Background suppression of electrical activity is a potential biomarker of subsequent brain injury in a rat model of neonatal hypoxia-ischemia.

Electrographic seizures and abnormal background activity in the neonatal electroencephalogram (EEG) may differentiate between harmful versus benign brain insults. Using two animal models of neonatal seizures, electrical activity was recorded in freely behaving rats and examined quantitatively during successive time periods with field-potential recordings obtained shortly after the brain insult (i.e., 0-4 days). Single-channel, differential recordings with miniature wireless telemetry were used to analyze spontaneous electrographic seizures and background suppression of electrical activity after 1) hypoxia-ischemia (HI), which is a model of neonatal encephalopathy that causes acute seizures and a large brain lesion with possible development of epilepsy, 2) hypoxia alone (Ha), which causes severe acute seizures without an obvious lesion or subsequent epilepsy, and 3) sham control rats. Background EEG exhibited increases in power as a function of age in control animals. Although background electrical activity was depressed in all frequency bands immediately after HI, suppression in the ß and γ bands was greatest and lasted longest. Spontaneous electrographic seizures were recorded, but only in a few HI-treated animals. Ha-treated rat pups were similar to sham controls, they had no subsequent spontaneous electrographic seizures after the treatment and background suppression was only briefly observed in one frequency band. Thus, the normal age-dependent maturation of electrical activity patterns in control animals was significantly disrupted after HI. Suppression of the background EEG observed here after HI-induced acute seizures and subsequent brain injury may be a noninvasive biomarker for detecting severe brain injuries and may help predict subsequent epilepsy.NEW & NOTEWORTHY Biomarkers of neonatal brain injury are needed. Hypoxia-ischemia (HI) in immature rat pups caused severe brain injury, which was associated with strongly suppressed background EEG. The suppression was most robust in the ß and γ bands; it started immediately after the HI injury and persisted for days. Thus, background suppression may be a noninvasive biomarker for detecting severe brain injuries and may help predict subsequent epilepsy.

Mechanisms of Neuronal Silencing After Cortical Spreading Depression.

Cortical spreading depression (CSD) is associated with migraine, stroke, and traumatic brain injury, but its mechanisms remain poorly understood. One of the major features of CSD is an hour-long silencing of neuronal activity. Though this silencing has clear ramifications for CSD-associated disease, it has not been fully explained. We used in vivo whole-cell recordings to examine the effects of CSD on layer 2/3 pyramidal neurons in mouse somatosensory cortex and used in vitro recordings to examine their mechanism. We found that CSD caused a reduction in spontaneous synaptic activity and action potential (AP) firing that lasted over an hour. Both pre- and postsynaptic mechanisms contributed to this silencing. Reductions in frequency of postsynaptic potentials were due to a reduction in presynaptic transmitter release probability as well as reduced AP activity. Decreases in postsynaptic potential amplitude were due to an inhibitory shift in the ratio of excitatory and inhibitory postsynaptic currents. This inhibitory shift in turn contributed to the reduced frequency of APs. Thus, distinct but complementary mechanisms generate the long neuronal silence that follows CSD. These cellular changes could contribute to wider network dysfunction in CSD-associated disease, while the pre- and postsynaptic mechanisms offer separate targets for therapy.

A rodent model of human organophosphate exposure producing status epilepticus and neuropathology.

Exposure to organophosphates (OPs) often results in seizures and/or status epilepticus (SE) that produce neural damage within the central nervous system (CNS). Early control of SE is imperative for minimizing seizure-related CNS neuropathology. Although standard therapies exist, more effective agents are needed to reduce OP-induced SE and neuronal loss, particularly therapies with efficacy when administered 10's of minutes after the onset of SE. To evaluate novel antiseizure compounds, animal models should simulate the CNS effects of OP exposure observed in humans. We characterized in rats the effects of the OP, diisopropyl flourophosphate (DFP) as a function of dose and route of administration of supporting agents (pyridostigmine, 2-PAM, atropine); outcome measures were mortality, electrographic seizure activity during SE, and subsequent CNS neuropathology. Doses of DFP between 3 and 7mg/kg consistently caused SE, and the latency to behavioral tremors and to subsequent initiation of SE were dose related. In distinction, all doses of DFP that resulted in electrographic SE (3-7mg/kg) produced seizures of similar intensity and duration, and similar CNS neuropathology (i.e., the effects were all-or-none). Although SE was similar across doses, mortality progressively increased with higher doses of DFP. Mortality was significantly lower when the route of administration of therapeutic agents was intramuscular compared to intraperitoneal. This rodent model of OP poisoning demonstrates pathological characteristics similar to those observed in humans, and thus begins to validate this model for investigating potential new therapeutic approaches.

Temporal progression of evoked field potentials in neocortical slices after unilateral hypoxia-ischemia in perinatal rats: Correlation with cortical epileptogenesis.

Infarcts of the neonatal cerebral cortex can lead to progressive epilepsy, which is characterized by time-dependent increases in seizure frequency after the infarct and by shifts in seizure-onset zones from focal to multi-focal. Using a rat model of unilateral perinatal hypoxia-ischemia (PHI), where long-term seizure monitoring had previously demonstrated progressive epilepsy, evoked field potentials (EFPs) were recorded in layers II/III of coronal neocortical slices to analyze the underlying time-dependent, network-level alterations ipsilateral vs. contralateral to the infarct. At 3weeks after PHI, EFPs ipsilateral to the infarct were normal in artificial cerebrospinal fluid (ACSF); however, after blocking GABAA receptors with bicuculline methiodide (BMI, 30µM), the slices with an infarct were more hyperexcitable than slices without an infarct. At 3weeks, contralateral PHI slices had responses indistinguishable from controls. Six months after PHI in normal ACSF, both ipsi- and contralateral slices from rats with cortical infarcts showed prolonged afterdischarges, which were only slightly augmented in BMI. These data suggest that the early changes after PHI are localized to the ipsilateral infarcted cortex and masked by GABA-mediated inhibition; however, after 6months, progressive epileptogenesis results in generation of robust bilateral hyperexcitability. Because these afterdischarges were only slightly prolonged by BMI, a time-dependent reduction of GABAergic transmission is hypothesized to contribute to the pronounced hyperexcitability at 6months. These changes in the EFPs coincide with the seizure semiology of the epilepsy and therefore offer an opportunity to study the mechanisms underlying this form of progressive pediatric epilepsy.

Ischemic injury suppresses hypoxia-induced electrographic seizures and the background EEG in a rat model of perinatal hypoxic-ischemic encephalopathy.

The relationship among neonatal seizures, abnormalities of the electroencephalogram (EEG), brain injury, and long-term neurological outcome (e.g., epilepsy) remains controversial. The effects of hypoxia alone (Ha) and hypoxia-ischemia (HI) were studied in neonatal rats at postnatal day 7; both models generate EEG seizures during the 2-h hypoxia treatment, but only HI causes an infarct with severe neuronal degeneration. Single-channel, differential recordings of acute EEG seizures and background suppression were recorded with a novel miniature telemetry device during the hypoxia treatment and analyzed quantitatively. The waveforms of electrographic seizures (and their behavioral correlates) appeared virtually identical in both models and were identified as discrete events with high power in the traditional delta (0.1-4 Hz) and/or alpha (8-12 Hz) bands. Although the EEG patterns during seizures were similar in Ha- and HI-treated animals at the beginning of the hypoxic insult, Ha caused a more severe electrographic seizure profile than HI near the end. Analyses of power spectral density and seizure frequency profiles indicated that the electrographic seizures progressively increased during the 2-h Ha treatment, while HI led to a progressive decrease in the seizures with significant suppression of the EEG background. These data show that 1) the hypoxia component of these two models drives the seizures; 2) the seizures during Ha are substantially more robust than those during HI, possibly because ongoing neuronal damage blunts the electrographic activity; and 3) a progressive decrease in background EEG, rather than the presence of electrographic seizures, indicates neuronal degeneration during perinatal HI.

Co-administration of subtherapeutic diazepam enhances neuroprotective effect of COX-2 inhibitor, NS-398, after lithium pilocarpine-induced status epilepticus.

RATIONALE: Seizures during status epilepticus (SE) cause neuronal death and induce cyclooxygenase-2 (COX-2). Pilocarpine-induced SE was used to determine if COX-2 inhibition with NS-398, when administered alone or with diazepam, decreases the duration and/or intensity of SE and/or reduces neuronal injury in the rat hippocampus. METHODS: Electroencephalogram (EEG) electrodes were implanted in male Sprague-Dawley rats. SE was induced with lithium-pilocarpine, and continuous EEG and video monitoring were performed for 24 h. Rats were divided into four groups (n=8-14 rats/group) and received NS-398, diazepam, NS-398 and diazepam, or vehicle 30 min after the first motor seizure. Six hours later, NS-398 injection was repeated in the NS-398 and in the NS-398+diazepam groups. The duration of SE (continuous spiking) and the EEG power in the γ-band were analyzed. FluoroJade B staining in the dorsal hippocampus at 24h after SE was analyzed semi-quantitatively in the CA1, CA3 and hilus. RESULTS: The duration and intensity of electrographic SE was not significantly different across the four groups. In rats treated with NS-398 alone, compared to vehicle-treated rats, neuronal damage was significantly lower compared to vehicle-treated rats in the CA3 (27%) and hilus (27%), but neuroprotection was not detected in the CA1. When NS-398 was administered with diazepam, decreased neuronal damage was further obtained in all areas investigated (CA1: 61%, CA3: 63%, hilus: 60%). CONCLUSIONS: NS-398, when administered 30 min after the onset of SE with a repeat dose at 6h, decreased neuronal damage in the hippocampus. Administration of diazepam with NS-398 potentiates the neuroprotective effect of the COX-2 inhibitor. These neuroprotective effects occurred with no detectable effect on electrographic SE.

Neuronal degeneration is observed in multiple regions outside the hippocampus after lithium pilocarpine-induced status epilepticus in the immature rat.

Although hippocampal sclerosis is frequently identified as a possible epileptic focus in patients with temporal lobe epilepsy, neuronal loss has also been observed in additional structures, including areas outside the temporal lobe. The claim from several researchers using animal models of acquired epilepsy that the immature brain can develop epilepsy without evidence of hippocampal neuronal death raises the possibility that neuronal death in some of these other regions may also be important for epileptogenesis. The present study used the lithium pilocarpine model of acquired epilepsy in immature animals to assess which structures outside the hippocampus are injured acutely after status epilepticus. Sprague-Dawley rat pups were implanted with surface EEG electrodes, and status epilepticus was induced at 20 days of age with lithium pilocarpine. After 72 h, brain tissue from 12 animals was examined with Fluoro-Jade B, a histochemical marker for degenerating neurons. All animals that had confirmed status epilepticus demonstrated Fluoro-Jade B staining in areas outside the hippocampus. The most prominent staining was seen in the thalamus (mediodorsal, paratenial, reuniens, and ventral lateral geniculate nuclei), amygdala (ventral lateral, posteromedial, and basomedial nuclei), ventral premammillary nuclei of hypothalamus, and paralimbic cortices (perirhinal, entorhinal, and piriform) as well as parasubiculum and dorsal endopiriform nuclei. These results demonstrate that lithium pilocarpine-induced status epilepticus in the immature rat brain consistently results in neuronal injury in several distinct areas outside of the hippocampus. Many of these regions are similar to areas damaged in patients with temporal lobe epilepsy, thus suggesting a possible role in epileptogenesis.

Recording EEG in immature rats with a novel miniature telemetry system.

Serial EEG recordings from immature rat pups are extremely difficult to obtain but important for analyzing animal models of neonatal seizures and other pediatric neurological conditions as well as normal physiology. In this report, we describe the features and applications of a novel miniature telemetry system designed to record EEG in rat pups as young as postnatal day 6 (P6). First, we have recorded electrographic seizure activity in two animal models of neonatal seizures, hypoxia- and kainate-induced seizures at P7. Second, we describe a viable approach for long-term continuous EEG monitoring of naturally reared rat pups implanted with EEG at P6. Third, we have used serial EEG recordings to record age-dependent changes in the background EEG signal as the animals matured from P7 to P11. The important advantages of using miniature wireless EEG technology are: 1) minimally invasive surgical implantation; 2) a device form-factor that is compatible with housing of rat pups with the dam and littermates; 3) serial recordings of EEG activity; and 4) low power consumption of the unit, theoretically allowing continuous monitoring for up to 2 yr without surgical reimplantation. The miniature EEG telemetry system provides a technical advance that allows researchers to record continuous and serial EEG recordings in neonatal rodent models of human neurological disorders, study the progression of the disease, and then assess possible therapies using quantitative EEG as an outcome measure. This new technical approach should improve animal models of human conditions that rely on EEG monitoring for diagnosis and therapy.

A comparative electrographic analysis of the effect of sec-butyl-propylacetamide on pharmacoresistant status epilepticus.

Better treatment of status epilepticus (SE), which typically becomes refractory after about 30 min, will require new pharmacotherapies. The effect of sec-butyl-propylacetamide (SPD), an amide derivative of valproic acid (VPA), on electrographic status epilepticus (ESE) was compared quantitatively to other standard-of-care compounds. Cortical electroencephalograms (EEGs) were recorded from rats during ESE induced with lithium-pilocarpine. Using a previously-published algorithm, the effects of SPD on ESE were compared quantitatively to other relevant compounds. To confirm benzodiazepine resistance, diazepam (DZP) was shown to suppress ESE when administered 15 min after the first motor seizure, but not after 30 min (100mg/kg). VPA (300 mg/kg) also lacked efficacy at 30 min. SPD (130 mg/kg) strongly suppressed ESE at 30 min, less after 45 min, and not at 60 min. At a higher dose (180 mg/kg), SPD profoundly suppressed ESE at 60 min, similar to propofol (100mg/kg) and pentobarbital (30 mg/kg). After 4-6h of SPD-induced suppression, EEG activity often overshot control levels at 7-12h. Valnoctamide (VCD, 180 mg/kg), an SPD homolog, was also efficacious at 30 min. SPD blocks pilocarpine-induced electrographic seizures when administered at 1h after the first motor seizure. SPD has a faster onset and greater efficacy than DZP and VPA, and is similar to propofol and pentobarbital. SPD and structurally similar compounds may be useful for the treatment of refractory ESE. Further development and use of automated analyses of ESE may facilitate drug discovery for refractory SE.

Pilocarpine-induced status epilepticus and subsequent spontaneous seizures: lack of effect on the number of gonadotropin-releasing hormone-positive neurons in a mouse model of temporal lobe epilepsy.

Women with temporal lobe epilepsy have a higher incidence of reproductive disorders, which have been linked to alterations in the pulsatile release of gonadotropin-releasing hormone (GnRH). These experiments tested the hypothesis that the number of GnRH neurons is reduced in an animal model of temporal lobe epilepsy. The effects of pilocarpine-induced status epilepticus (SE) and the subsequent spontaneous recurrent eizures on the number of GnRH-positive neurons were studied in adult female mice. Systemic injections of pilocarpine were used to induce SE, and diazepam was administered 90 min after the first seizure. Control mice received all drugs except pilocarpine. The mice were euthanized either 1 week or 3 months after SE (i.e. after spontaneous recurrent seizures were observed). Even though the estrous cycle was disrupted after SE, and hippocampal damage was detected in both the CA1 and CA3 regions, pilocarpine-treated mice did not show a significant decrease in total or regional numbers of GnRH-immunopositive neurons. Therefore, these data do not support the hypothesis that a reduction in the number of GnRH neurons is responsible for the disruption of the estrous cycle after pilocarpine-induced epilepsy, which suggests that other mechanisms contribute to female reproductive disorders associated with chronic epilepsy.

Lithium pilocarpine-induced status epilepticus in postnatal day 20 rats results in greater neuronal injury in ventral versus dorsal hippocampus.

Many quantitative animal studies examining the possible relationship between hippocampal neuronal loss and the development of epilepsy have examined only the dorsal hippocampus. The ventral hippocampus, however, represents the more homologous structure to the anterior hippocampus in humans, which is the area associated with the maximal damage in patients with temporal lobe epilepsy. This study tested the hypothesis that the ventral hippocampus has greater neuronal injury than the dorsal hippocampus in an animal model of chemoconvulsant-status epilepticus at postnatal day 20. Status epilepticus was induced in postnatal day 20 Sprague-Dawley rat pups with the chemoconvulsant lithium-pilocarpine and brain tissue was examined with Fluoro-Jade B. Horizontal sections (n=7) favoring a visualization of the ventral hippocampus showed marked Fluoro-Jade B staining in CA1, CA3, and hilar region. Coronal sections favoring a visualization of the dorsal hippocampus did not consistently show as robust a staining pattern in these regions. In coronal sections where both the dorsal and ventral hippocampus could be viewed, greater staining was always seen in ventral versus dorsal hippocampus. Quantitative analysis of cell counts demonstrated a significant difference between ventral and dorsal hippocampus in CA1 and CA3, but not hilus. These results demonstrate that in ventral hippocampus, lithium pilocarpine-induced status epilepticus consistently results in hippocampal neuronal injury in postnatal day 20 rats. This study shows the importance of including the ventral hippocampus in any analysis of seizure-induced hippocampal neuronal injury, and raises concerns about the accuracy of studies quantifying hippocampal neuronal loss when only the dorsal hippocampus is examined.

A potential model of pediatric posttraumatic epilepsy.

Preclinical models of pediatric posttraumatic epilepsy (PTE) are lacking. We hypothesized that traumatic brain injury (TBI), induced by controlled cortical impact, in immature rats would cause electroencephalographic (EEG) epileptiform activity and behavioral seizures. TBI or sham craniotomy was performed on postnatal day 17. Using video-EEG monitoring 4-11 months post-TBI, most TBI rats (87.5%) showed EEG spiking and one had spontaneous, recurrent seizures. Controls showed neither EEG spikes nor electrographic/behavioral seizures. Late seizures were rare after TBI, but EEG spiking was common and may represent a surrogate for PTE.

A simple quantitative method for analyzing electrographic status epilepticus in rats.

Electrographic status epilepticus (ESE) is a medical emergency consisting of repetitive seizures and may result in death or severe brain damage. Epilepsy can develop following ESE. The properties of ESE (e.g., duration and intensity) are variable, as are the effects of putative therapeutic treatments. Therefore a straightforward method to quantify different components of ESE would be beneficial for both researchers and clinicians. A frequency range close to the gamma band was selected for extraction of seizure-related activity from the EEG. This filtering strategy reduced motion artifacts and other noise sources in the electrophysiological recordings, thus increasing the signal-to-noise ratio of the EEG spike activity. EEG spiking was quantified using an energy operator and modeled by an eighth-order polynomial. In a benzodiazepine-resistant rat model of pilocarpine-induced ESE, the efficacy of various pharmaceutical agents at suppressing ESE was analyzed with this and other methods on data collected for < or =24 h after ESE induction. This approach allows for the objective, quantitative, and rapid assessment of the effects of both short- and long-lasting pharmacological manipulations on ESE and other forms of prolonged repetitive electrical activity.

A chronic histopathological and electrophysiological analysis of a rodent hypoxic-ischemic brain injury model and its use as a model of epilepsy.

Ischemic brain injury is one of the leading causes of epilepsy in the elderly, and there are currently no adult rodent models of global ischemia, unilateral hemispheric ischemia, or focal ischemia that report the occurrence of spontaneous motor seizures following ischemic brain injury. The rodent hypoxic-ischemic (H-I) model of brain injury in adult rats is a model of unilateral hemispheric ischemic injury. Recent studies have shown that an H-I injury in perinatal rats causes hippocampal mossy fiber sprouting and epilepsy. These experiments aimed to test the hypothesis that a unilateral H-I injury leading to severe neuronal loss in young-adult rats also causes mossy fiber sprouting and spontaneous motor seizures many months after the injury, and that the mossy fiber sprouting induced by the H-I injury forms new functional recurrent excitatory synapses. The right common carotid artery of 30-day old rats was permanently ligated, and the rats were placed into a chamber with 8% oxygen for 30 min. A quantitative stereologic analysis revealed that the ipsilateral hippocampus had significant hilar and CA1 pyramidal neuronal loss compared with the contralateral and sham-control hippocampi. The septal region from the ipsilateral and contralateral hippocampus had small but significantly increased amounts of Timm staining in the inner molecular layer compared with the sham-control hippocampi. Three of 20 lesioned animals (15%) were observed to have at least one spontaneous motor seizure 6-12 months after treatment. Approximately 50% of the ipsilateral and contralateral hippocampal slices displayed abnormal electrophysiological responses in the dentate gyrus, manifest as all-or-none bursts to hilar stimulation. This study suggests that H-I injury is associated with synaptic reorganization in the lesioned region of the hippocampus, and that new recurrent excitatory circuits can predispose the hippocampus to abnormal electrophysiological activity and spontaneous motor seizures.

Connexin36 vs. connexin32, "miniature" neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmatic nucleus.

Suprachiasmatic nucleus (SCN) neurons generate circadian rhythms, and these neurons normally exhibit loosely-synchronized action potentials. Although electrotonic coupling has long been proposed to mediate this neuronal synchrony, ultrastructural studies have failed to detect gap junctions between SCN neurons. Nevertheless, it has been proposed that neuronal gap junctions exist in the SCN; that they consist of connexin32 or, alternatively, connexin36; and that connexin36 knockout eliminates neuronal coupling between SCN neurons and disrupts circadian rhythms. We used confocal immunofluorescence microscopy and freeze-fracture replica immunogold labeling to examine the distributions of connexin30, connexin32, connexin36, and connexin43 in rat and mouse SCN and used whole-cell recordings to re-assess electrotonic and tracer coupling. Connexin32-immunofluorescent puncta were essentially absent in SCN but connexin36 was relatively abundant. Fifteen neuronal gap junctions were identified ultrastructurally, all of which contained connexin36 but not connexin32, whereas nearby oligodendrocyte gap junctions contained connexin32. In adult SCN, one neuronal gap junction was >600 connexons, whereas 75% were smaller than 50 connexons, which may be below the limit of detectability by fluorescence microscopy and thin-section electron microscopy. Whole-cell recordings in hypothalamic slices revealed tracer coupling with neurobiotin in <5% of SCN neurons, and paired recordings (>40 pairs) did not reveal obvious electrotonic coupling or synchronized action potentials, consistent with few neurons possessing large gap junctions. However, most neurons had partial spikes or spikelets (often <1 mV), which remained after QX-314 [N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide] had blocked sodium-mediated action potentials within the recorded neuron, consistent with spikelet transmission via small gap junctions. Thus, a few "miniature" gap junctions on most SCN neurons appear to mediate weak electrotonic coupling between limited numbers of neuron pairs, thus accounting for frequent detection of partial spikes and hypothetically providing the basis for "loose" electrical or metabolic synchronization of electrical activity commonly observed in SCN neuronal populations during circadian rhythms.

Persistent calcium current in rat suprachiasmatic nucleus neurons.

The suprachiasmatic nuclei contain the primary circadian clock, and suprachiasmatic nuclei neurons exhibit a diurnal modulation of spontaneous firing rate. The present study examined the voltage-gated persistent Ca(2+) current, in acutely isolated rat suprachiasmatic nuclei neurons using a ramp-type voltage-clamp protocol. Slow triangular voltage-clamp commands from a holding potential of -85 mV to +5 mV elicited inward current (100-400 pA) that was completely blocked by Cd(2+). This current showed little or no hysteresis, and was identified as persistent Ca(2+) current. The threshold for persistent Ca(2+) current ranged between -60 and -45 mV, and it was maximal at about -8 mV. Nifedipine at 10-20 microM blocked 80-100%. To assess the role of persistent Ca(2+) current in the generation of spontaneous action potentials in both acutely isolated and intact suprachiasmatic nuclei neurons, the effect of Cd(2+) and nifedipine on firing rate was studied using on-cell recording. Application of Cd(2+) exerted a weak excitatory effect and nifedipine had no significant effect on the spontaneous firing rate of isolated suprachiasmatic nuclei neurons. In all intact suprachiasmatic nuclei neurons in slice preparations (n=15), Cd(2+) slowly inhibited spontaneous firing; in high-frequency firing cells (four of 15), a transient increase of firing rate preceded inhibition. No significant effect of nifedipine on firing rate of intact suprachiasmatic nuclei neurons was found. Therefore, persistent Ca(2+) current itself (as carrier of charge) does not appear to contribute significantly to spontaneous firing of suprachiasmatic nuclei neurons. A slowly developing inhibitory effect of Cd(2+) on spontaneous firing of intact suprachiasmatic nuclei neurons in slice preparations may be due to penetration of Cd(2+) through Ca(2+) channels, and its subsequent effect on intracellular mechanisms, while the transient increase of firing rate in high-frequency firing neurons is probably due to inhibition of Ca(2+)-activated K(+) current.

Spike-dependent depolarizing afterpotentials contribute to endogenous bursting in gonadotropin releasing hormone neurons.

Pulsatile secretion of gonadotropin releasing hormone in mammals is thought to depend on repetitive and prolonged bursts of action potentials in specific neuroendocrine cells. We have previously described episodes of electrical activity in isolated gonadotropin releasing hormone neurons, but the intrinsic mechanisms underlying the generation of spike bursts are unknown. In acutely isolated gonadotropin releasing hormone neurons, which had been genetically targeted to express enhanced green fluorescent protein, current pulses generated spike-mediated depolarizing afterpotentials in 69% of cells. Spike-dependent depolarizing afterpotentials could evoke bursts of action potentials that lasted for tens of seconds. Brief pulses of glutamate (as short as 1 ms), which simulated excitatory postsynaptic potentials, also triggered spike-mediated depolarizing afterpotentials and episodic activity. These data indicate that spike-dependent depolarizing afterpotentials, an endogenous mechanism in gonadotropin releasing hormone neurons, likely contribute to the episodic firing thought to underlie pulsatile secretion of gonadotropin releasing hormone. Furthermore, fast excitatory postsynaptic potentials mediated by glutamate can activate this intrinsic mechanism.

Persistent subthreshold voltage-dependent cation single channels in suprachiasmatic nucleus neurons.

The hypothalamic suprachiasmatic nucleus (SCN) contains the primary circadian pacemaker in mammals, and transmits circadian signals by diurnal modulation of neuronal firing frequency. The ionic mechanisms underlying the circadian regulation of firing frequency are unknown, but may involve changes in membrane potential and voltage-gated ion channels. Here we describe novel tetrodotoxin- and nifedipine-resistant subthreshold, voltage-dependent cation (SVC) channels that are active at resting potential of SCN neurons and increase their open probability (P(o)) with membrane depolarization. The increased P(o) reflects changes in the kinetics of the slow component of the channel closed-time, but not the channel open-time or fast closed-time. This study provides a background for investigation of the possible role of SVC channels in regulation of circadian oscillations of membrane excitability in SCN neurons.

High-resolution proteomic mapping in the vertebrate central nervous system: close proximity of connexin35 to NMDA glutamate receptor clusters and co-localization of connexin36 with immunoreactivity for zonula occludens protein-1 (ZO-1).

Combined confocal microscopy and freeze-fracture replica immunogold labeling (FRIL) were used to examine the connexin identity at electrical synapses in goldfish brain and rat retina, and to test for "co-localization" vs. "close proximity" of connexins to other functionally interacting proteins in synapses of goldfish and mouse brain and rat retina. In goldfish brain, confocal microscopy revealed immunofluorescence for connexin35 (Cx35) and NMDA-R1 (NR1) glutamate receptor protein in Mauthner Cell/Club Ending synapses. By FRIL double labeling, NR1 glutamate receptors were found in clusters of intramembrane particles in the postsynaptic membrane extraplasmic leaflets, and these distinctive postsynaptic densities were in close proximity (0.1-0.3 microm) to neuronal gap junctions labeled for Cx35, which is the fish ortholog of connexin36 (Cx36) found at neuronal gap junctions in mammals. Immunogold labeling for Cx36 in adult rat retina revealed abundant gap junctions, including several previously unrecognized morphological types. As in goldfish hindbrain, immunogold double labeling revealed NR1-containing postsynaptic densities localized near Cx36-labeled gap junction in rat inferior olive. Confocal immunofluorescence microscopy revealed widespread co-localization of Cx36 and ZO-1, particularly in the reticular thalamic nucleus and amygdala of mouse brain. By FRIL, ZO-1 immunoreactivity was co-localized with Cx36 at individual gap junction plaques in rat retinal neurons. As cytoplasmic accessory proteins, ZO-1 and possibly related members of the membrane-associated guanylate kinase (MAGUK) family represent scaffolding proteins that may bind to and regulate the activity of many neuronal gap junctions. These data document the power of combining immunofluorescence confocal microscopy with FRIL ultrastructural imaging and immunogold labeling to determine the relative proximities of proteins that are involved in short- vs. intermediate-range molecular interactions in the complex membrane appositions at synapses between neurons.