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Seizure-induced neuronal death may involve engagement of the BCL-2 family of apoptosis-regulating proteins. In the present study we examined the activation of proapoptotic BAD in cultured hippocampal neurons following seizures induced by removal of chronic glutamatergic transmission blockade. Kynurenic acid withdrawal elicited an increase in seizure-like electrical activity, which was inhibited by blockers of AMPA (CNQX) and NMDA (MK801 and AP5) receptor function. However, only NMDA receptor antagonists inhibited calcium entry as assessed by fura-2, and cell death of hippocampal neurons. Seizures increased proteolysis of caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) of cells. Seizure-like activity induced dephosphorylation of BAD and the disruption of its constitutive interaction with 14-3-3 proteins. In turn, BAD dimerized with antiapoptotic BCL-Xl after seizures. However, the absence of neuroprotective effects of pathway intervention suggests that BAD may perform a reinforcement rather than instigator role in cell death following seizures in vitro.

Acid-sensing ion channels (ASICs) are expressed in various sensory and central neurons. The functional role of these channels remains elusive. Complex subunit combinations and lack of specific blockers for native receptors are likely to contribute to the difficulty of resolving the function of ASICs. Finding a neuronal cell line, which expresses a single population of ASICs, should prove to be useful in delineating the function of individual ASICs. Using patch-clamp, Ca(2+)-imaging, and RT-PCR techniques, we have explored the existence of ASICs in PC12 cells, a clonal neuronal cell line. Fast drops of extracellular pH activated transient inward currents in PC12 cells with pH(0.5) at 6.0-6.2. The ASICs in PC12 cells were selective for Na(+) with significant Ca(2+) permeability. Currents in PC12 cells were blocked by the nonselective ASIC blocker amiloride. PcTX1, a specific homomeric ASIC1a blocker, also blocked the ASIC currents with an IC(50) of approximately 1.5 nM. RT-PCR demonstrated the existence of ASIC1a transcript in both undifferentiated and nerve growth factor-differentiated PC12 cells. Our data suggest that PC12 cells likely contain a single population of functional proton-gated channel-homomeric ASIC1a. It might be an ideal neuronal cell line for the study of physiological and potential pathological roles of this key subunit of ASICs.

Concentrations of extracellular calcium ([Ca(2+)](e)) in the CNS decrease substantially during seizure activity. We have demonstrated previously that decreases in [Ca(2+)](e) activate a novel calcium-sensing nonselective cation (csNSC) channel in hippocampal neurons. Activation of csNSC channels is responsible for a sustained membrane depolarization and increased neuronal excitability. Our study has suggested that the csNSC channel is likely involved in generating and maintaining seizure activities. In the present study, the effects of anti-epileptic agent lamotrigine (LTG) on csNSC channels were studied in cultured mouse hippocampal neurons using patch-clamp techniques. At a holding potential of -60 mV, a slow inward current through csNSC channels was activated by a step reduction of [Ca(2+)](e) from 1.5 to 0.2 mM. LTG decreased the amplitude of csNSC currents dose dependently with an IC(50) of 171 +/- 25.8 (SE) microM. The effect of LTG was independent of membrane potential. In the presence of 300 microM LTG, the amplitude of csNSC current was decreased by 31 +/- 3% at -60 mV and 29 +/- 2.9% at +40 mV (P > 0.05). LTG depressed csNSC current without affecting the potency of Ca(2+) block of the current (IC(50) for Ca(2+) block of csNSC currents in the absence of LTG: 145 +/- 18 microM; in the presence of 300 microM LTG: 136 +/- 10 microM. n = 5, P > 0.05). In current-clamp recordings, activation of csNSC channel by reducing the [Ca(2+)](e) caused a sustained membrane depolarization and an increase in the frequency of spontaneous firing of action potentials. LTG (300 microM) significantly inhibited csNSC channel-mediated membrane depolarization and the excitation of neurons. Fura-2 ratiometric Ca(2+) imaging experiment showed that LTG also inhibited the increase in intracellular Ca(2+) concentration induced by csNSC channel activation. The effect of LTG on csNSC channels may partially contribute to its broad spectrum of anti-epileptic actions.

The classic opioid peptide, enkephalin, and the novel member of the opioid family, nociceptin/orphanin FQ, inhibit the spontaneous electrical activity of neurons recorded from the rostral ventrolateral medulla, presumably cardiovascular neurons. In this study, the putative effects of endomorphin-1 and endomorphin-2, the newly discovered endogenous ligands for the micro-opioid receptor, on the electrical activity of rostral ventrolateral medulla neurons were investigated in rat brain slices in vitro. Like enkephalin and nociceptin, perfusion of endomorphin-1 or endomorphin-2 profoundly inhibited spontaneous discharges of 43% and 38% of the medullary neurons, respectively. No excitatory response to perfusion of either endomorphin was found in all neurons surveyed. Both endomorphins produced concentration-dependent inhibition. However, endomorphin-1 was more potent than endomorphin-2 for production of the inhibition, as demonstrated by the greater and longer suppression induced by endomorphin-1 than that induced by endomorphin-2 at the same concentration. Among the four opioid agonists tested, EC50 values (in nM) were 3.17 (endomorphin-1), 3.02 (nociceptin), 10.1 (endomorphin-2) and 150.0 (enkephalin). The non-selective opioid receptor antagonist, naloxone, blocked the inhibitory responses of the neurons to endomorphin-1, endomorphin-2 and enkephalin, but not to nociceptin. The selective mu antagonist, beta-funaltrexamine, prevented the neuronal inhibition induced by endomorphins, but not by enkephalin and nociceptin. Neither naloxone nor beta-funaltrexamine alone had a significant effect on the firing rate of the neurons. These results demonstrate that endomorphin-1 and, to a lesser extent, endomorphin-2 exert an inhibitory modulation of the electrical activity of rostral ventrolateral medulla neurons, which is mediated through the stimulation of mu-opioid receptors.

Extracellular single-unit discharges were obtained from 106 spontaneously active neurons in the region of the rostral ventrolateral medulla (RVLM) by glass microelectrode from 73 brain alices of Sprague-Dawley rats. Exogenous gamma-aminobutyric acid (GABA, 0.1-3.0 mmol/L) inhibited the electrical activity of 84 out of 106 RVLM neurons dose-dependently. The inhibitory effect of GABA could be blocked by GABAA receptor antagonist bicuculline methiodide (BMI) and Cl- channel blocker picrotoxin (PTX). When perfused with BMI or PTX alone, the firing rates of most of the RVLM neurons were significantly increased. In 41 neurons responding to GABA, baclofen (0.1-3.0 mumol/L), a GABAB receptor selective agonist, inhibited the discharges of 33 of the neurons dose-dependently. GABAB receptor antagonist CGP35348 (n = 13) blocked the inhibition due to baclofen (n = 21). After perfused with CGP35348 alone, the firing rates of most of the RVLM neurons were significantly increased. Taken together, the inhibition of GABA on RVLM neurons is mediated through either GABAA or GABAB receptors and some intrinsic GABA neurons exert tonic inhibition activity.

Extracellular single-unit discharges were obtained from 165 spontaneously active neurons within the region of the rostral ventrolateral medulla (RVLM) by glass microelectrode from 89 brain slices of the Sprague-Dawley rats. The units could be divided into three types: regular (61.8%), irregular (24.2%) and silent (14%). Acetylcholine (ACh, 0.1, 0.3 mumol/L) showed four kinds of effects on spontaneous discharges of RVLM neurons: excitatory, inhibitory, biphasic and non-responsive, counting respectively 41.8%, 20%, 3% and 35.2% of the neurons tested. The excitatory effect of ACh was dose-dependent. The effects, either excitatory or inhibitory, of ACh (n = 49) were mostly blocked by atropine (0.3 mumol/L, n = 42). The excitatory effect of ACh (n = 14) could be blocked mainly by selective antagonist of M1 receptor, pirenzepine (PZ, 30 nmol/L, n = 9), but not by selective antagonist of M2 receptor, methoctramine (MT) and AFDX-116. The inhibitory effect of ACh (n = 10) could be blocked mostly by M2 receptor antagonist MT (30 nmol/L, n = 7); and this inhibitory effect (n = 9) could be blocked mostly by another M2 receptor antagonist AFDX-116 (30 nmol/L, n = 6), but not by M1 receptor antagonist PZ.