary with survival interval (3 h, and 1, 3, and 7 days); however, they were significantly decreased at each time point following moderate injury (p0.05; 0.51 +/- 0.11, 0.49 +/- 0.08, 0.46 +/- 0.10, and 0.75 +/- 0.13 mV, respectively). The CAPA at 7 days in the injured group were higher than at 3 h, and 1 and 3 days. HE and amyloid precursor protein (APP) light microscopic analysis confirmed previously reported trauma-induced axonal injury in the corpus callosum seen after fluid percussion injury. Increased APP expression was confirmed using Western blotting showing significant accumulation at 1 day (IOD 913.0 +/- 252.7; n = 3; p = 0.05), 3 days (IOD 753.1 +/- 159.1; n = 3; p = 0.03), and at 7 days (IOD 1093.8 = 105.0; n = 3; p = 0.001) compared to shams (IOD 217.6 +/- 20.4; n = 3). Thus, we report the characterization of white matter axonal dysfunction in the corpus callosum following TBI. This novel method was easily applied, and the results were consistent and reproducible. The electrophysiological changes were sensitive to the early effects of impact intensity, as well as to delayed changes occurring several days following injury. They also indicated a greater degree of attenuation than predicted by APP expression changes alone.

This presentation focuses on the application of methods to determine functional connections between neurons in the respiratory network of adult decerebrate rats. We employ a general network investigation paradigm that first examines the intracellular recordings of a respiratory neuron and then determines which neurons synapse with it to produce the observed membrane potential changes. It is used to pursue the source of respiratory excitation and inhibition from its arrival at phrenic motoneurons to respiratory neurons in the medulla, and then examine some of the interactions among these neurons that shape their patterns of activity. Findings include a demonstration that phrenic motoneuron activity is determined by excitation from medullary inspiratory premotor neurons and inhibition by Bötzinger complex expiratory neurons, and that the latter neurons inhibit both medullary inspiratory premotor neurons and themselves. We conclude that these functional interconnections explain the activity patterns of some respiratory neurons, but the connections between neurons thought to be involved in rhythm generation remain to be demonstrated in adult rats.

We examined the respiratory rhythm of two in-vitro preparations from neonatal rats, the brainstem-spinal cord and transverse brainstem slice, recording the bursting activity of phrenic and hypoglossal nerves, respectively at 1 degree C intervals from 25 to 35 degrees C at two pH's, 7.4 and 7.1. In both preparations at either pH, burst frequency increased with temperature, burst duration declined and burst amplitude reached a peak at 30 degrees C. The shapes of the bursts changed from a decrementing pattern at low temperatures to a bell-shaped pattern at high temperatures. At reduced pH, frequency increased for temperatures between 25 and 32 degrees C in the brainstem-spinal cord but not in the slice. Burst duration was increased at reduced pH for temperatures between 27 and 29 degrees C in the brainstem-spinal cord, but not in the slice. Burst amplitude only changed with pH at the lower temperatures, decreasing at the lower pH in the brainstem-spinal cord and increasing in the slice. With respect to the effects of temperature, we concluded that both preparations were similarly affected, and that an increase in temperature alters the in-vitro burst pattern towards that observed in-vivo. With respect to the effects of pH, we concluded that effects differ between these in-vitro preparations and from in-vivo preparations, and that the difference between in-vivo and in-vitro preparations in their response to decreasing pH is not due to differences in temperature.

Perfusion of rat brain slices with low millimole CsCl elicits slow oscillations of ≤1 Hz in hippocampal CA1 pyramidal neurons. These oscillations are GABAAreceptor-mediated hyperpolarizations that permit a coherent fire–pause pattern in a population of CA1 neurons. They can persist without the activation of ionotropic glutamate receptors but require adenosine-dependent inhibition of glutamate transmission. In response to external Cs+, multiple interneurons in the CA1 region display rhythmic discharges that correlate with the slow oscillations in CA1 pyramidal neurons. The interneuronal discharges arise spontaneously from the resting potential, and their rhythmicity is regulated by periodic, GABAAreceptor-mediated hyperpolarizations. In addition, interneurons show periodic partial spikes and neurobiotin coupling, and applications of known gap junctional uncouplers interrupt the Cs+-induced slow rhythm in both CA1 pyramidal neurons and interneurons. We propose that these slow oscillations originate from a GABAergic interneuronal network that interacts through reciprocal inhibition and possibly gap junctional connection.

We examined the synaptic connections between ventral-group, bulbospinal inspiratory neurons in 27 vagotomized, paralyzed, ventilated, and decerebrated rats using cross-correlation and spike-triggered averaging of intracellular potentials. The neurons were recorded in the medulla about the level of the obex and identified by their inspiratory firing pattern and antidromic activation from the spinal cord at C7. Whole C5 phrenic nerve recordings were made using bipolar electrodes from the central cut ends of the nerve. Most (108/137, 79%) inspiratory neurons discharged only during inspiration but some (29/137, 21%) also discharged during early expiration. Their intracellular membrane potentials displayed a pattern of depolarization during inspiration, repolarization during early expiration, and hyperpolarization during late expiration. Intracellular chloride iontophoresis changed the inspiratory membrane potential trajectories from augmenting to decrementing in 11 of 19 neurons tested (58%), and demonstrated the presence of both early-decrementing and late-augmenting waves of inhibitory postsynaptic potentials during expiration in 11 of 19 neurons tested (58%). Cross-correlation histograms were computed between pairs of extracellularly recorded neurons to detect short time scale synchronizations indicative of synaptic connections (26 ipsilateral; 23 contralateral). While none of the cross-correlation histograms for contralateral pairs showed peaks, most (23, 88%) of those for ipsilateral pairs showed peaks (mean half-amplitude width +/- SD = 1.3 +/- 0.4 ms) at time zero suggestive of common activation. Some of the latter (6, 23%) showed troughs superimposed on the central peaks (mean half-amplitude width +/- SD = 0.9 +/- 0.2 ms) at short latencies (mean latency +/- SD = 1.8 +/- 1.9 ms) suggestive of inhibition; others (8, 31%) had asymmetrical central peaks and two had bilateral peaks suggesting more complex interconnections. Averages of intracellular membrane potentials of inspiratory neurons (n = 24), triggered by action potentials of a nearby extracellularly recorded inspiratory neuron, were computed to detect synchronized postsynaptic potentials. Over half (16, 67%) showed postsynaptic potentials (mean amplitude +/- SD = 201 +/- 176 microV; mean half-amplitude width +/- SD = 2.3 +/- 0.8 ms) confirming the cross-correlation findings of common excitation. We conclude that in decerebrated rats, ventral-group inspiratory neurons projecting to the C7 spinal segment share powerful, ipsilaterally distributed excitatory inputs which enhance their synchronous activity during inspiration. They also receive inhibition during inspiration and early-decrementing and late-augmenting inhibitory inputs during expiration.

Spinal cord injury (SCI) is often complicated by secondary injury as a result of the innate inflammatory response to tissue trauma and swelling. Previous studies have shown that excessive ATP release from peritraumatic regions contributes to the inflammatory response to SCI by activation of low-affinity P2X7 receptors. Because connexin hemichannels constitute an important route for astrocytic ATP release, we here evaluated the impact on post-traumatic ATP release of deletion of connexins (Cx30/Cx43) in astrocytes.In vivobioluminescence imaging showed a significant reduction in ATP release after weight-drop injury in mice with deletion of Cx43 compared with Cx43-expressing littermates, both on a Cx30 knockout background. Moreover, astrogliosis and microglia activation were reduced in peritraumatic areas of those mice lacking Cx43; motor recovery was also significantly improved, and the traumatic lesion was smaller. Combined, these observations are consistent with a contribution by astrocytic hemichannels to post-traumatic ATP release that aggravates secondary injury and restrains functional recovery after experimental spinal cord injury. Connexins may thereby constitute a new therapeutic target in spinal cord injury.

Gliosis is a pathological hallmark of posttraumatic epileptic foci, but little is known about these reactive astrocytes beyond their high glial fibrillary acidic protein (GFAP) expression. Using diolistic labeling, we show that cortical astrocytes lost their nonoverlapping domain organization in three mouse models of epilepsy: posttraumatic injury, genetic susceptibility, and systemic kainate exposure. Neighboring astrocytes in epileptic mice showed a 10-fold increase in overlap of processes. Concurrently, spine density was increased on dendrites of excitatory neurons. Suppression of seizures by the common antiepileptic, valproate, reduced the overlap of astrocytic processes. Astrocytic domain organization was also preserved in APP transgenic mice expressing a mutant variant of human amyloid precursor protein despite a marked upregulation of GFAP. Our data suggest that loss of astrocytic domains was not universally associated with gliosis, but restricted to seizure pathologies. Reorganization of astrocytes may, in concert with dendritic sprouting and new synapse formation, form the structural basis for recurrent excitation in the epileptic brain.

Summary Congenitally hypomyelinated shiverer mice fail to generate compact myelin and die by 18–21 weeks of age. Using multifocal anterior and posterior fossa delivery of sorted fetal human glial progenitor cells into neonatal shiverer × rag2 −/− mice, we achieved whole neuraxis myelination of the engrafted hosts, which in a significant fraction of cases rescued this otherwise lethal phenotype. The transplanted mice exhibited greatly prolonged survival with progressive resolution of their neurological deficits. Substantial myelination in multiple regions was accompanied by the acquisition of normal nodes of Ranvier and transcallosal conduction velocities, ultrastructurally normal and complete myelination of most axons, and a restoration of a substantially normal neurological phenotype. Notably, the resultant mice were cerebral chimeras, with murine gray matter but a predominantly human white matter glial composition. These data demonstrate that the neonatal transplantation of human glial progenitor cells can effectively treat disorders of congenital and perinatal hypomyelination.

Although astrocytes are the most abundant cell type in the brain, evidence for their activation during physiological sensory activity is lacking. Here we show that whisker stimulation evokes increases in astrocytic cytosolic calcium (Ca(2+)) within the barrel cortex of adult mice. Increases in astrocytic Ca(2+) were a function of the frequency of stimulation, occurred within several seconds and were inhibited by metabotropic glutamate receptor antagonists. To distinguish between synaptic input and output, local synaptic activity in cortical layer 2 was silenced by iontophoresis of AMPA and NMDA receptor antagonists. The antagonists did not reduce astrocytic Ca(2+) responses despite a marked reduction in excitatory postsynaptic currents in response to whisker stimulation. These findings indicate that astrocytes respond to synaptic input, by means of spillover or ectopic release of glutamate, and that increases in astrocytic Ca(2+) occur independently of postsynaptic excitatory activity.

Local increase in blood flow during neural activity forms the basis for functional brain imaging, but its mechanism remains poorly defined. Here we show that cortical astrocytes in vivo possess a powerful mechanism for rapid vasodilation. We imaged the activity of astrocytes labeled with the calcium (Ca(2+))-sensitive indicator rhod-2 in somatosensory cortex of adult mice. Photolysis of caged Ca(2+) in astrocytic endfeet ensheathing the vessel wall was associated with an 18% increase in arterial cross-section area that corresponded to a 37% increase in blood flow. Vasodilation occurred with a latency of only 1-2 s, and both indomethacin and the cyclooxygenase-1 inhibitor SC-560 blocked the photolysis-induced hyperemia. These observations implicate astrocytes in the control of local microcirculation and suggest that one of their physiological roles is to mediate vasodilation in response to increased neural activity.

Cerebral ischemic damage is an important cause of morbidity and mortality. However, there is conflicting evidence regarding the effect of the extracellular glucose concentration in focal and global ischemic injury. This study was designed to investigate this effect in ischemia-induced synaptic transmission damage in rat hippocampal slices. Slices were superfused with artificial cerebrospinal fluid (ACSF) containing various concentrations of glucose before and after ischemia. The evoked somatic postsynaptic population spike (PS) and dendritic field excitatory postsynaptic potential (fEPSP) were extracellularly recorded in the CA1 stratum pyramidal cell layer and s. radiatum after stimulation of the Schaeffer collaterals, respectively. The glucose concentration in ACSF before and after ischemia determined the duration of ischemia tolerated by synaptic transmission as demonstrated by complete recovery of the somatic PS and dendritic fEPSP. Specifically, the somatic PS and dendritic fEPSP completely recovered following 3, 4, and 5 min of ischemia only when slices were superfused with ACSF containing 4, 10, and 20 mM glucose before and after ischemia, respectively. The latencies of the somatic and dendritic ischemic depolarization (ID) occurrence in the CA1 s. pyramidal cell layer and s. radiatum were significantly longer with 10 than 4 mM glucose in ACSF before ischemia and significantly longer with 20 than 10 mM glucose in ACSF before ischemia. Regardless of the glucose concentration in ACSF before and after ischemia, the somatic PS and dendritic fEPSP only partially recovered when ischemia was terminated at the occurrence of ID. These results indicate that high glucose in ACSF during the period before and after ischemia significantly protects CA1 synaptic transmission against in vitro ischemia-induced damage through postponing the occurrence of ID.

Prolonged anoxia can cause permanent damage to synaptic transmission in the mammalian CNS. We tested the hypothesis that lack of glucose is the major cause of irreversible anoxic transmission damage, and that anoxic synaptic transmission damage could be prevented by glycolysis in rat hippocampal slices. The evoked population spike (PS) was extracellularly recorded in the CA1 pyramidal cell layer after stimulation of the Schaffer collaterals. When the slice was superfused with artificial cerebrospinal fluid (ACSF) containing 4 mM glucose, following 10 min anoxia, the evoked PS did not recover at all after 60 min reoxygenation. When superfusion ACSF contained 10 mM glucose with or without 0.5 mM α-cyano-4-hydroxycinnate (4-CIN), after 60 min reoxygenation the evoked PS completely recovered following 10 min anoxia. When superfusion ACSF contained 20 mM glucose with or without 1 mM sodium cyanide (NaCN), after 60 min reoxygenation the evoked PS completely recovered even following 120 min anoxia. In contrast, when superfusion ACSF contained 4 mM glucose, following 10 min 1 mM NaCN chemical anoxia alone, without anoxic anoxia, the evoked PS displayed no recovery after 60 min reoxygenation. Moreover, when 16 mM mannitol and 16 sodiuml-lactate were added into 4 mM glucose ACSF, following 10 min anoxia the evoked PS failed to recover at all after 60 min reoxygenation. The results indicate that elevated glucose concentration powerfully protected the synaptic transmission against anoxic damage, and the powerful protection is due to anaerobic metabolism of glucose and not a result of the higher osmolality in higher glucose ACSF. We conclude that lack of glucose is the major cause of anoxia-induced synaptic transmission damage, and that if sufficient glucose is supplied, glycolysis could prevent this damage in vitro.

The present experiments were conducted to determine the time course of synaptic dysfunction in the vulnerable regions of the post-ischemia hippocampus. Following transient cerebral ischemia, neurons in the CA1 subfield of the hippocampus undergo a delayed degeneration that develops about 48 h after reperfusion. We have shown previously that CA1 glutamatergic transmission is decreased in the CA1 subfield well before any morphological deterioration of the CA1 cells is visible under the light microscope. However, it is unknown whether a time window exists after insult in which attenuated synaptic activity may be restored to normal levels. We show here that evoked CA1 somatic population spikes and dendritic field potential responses decline progressively after reperfusion in the CA1 subfield, such that by 72 h post-insult, the challenged neurons are unable to elicit evoked excitatory responses. This attenuation of synaptic transmission was confined to the vulnerable neurons of the hippocampus, however, as the evoked responses in the dentate gyrus displayed amplitudes that were not significantly diminished from sham control after challenge. In brain slices obtained from 24 h post-ischemia rats with significantly impaired CA1 somatic responses, the application of 5 or 50 μM of the potassium channel blocker 4-aminopyridine (4-AP) restored the magnitude of the evoked excitatory response to control values. At 36 h post-ischemia, the decreased CA1 evoked responses could be partially improved by 4-AP, but not to control levels. Based upon these results, we conclude that the decreased CA1 synaptic activity at 24 h post-ischemia is potentially reversible, and suggest that 4-AP improves the CA1 synaptic responses at least in part by improving transmitter release at post-ischemia glutamatergic synapses.

Extracellularly recorded action potentials from 49 Bötzinger-complex, bulbospinal expiratory neurones were used as triggers to compute 162 spike-triggered averages (STAs) of intracellular potentials recorded from 167 respiratory neurones in the ventral respiratory group (VRG) near the obex in 15 vagotomized, paralysed, ventilated and decerebrated rats. All of the Bötzinger-complex expiratory neurones were antidromically activated from the ipsilateral border between the C2/C3 segments of the spinal cord and discharged only during the late part of expiration with an augmenting pattern. We found evidence for monosynaptic inhibitory post-synaptic potentials (IPSPs) in 74 (approximately 44%) of the STAs computed using 34 (approximately 69%) of the trigger neurones. For vagal motoneurones, IPSPs were found in 24 of the 53 STAs of expiratory motoneurones, but in none of the 12 STAs of inspiratory motoneurones. For inspiratory neurones, IPSPs were found in 23 of the 33 STAs of bulbospinal neurones and in 6 of the 26 STAs of not antidromically activated (NAA) neurones. For expiratory neurones, IPSPs were found in one of the two STAs of bulbospinal neurones and in 20 of the 36 STAs of NAA neurones. We conclude that Bötzinger-complex, bulbospinal expiratory neurones monosynaptically inhibit bulbospinal inspiratory neurones, expiratory vagal motoneurones and other unidentified inspiratory and expiratory neurones in the VRG of rats during the late part of expiration.

We examined respiratory neurons in the Bötzinger complex of the medulla oblongata in 18 vagotomized, paralyzed, ventilated, and decerebrated rats and tested the hypothesis that bulbospinal expiratory neurons in this region monosynaptically inhibit phrenic motoneurons. First, we surveyed the types of respiratory neurons found in the Bötzinger complex; only 11 of the 98 (approximately 11%) examined were bulbospinal, and all discharged only during late expiration (E2), usually with an augmenting discharge frequency (AUG). Then, we examined the spinal projections of 34 E2-AUG neurons using antidromic activation and found that all projected as far as the C4 or C5 segments of the spinal cord but no further caudally. Most (30, approximately 88%) had only unilateral projections, the majority (25, approximately 83%) ipsilateral, but 4 neurons (approximately 12%) had bilateral projections. Their axons could be antidromically activated at low currents (less than 10 microA) in the dorsal-lateral part of the spinal cord at the C2-3 border; 0.5-1.2 mm (mean+/-SD 0.84+/-0.23 mm) below the dorsal surface and 0.7-1.5 mm (1.19+/-0.25 mm) lateral from the midline. We sought evidence for connections from bulbospinal E2-AUG neurons to 118 phrenic motoneurons by computing spike-triggered averages (STAs) of their intracellular potentials triggered by the action potentials of 38 unilaterally-projecting E2-AUG neurons. Resting phrenic motoneuron membrane potentials ranged from -40 to -75 mV (-56+/-8 mV) and fluctuations with the respiratory cycle from 7 to 20 mV (14+/-4 mV). Of the 118 STAs computed, hyperpolarizations were evident in 18 (approximately 15%) STAs, evoked by 11 of 38 (approximately 29%) E2-AUG neurons. Their amplitudes varied from 35 to 550 microV (105+/-113 microV), 10-90% fall times from 0.4 to 0.9 ms (0.63+/-0.17 ms), and half-amplitude widths from 1.3 to 3.2 ms (2.0+/-0.52 ms). Most (16/95, approximately 17%) of the STAs that displayed hyperpolarizations were associated with ipsilateral trigger neurons but some (2/23, approximately 9%) resulted from contralateral trigger neurons. We conclude that Bötzinger-complex, expiratory neurons project to the C4 and/or C5 segments of the cervical spinal cord but no further caudal. Their axons are located dorsolaterally in the upper cervical segments of the spinal cord, and they monosynaptically inhibit phrenic motoneurons during the late part of expiration.

We examined the role of dorsal respiratory group (DRG) inspiratory neurons as transmitters of respiratory drive to phrenic and intercostal motoneurons and as relays of afferent information to ventral respiratory group (VRG) bulbospinal, inspiratory neurons. Attempts to antidromically activate 76 DRG neurons from the spinal cord at the C7 segment resulted in only 4 (5.3%) successes (3 contralateral, 1 ipsilateral). Cross-correlating DRG neuron discharge with that of the ipsilateral (56) and contralateral (20) phrenic nerve detected common activation peaks in 2 and 3 cases respectively, with no evidence for monosynaptic connections. Cross-correlating DRG neuron discharge with that of bulbospinal, inspiratory VRG neurons found some evidence for interaction. Peaks in 7 of 73 (10%) cross-correlation histograms were attributed to a monosynaptic excitation of DRG neurons by VRG neurons, although a common activation cannot be ruled out; troughs, some with an accompanying peak, in 9 (12.3%) histograms were interpreted as a combined excitation of the DRG neuron and delayed inhibition of the VRG neuron. In addition, 2 cross-correlation histograms showed peaks with latencies and half-amplitude widths consistent with a disynaptic excitation of a DRG neuron by a bulbospinal inspiratory VRG neuron. Cross-correlating the discharge of 57 pairs of DRG inspiratory neurons (6 contralateral) detected common activation peaks in 7 (12.3%) cases (none contralateral) and one case interpreted as evidence for a disynaptic excitation. These findings suggest that the role of the DRG inspiratory neurons in rats differs from that in cats, primarily because they do not act to transmit respiratory rhythmic drive directly to phrenic and intercostal motoneurons. The results offer some support for an excitation of DRG neurons by VRG inspiratory neurons, but no support for a role of DRG inspiratory neurons as mediators of afferent information transfer to VRG bulbospinal inspiratory neurons.

In rats, respiratory neurons in the medulla oblongata are arranged in longitudinally distributed groups that are duplicated on each side of the neuraxis. Our aim was to determine whether respiratory rhythm is generated independently by each side. We made a complete mid-saggital section of the medulla oblongata, 3.5 mm rostral and 3.5 mm caudal to the obex, in decerebrate, vagotomized, and paralysed adult rats. Respiratory rhythm, monitored by recording the activity of both left and right phrenic nerves, was maintained and became asynchronous between the left and right sides. We concluded that in the adult rat each half of the medulla oblongata is capable of generating respiratory rhythm independently.

We examined the synaptic connections from ventral-group bulbospinal inspiratory neurons to upper-cervical inspiratory neurons and phrenic and intercostal motoneurons in decerebrate rats using cross-correlation. Inspiratory neurons were recorded in the medulla (n = 28) at the level of the obex and from the upper-cervical segments (C1 and C2) of the spinal cord (n = 29) in 18 vagotomized, paralyzed, ventilated, and decerebrated rats. The neurons were identified by their inspiratory firing pattern and antidromic activation from the spinal cord at C7. Whole-nerve recordings were made using bipolar electrodes from the central cut ends of the C5 phrenic nerve and the external and internal intercostal nerves at various thoracic levels. Cross-correlation histograms were computed between these recordings to detect short time scale synchronizations indicative of synaptic connections. Cross-correlation histograms (n = 20), computed between the activities of ventral-group bulbospinal inspiratory neurons and the phrenic nerve, all showed peaks (mean half-amplitude width +/- SD, 1.1 +/- 0.3 ms) at short latencies (mean latency +/- SD, 2.0 +/- 0.6 ms) suggestive of monosynaptic excitation. Cross-correlation histograms (n = 33), computed between the activities of ventral-group bulbospinal inspiratory neurons and upper-cervical inspiratory neurons, displayed four (12%) peaks (mean half-amplitude width +/- SD, 0.9 +/- 0.1 ms) at short latencies (mean latency +/- SD, 1.8 +/- 0.6 ms) suggestive of monosynaptic excitation, and six (18%) peaks (mean half-amplitude width +/- SD, 1.4 +/- 0.4 ms) at latencies near zero suggestive of excitation from a common source. Cross-correlation histograms (n = 34), computed between the activities of ventral-group bulbospinal inspiratory neurons and the internal and external intercostal nerves at various thoracic levels (T2-8), showed six (18%) peaks (mean half-amplitude width +/- SD, 2.5 +/- 0.5 ms) at short latency (mean latency +/- SD, 4.5 +/- 1.1 ms) suggestive of oligosynaptic connections. Cross-correlation histograms (n = 42) computed between activities of intercostal nerves at various levels of the thoracic spinal cord showed central peaks suggestive of excitation from a common source. Although the size of the peaks decreased with segmental separation, the displacement of the peaks from time zero did not increase with segmental separation (mean displacement +/- SD, 0.6 +/- 0.6 ms) as would be expected if the common excitation resulted from a descending monosynaptic excitation by a source such as the ventral-group bulbospinal inspiratory neurons. We conclude that all ventral-group bulbospinal inspiratory neurons make monosynaptic connections to phrenic motoneurons, a few make monosynaptic connections to upper-cervical, inspiratory neurons, but connections to intercostal motoneurons are made via interneurons.

We examined the synaptic connections from upper cervical inspiratory neurons to phrenic and intercostal motoneurons in decerebrate rats using cross-correlation. Upper cervical inspiratory neurons (n = 79) were recorded from the C1 and C2 segments of the spinal cord in 38 vagotomized, paralyzed, ventilated, and decerebrate rats. The neurons were identified by their inspiratory firing pattern and antidromic activation from the ipsilateral spinal cord at C7. Whole-nerve recordings were made using bipolar electrodes from the central cut ends of the C5 phrenic nerve and the external and internal intercostal nerves at various thoracic levels. Cross-correlation histograms were computed between these recordings to detect short time-scale synchronizations indicative of synaptic connections. The 55 cross-correlation histograms computed between the upper cervical inspiratory neurons and the ipsilateral phrenic nerve showed seven (13%) narrow peaks (mean half-amplitude width +/- SD, 1.09 +/- 0.15 ms) at short latencies (mean latency +/- SD, 1.29 +/- 0.26 ms) suggestive of monosynaptic excitation, and four (7%) broader peaks (mean half-amplitude width +/- SD, 1.50 +/- 0.17 ms) at short latencies (mean latency +/- SD, 1.40 +/- 0.24 ms) suggestive of oligosynaptic excitation. Another 14 (25%) cross-correlation histograms displayed a central broad peak (mean half-amplitude width +/- SD, 1.59 +/- 0.23 ms) suggestive of common activation. The eight cross-correlation histograms computed between the upper cervical inspiratory neurons and the contralateral phrenic nerve were featureless. The 77 cross-correlation histograms computed between the upper cervical inspiratory neurons and the internal and external intercostal nerves at various thoracic levels (T2-8) showed no peaks suggestive of synaptic connections. We conclude that some upper cervical inspiratory neurons make monosynaptic and paucisynaptic connections to phrenic motoneurons but not to intercostal motoneurons.