Two distinct subgroups of cholecystokinin-immunoreactive cortical interneurons

Sustained high levels of corticosterone (CORT), one of the major stress-induced hormones in the rat, were suggested as generating 'accelerated brain aging' and were shown to induce both specific brain changes in the hippocampus and learning impairments in young and middle-aged Fischer-344 rats. Evidence that altered calcium (Ca) homeostasis may play a major role in brain aging has accumulated over the last decade. Recently, new data established a connection between glucocorticoids and voltage-activated Ca influx in aged hippocampal neurons. In the present study, an attempt was made to block the CORT-induced 'accelerated aging' by the simultaneous administration of the L-type Ca channel blocker nimodipine. CORT or placebo sustained-release (SR) pellets were implanted subcutaneously in 3 months old Fischer male rats. Each group was further sub-divided between nimodipine and placebo SR treatments. Characteristic CORT-induced morphological changes were observed in pyramidal hippocampal cells, such as at the CA1 and CA4 sub-regions (22.2% +/- 7.7 and 28.6% +/- 8.4 of pyknotic cells without clear nuclei, respectively). Concomitant treatment with nimodipine conferred full protection against CORT-induced morphological changes (e.g. 3.2% +/- 0.8 and 2.1% +/- 1.9 of pyknotic cells in CA1 and CA4, n = 7 rats in each group; P < 0.04). The neuroprotective efficacy of nimodipine supports the theory of Ca involvement in CORT related 'accelerated brain aging'.     

Modulation of voltage-activated calcium currents by mechanical stimulation in rat sensory neurons

We examined the effects of mechanical stress, induced by a stream of bath solution, on evoked action potentials, electrical excitability, and Ca2+ currents in rat dorsal root ganglion neurons in culture with the use of the whole cell patch-clamp technique. Action-potential duration was altered reversibly by flow in 39% of the 51 neurons tested, but membrane potential and excitability were unaffected. The flow-induced increases and decreases in action-potential duration were consistent with the different effects of flow on two types of Ca2+ channel, determined by voltage-clamp recordings of Ba2+ currents. Current through omega-conotoxin-sensitive (N-type) Ca2+ channels increased by an estimated 74% with flow, corresponding to 23% increase in the total high voltage-activated current, whereas current through low-threshold voltage-activated (T-type) channels decreased by 14%. We conclude that modulation of voltage-activated Ca2+ currents constitutes a route by which mechanical events can regulate Ca2+ influx in sensory neurons.     

Blockade by ifenprodil of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones: comparison with N-methyl-D-aspartate receptor antagonist actions

1. The block by ifenprodil of voltage-activated Ca2+ channels was investigated in intracellular free calcium concentration ([Ca2+]i) evoked by 50 mM K+ (high-[K+]o) in Fura-2-loaded rat hippocampal pyramidal neurones in culture and on currents carried by Ba2+ ions (IBa) through Ca2+ channels in mouse cultured hippocampal neurones under whole-cell voltage-clamp. The effects of ifenprodil on voltage-activated Ca2+ channels were compared with its antagonist actions on N-methyl-D-aspartate- (NMDA) evoked responses in the same neuronal preparations. 2. Rises in [Ca2+]i evoked by transient exposure to high-[K+]o in our preparation of rat cultured hippocampal pyramidal neurones are mediated predominantly by Ca2+ flux through nifedipine-sensitive Ca2+ channels, with smaller contributions from nifedipine-resistant, omega-conotoxin GVIA-sensitive Ca2+ channels and Ca2+ channels sensitive to crude funnel-web spider venom (Church et al., 1994). Ifenprodil (0.1-200 microM) reversibly attenuated high-[K+]o-evoked rises in [Ca2+]i with an IC50 value of 17 +/- 3 microM, compared with an IC50 value of 0.7 +/- 0.1 microM for the reduction of rises in [Ca2+]i evoked by 20 microM NMDA. Tested in the presence of nifedipine 10 microM, ifenprodil (1-50 microM) produced a concentration-dependent reduction of the dihydropyridine-resistant high-[K+]o-evoked rise in [Ca2+]i with an IC50 value of 13 +/- 4 microM. The results suggest that ifenprodil blocks Ca2+ flux through multiple subtypes of high voltage-activated Ca2+ channels. 3. Application of the polyamine, spermine (0.25-5 mM), produced a concentration-dependent reduction of rises in [Ca2+]i evoked by high-[K+]o. The antagonist effects of ifenprodil 20 micro M on high-[K+]0-evoked rises in [Ca2+]. were attenuated by spermine 0.25 mM but not by putrescine 1 or 5 mM. In contrast,spermine 0.1 mM increased rises in [Ca2+]i evoked by NMDA and enhanced the ifenprodil (5 micro M) block of NMDA-evoked rises in [Ca2+]i.4. Similar results were obtained in mouse cultured hippocampal pyramidal neurones under whole-cell voltage-clamp. Ifenprodil attenuated both the peak and delayed whole-cell IB. with an IC% value of 18 +/- 2 micro M, whilst it attenuated steady-state NMDA-evoked currents with an IC50 of 0.8 +/- 0.2 micro M. Block of IBa by ifenprodil 10 JaM was rapid in onset, fully reversible and occurred without change in thecurrent-voltage characteristics of Ba. The ifenprodil block of IBa was enhanced on membrane depolarization and was weakly dependent on the frequency of current activation. Spermine 0.1 mM potentiated control NMDA-evoked currents but attenuated IB,. In agreement with the microspectrofluorimetric studies, co-application of spermine produced a small enhancement of the inhibitory effect of ifenprodil 10 micro M on NMDA-evoked responses whereas the reduction of I4 by ifenprodil 10 micro M in the presence of spermine was less than expected if the inhibitory effects of ifenprodil and spermine on IBa were simply additive.5. The results indicate that ifenprodil blocks high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones. Although the Ca2+ channel blocking actions of ifenprodil are observed at higher concentrations than those associated with NMDA antagonist activity, Ca2+ channel blockade may contribute, at least in part, to the established neuroprotective and anticonvulsant properties of the compound.     

Blockade by sigma site ligands of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones

1. The effects of a series of structurally-dissimilar sigma site ligands were examined on high voltage-activated Ca2+ channel activity in two preparations of cultured hippocampal pyramidal neurones. 2. In mouse hippocampal neurones under whole-cell voltage-clamp, voltage-activated Ca2+ channel currents carried by barium ions (IBa) were reduced with the rank order (IC50 values in microM): 1S,2R-(-)-cis-N-methyl-N-[2-(3,4-dichlorophenyl)ethyl]- 2-(1-pyrrolidinyl)cyclohexylamine (7.8) > rimcazole (13) > haloperidol (16) > ifenprodil (18) > opipramol (32) > carbetapentane (40) = 1-benzylspiro[1,2,3,4-tetrahydronaphthalene-1,4-piperidine] (42) > caramiphen (47) > dextromethorphan (73). At the highest concentrations tested, the compounds almost abolished IBa in the absence of any other pharmacological agent. 3. The current-voltage characteristics of the whole-cell IBa were unaffected by the test compounds. The drug-induced block was rapid in onset and offset, with the exceptions of carbetapentane and caramiphen where full block was achieved only after two to three voltage-activated currents and was associated with an apparent increase in the rate of inactivation of IBa. 4. In rat hippocampal neurones loaded with the Ca(2+)-sensitive dye Fura-2, rises in intracellular free Ca2+ concentration evoked by transient exposure to 50 mM K(+)-containing medium, either in the absence or in the presence of 10 microM nifedipine (to block L-type high voltage-activated Ca2+ channels), were also reversibly attenuated by the sigma ligands. The rank order potencies for the compounds in these experimental paradigms were similar to that observed for blockade of IBa in the electrophysiological studies. 5. These results indicate that, at micromolar concentrations, the compounds tested block multiple subtypes of high voltage-activated Ca2+ channels. These actions, which do not appear to be mediated by high-affinity sigma binding sites, may play a role in some of the functional effects previously described for the compounds.     

Inhibition of neuronal high voltage-activated calcium channels by insect peptides: a comparison with the actions of omega-conotoxin GVIA

The whole cell variant of the patch clamp technique was used to investigate the actions of two novel insect peptides on high voltage-activated Ca2+ currents in cultured dorsal root ganglion (DRG) neurones. The insect peptides (PMP-D2 and PMP-C) were isolated originally from insect brains and fat bodies, and have been found to have similar three-dimensional structures to the N-type Ca2+ channel inhibitor omega-conotoxin GVIA (omega-CgTx GVIA). High voltage-activated Ca2+ currents were activated from a holding potential of -90 mV by depolarizing step commands to 0 mV. Extracellular application of synthetic PMP-D2 or PMP-C (1 microM) attenuated high voltage-activated Ca2+ currents. The effects of PMP-C were strongly dependent on the frequency of current activation, but inhibition was apparent and reached a steady state after 20 steps when currents were evoked for 30 msec at 0.1 Hz. The actions of the two insect peptides overlapped both with each other and with omega-CgTx GVIA, suggesting that N-type Ca2+ current was predominantly sensitive to these peptides. Low voltage-activated T-type current and 1,4-dihydropyridine sensitive L-type Ca2+ currents were insensitive to 1 microM PMP-D2 and PMP-C, which indicates a degree of selectivity. The presence of a fucose group on PMP-C abolished the ability of this peptide to attenuate high voltage-activated Ca2+ currents, which may reflect a mechanism by which peptide function could be regulated in insects. The electrophysiological data are supported by studies on 45Ca2+ influx into rat cerebrocortical synaptosomes. Both PMP-D2 (10 microM), PMP-C (10 microM) and omega-CgTx GVIA (1 microM) attenuated a proportion of 45Ca2+ influx into the synaptosomes, but additive effects of these peptides were not observed. We conclude that these naturally occurring peptides obtained from invertebrate preparations have inhibitory effects on N-type Ca2+ channels. Although the peptides have related three-dimensional structures, they have distinct amino acid sequences and appear to have different mechanisms of action to produce inhibition of mammalian neuronal high voltage-activated Ca2+ channels.     

Regional difference of high voltage-activated Ca2+ channels in rat CNS neurones

The diversity of high voltage-activated (HVA) Ca2+ channels in rat CNS neurones was investigated with the nystatin perforated patch recording configuration. The neurones were freshly dissociated from rat substantia nigra, ventromedial hypothalamus, tuberomammillary nucleus, nucleus tractus solitarius, hippocampal CA1 region and cerebellum. Five different types of HVA Ca2+ channels were distinguished pharmacologically; dihydropyridine sensitive L-type, omega-conotoxin-GVIA sensitive N-type, omega-agatoxin-IVA sensitive P-type, omega-conotoxin-MVIIC sensitive Q-type, and R-type which is insensitive to these organic Ca2+ antagonists. The results showed clearly that the five subtypes of HVA Ca2+ channels differ considerably in their distribution among various CNS regions.     

Regulation of N-methyl-D-aspartate receptor function by constitutively active protein kinase C

The ability of the constitutively active fragment of protein kinase C (PKM) to modulate N-methyl-D-aspartate (NMDA)-activated currents in cultured mouse hippocampal neurons and acutely isolated CA1 hippocampal neurons from postnatal rats was studied using patch-clamp techniques. The responses of two heterodimeric combinations of recombinant NMDA receptors (NR1a/NR2A and NR1a/NR2B) expressed in human embryonic kidney 293 cells were also examined. Intracellular applications of PKM potentiated NMDA-evoked currents in cultured and isolated CA1 hippocampal neurons. This potentiation was observed in the absence or presence of extracellular Ca2+ and was prevented by the coapplication of the inhibitory peptide protein kinase inhibitor(19-36). Furthermore, the PKM-induced potentiation was not a consequence of a reduction in the sensitivity of the currents to voltage-dependent blockade by extracellular Mg2+. We also found different sensitivities of the responses of recombinant NMDA receptors to the intracellular application of PKM. Some potentiation was observed with the NR1a/NR2A subunits, but none was observed with the NR1a/NR2B combination. Applications of PKM to inside-out patches taken from cultured neurons increased the probability of channel opening without changing single-channel current amplitudes or channel open times. Thus, the activation of protein kinase C is associated with potentiation of NMDA receptor function in hippocampal neurons largely through an increase in the probability of channel opening.     

Whole cell recording of sodium, potassium and calcium currents of cultured neonatal rat sympathetic neurons

Na, K and Ca currents and other electrophysiological characteristics of cultured neonatal rat superior cervical sympathetic neurons were studied using whole cell clamp technique. The mean passive and active membrane properties measured are as follows: resting membrane potential, -51 +/- 6 mV; input resistance, 1432 +/- 389 M omega; time constant, 130 +/- 32 ms; amplitude of action potential, 96 +/- 10 mV; overshoot, 42 +/- 6 mV. Na, K and Ca currents were isolated upon pharmacological manipulations. The predominant type of K current was a noninactivating delayed rectifier. Voltage-clamp studies also showed the presence of a high voltage-activated sustained inward Ca current, while low voltage could not elicit any transient Ca current.     

Voltage-activated intracellular calcium transients in thalamic relay cells and interneurons

The dynamics of intracellular calcium concentration ([Ca2+]i) following activation of low voltage-activated (LVA) and high voltage-activated (HVA) Ca2+ currents were studied in identified relay neurons and interneurons of the rat dorsal lateral geniculate nucleus (LGNd) in situ using Ca2+ imaging and patch-clamp techniques. In relay neurons, [Ca2+]i transients associated with the LVA Ca2+ current showed a fairly homogeneous somatodendritic distribution, whereas HVA transients significantly decreased to 65% of the somatic value at 60 microns dendritic distance. In interneurons, LVA transients significantly increased to 239% of the somatic value at 60 microns dendritic distance, whereas HVA transients were not significantly different in the soma and dendrites. These results indicate differences in [Ca2+]i dynamics, which may reflect a heterogeneous distribution of Ca2+ channels contributing to subcellular compartmentation in the two types of thalamic neurons.     

HIV-1 envelope proteins gp120 and gp160 potentiate NMDA-induced [Ca2+]i increase, alter [Ca2+]i homeostasis and induce neurotoxicity in human embryonic neurons

The envelope glycoprotein gp120 of the human immunodeficiency virus HIV-1 has been proposed to cause neuron death in developing murine hippocampal cultures and rat retinal ganglion cells. In the present study, cultured human embryonic cerebral and spinal neurons from 8- to 10-week-old embryos were used to study the neurotoxic effect of gp120 and gp160. Electrophysiological properties as well as N-methyl-D-aspartate (NMDA)-induced current were recorded from neurons maintained in culture for 10-30 days. Neither voltage-activated sodium or calcium currents nor NMDA-induced currents were affected by exposure of neurons to 250 pM gp120 or gp160. In contrast, when neurons were subjected to photometric measurements using the calcium dye indo-1 to monitor the intracellular free Ca2+ concentration ([Ca2+])i, gp120 and gp160 (20-250 pM) potentiated the large rises in [Ca2+]i induced by 50 microM NMDA. The potentiation of NMDA-induced Ca2+ responses required the presence of Ca2+ in the medium, and was abolished by the NMDA antagonist D-2-amino-5-phosphonovalerate (AP5) and the voltage-gated Ca2+ channel inhibitor nifedipine. Moreover, exposure of a subpopulation of spinal neurons (25% of the cells tested) to 20-250 pM gp120 or gp160 resulted in an increase in [Ca2+]i that followed three patterns: fluctuations not affected by AP5, a single peak, and the progressive and irreversible rise of [Ca2+]i. The neurotoxicity of picomolar doses of gp120 and gp160 cultures was estimated by immunofluorescence and colorimetric assay. Treatment of cultures with AP5 or nifedipine reduced gp120-induced toxicity by 70 and     

Effect of adrenalectomy in kindled rats

It has previously been observed that adrenalectomy (ADX) increases the amplitude of transient Ca currents in CA1 hippocampal neurons. In the present study, the effect of ADX on cellular properties of CA1 neurons was investigated under conditions that challenge the CA1 network, i.e. in fully kindled rats, 5-6 weeks after the last kindling session. At this time, adrenally intact kindled rats showed, in comparison to non-kindled controls, a reduced population spike amplitude evoked in the CA1 area by Schaffer/commissural fiber stimulation, an increased amplitude of low-threshold, transient Ca currents and a decrease of the high-threshold, sustained Ca currents. As observed before, ADX in non-kindled rats increased the amplitude of transient Ca currents while sustained Ca currents were not affected. In kindled rats, ADX did not increase the transient Ca current amplitude beyond the level reached by kindling alone. The kindling-induced reduction of the sustained Ca current was partly normalized after ADX. Field responses of kindled rats were not affected by ADX. These data support the view that lack of adrenal hormones does not exacerbate but, to some extent, normalizes the kindling-induced changes in cellular properties and network function of the CA1 area. The hormonal effects on kindling may be reciprocal in nature, since kindling, in turn, was found to affect the stress response and the corticosteroid receptor mRNA expression in the hippocampus.     

Reversal of age-related alterations in synaptic plasticity by blockade of L-type Ca2+ channels

The role of L-type Ca2+ channels in the induction of synaptic plasticity in hippocampal slices of aged (22-24 months) and young adult (4-6 months) male Fischer 344 rats was investigated. Prolonged 1 Hz stimulation (900 pulses) of Schaffer collaterals, which normally depresses CA3/CA1 synaptic strength in aged rat slices, failed to induce long-term depression (LTD) during bath application of the L-channel antagonist nifedipine (10 microM). When 5 Hz stimulation (900 pulses) was used to modify synaptic strength, nifedipine facilitated synaptic enhancement in slices from aged, but not young, adult rats. This enhancement was pathway-specific, reversible, and impaired by the NMDA receptor (NMDAR) antagonist DL-2-amino-5-phosphonopentanoic acid (AP5). Induction of long-term potentiation (LTP) in aged rats, using 100 Hz stimulation, occluded subsequent synaptic enhancement by 5 Hz stimulation, suggesting that nifedipine-facilitated enhancement shares mechanisms in common with conventional LTP. Facilitation of synaptic enhancement by nifedipine likely was attributable to a reduction ( approximately 30%) in the Ca2+-dependent K+-mediated afterhyperpolarization (AHP), because the K+ channel blocker apamin (1 microM) similarly reduced the AHP and promoted synaptic enhancement by 5 Hz stimulation. In contrast, apamin did not block LTD induction using 1 Hz stimulation, suggesting that, in aged rats, the AHP does not influence LTD and LTP induction in a similar way. The results indicate that, during aging, L-channels can (1) facilitate LTD induction during low rates of synaptic activity and (2) impair LTP induction during higher levels of synaptic activation via an increase in the Ca2+-dependent AHP.     

Amyloid beta-protein impairs astrocyte-mediated differentiation of hippocampal neurons

Embryonic rat hippocampal neurons were cultured on poly-D-lysine (PDL) or on cortical astrocytes, some of which had been pretreated for 24 h with amyloid beta-protein (beta-AP). Amino acid-induced currents were quantified. Membrane capacitance (Cm), as well as the amplitude and density of amino acid-evoked currents recorded in neurons cultured on untreated astrocytes were all statistically greater than those recorded in neurons grown on PDL. However, compared to untreated astrocytes, those treated with beta-AP led to significantly lower values in neurons for Cm and GABA, kainate- and NMDA-induced currents, while glycine-activated current values were not significantly different. Furthermore, beta-AP treatment abolished spontaneous Cac2+ fluctuations in astrocytes, which may account for their impaired ability to promote the expression of functional transmitter receptors in neurons.     

Multiple actions of methohexital on hippocampal CA1 and cortical neurons of rat brain slices

To explore the mechanism by which methohexital (MTH) activates epileptiform activity in patients with epilepsy, we examined the effects of MTH on hippocampal CA1 and neocortical neurons via extracellular and whole-cell patch-clamp recordings in rat brain slices. Perfusion of slices with 10 to 100 microM MTH caused no significant change in glutamatergic transmission in the hippocampal CA1 region, but enhanced gamma-aminobutyric acid (GABA)A-mediated inhibitory postsynaptic currents and induced spontaneous inhibitory postsynaptic currents in neocortical and hippocampal CA1 neurons. In addition, MTH induced a tonic, bicuculline-sensitive hyperpolarization in association with increases in membrane conductance, suggesting a direct stimulation of GABAA receptors by MTH. Spontaneous epileptiform activity was not observed in the neocortex and hippocampus after exposure of slices to MTH, neither in the standard in vitro condition nor in the presence of 4-aminopyridine, which promotes rhythmic synaptic activities. We suggest that the activation of epileptiform activity in vivo by MTH may result from increased neuronal synchrony via the potentiation of GABAA-mediated synaptic inhibition.     

Spreading depression determines acute cellular damage in the hippocampal slice during oxygen/glucose deprivation

During ischaemia neurons depolarize and release the neurotransmitter L-glutamate, which accumulates extracellularly and binds to postsynaptic receptors. This initiates a sequence of events thought to culminate in immediate and delayed neuronal death. However, there is growing evidence that during ischaemia the development of spreading depression (SD) can be an important determinant of the degree and extent of ischaemic damage. In contrast, SD without metabolic compromise (as occurs in migraine aura) causes no discernible damage to brain tissue. SD is a profound depolarization of neurons and glia that propagates like a wave across brain tissue. Brain cell swelling, an early event of both the excitotoxic process and of SD, can be assessed by imaging associated intrinsic optical signals (IOSs). We demonstrate here that IOS imaging clearly demarcates the ignition site and migration of SD across the submerged hippocampal slice of the rat. If SD is induced by elevating [K+]O, the tissue fully recovers, but in slices that are metabolically compromised at 37.5 degrees C by oxygen/glucose deprivation (OGD) or by ouabain exposure, cellular damage develops only where SD has propagated. Specifically, the evoked CA1 field potential is permanently lost, the cell bodies of involved neurons swell and their dendritic regions increase in opacity. In contrast to OGD, bath application of L-glutamate (6-10 mM) at 37.5 degrees C evokes a non-propagating LT increase in CA1 that reverses without obvious cellular damage. Moreover, application of 2-20 mM glutamate or various glutamate agonists fail to evoke SD in the submerged hippocampal slice. We propose that SD and OGD together (but not alone) constitute a 'one-two punch', causing acute neuronal death in the slice that is not replicated by elevated glutamate. These findings support the proposal that SD generation during stroke promotes and extends acute ischaemic damage.     

Reduction of voltage-dependent Mg2+ blockade of NMDA current in mechanically injured neurons

Activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors is implicated in the pathophysiology of traumatic brain injury. Here, the effects of mechanical injury on the voltage-dependent magnesium (Mg2+) block of NMDA currents in cultured rat cortical neurons were examined. Stretch-induced injury was found to reduce the Mg2+ blockade, resulting in significantly larger ionic currents and increases in intracellular free calcium (Ca2+) concentration after NMDA stimulation of injured neurons. The Mg2+ blockade was partially restored by increased extracellular Mg2+ concentration or by pretreatment with the protein kinase C inhibitor calphostin C. These findings could account for the secondary pathological changes associated with traumatic brain injury.     

Neuregulins stimulate the functional expression of Ca2+-activated K+ channels in developing chicken parasympathetic neurons

The developmental expression of macroscopic Ca2+-activated K+ currents (IK[Ca]) in chicken ciliary ganglion (CG) neurons is dependent in part on trophic factors released from preganglionic nerve terminals. Neuregulins are expressed in the preganglionic neurons that innervate the chicken CG and are therefore plausible candidates for this activity. Application of 1 nM beta1-neuregulin peptide for 12 hr evokes a large (7- to 10-fold) increase in IK[Ca] in embryonic day 9 CG neurons, even in the presence of a translational inhibitor. A similar posttranslational effect is produced by high concentrations (10 nM) of epidermal growth factor and type alpha transforming growth factor but not by 10 nM alpha2-neuregulin peptide or by neurotrophins at 40 ng.ml-1. beta1-neuregulin treatment for 12 hr also confers Ca2+ sensitivity onto large-conductance (285 pS) K+ channels observed in inside-out patches. beta-Neuregulins have no effect on voltage-activated Ca2+ currents of CG neurons. These data support the hypothesis that beta-neuregulins mediate the trophic effects of preganglionic nerve terminals on the electrophysiological differentiation of developing CG neurons.     

Calcium currents of rhythmic neurons recorded in the isolated respiratory network of neonatal mice

To obtain a quantitative characterization of voltage-activated calcium currents in respiratory neurons, we performed voltage-clamp recordings in the transverse brainstem slice of mice from neurons located within the ventral respiratory group. It is assumed that this medullary region contains the neuronal network responsible for generating the respiratory rhythm. This study represents one of the first attempts to analyze quantitatively the currents in respiratory neurons. The inward calcium currents of VRG neurons consisted of two components: a high voltage-activated (HVA) and a low voltage-activated (LVA) calcium current. The activation threshold of the HVA current was at -40 mV. It was fully activated (peak voltage) between -10 and 0 mV. The half-maximal activation (V50) was at -27. 29 mV +/- 1.15 (n = 24). The HVA current was inactivated completely at a holding potential of -35 mV and fully deinactivated at a holding potential of -65 mV (V50, -52.26 mV +/- 0.27; n = 18). The threshold for the activation of the LVA current was at -65 mV. This current had its peak voltage between -50 and -40 mV (mean, V50 = -59. 15 mV +/- 0.21; n = 15). The LVA current was inactivated completely at a holding potential of -65 mV and deinactivated fully at a holding potential of -95 mV (mean, V50 = -82.40 mV +/- 0.32; n = 38). These properties are consistent with other studies suggesting that the LVA current is a T-type current. The properties of these inward currents are discussed with respect to their role in generating Ca2+ potentials that may contribute to the generation of the mammalian respiratory rhythm.     

Developmental change of the inhibition by lead of NMDA-activated currents in cultured hippocampal neurons

The inhibition of N-methyl-D-aspartate (NMDA)-activated current in cultured fetal rat hippocampal neurons by Pb2+ was investigated at various stages of cell development. Pb2+ selectively inhibited NMDA currents recorded from young cultured neurons. In the first week of culture, Pb2+ showed the most prominent inhibition, which was gradually attenuated in the following weeks. Pb2+'s action was selective for NMDA- as opposed to either kainate- or quisqualate-induced currents. The current-voltage relationship for NMDA-induced currents in the presence of Pb2+ revealed that the effect of this cation was voltage-independent, which suggested that the site of interaction of Pb2+ with the NMDA receptor/channel is located outside the membrane electric field. Single channel studies showed that Pb2+ reduced the frequency but not the lifetime of the NMDA-activated single channel currents. Further evaluation of the mechanism of action of Pb2+ on the NMDA receptor demonstrated that this cation is a noncompetitive antagonist of both NMDA and glycine. We have demonstrated that the NMDA-induced whole cell currents change along with cell development, and the effects of Pb2+ are also dependent upon age of culture. The NMDA-induced currents in cultured rat hippocampal neurons had two components, one that decayed rapidly and another that decayed slowly. The fast component was clearly observed at concentrations of glycine higher than 1 microM, whereas the slow component reached its maximum amplitude at the glycine concentration of 1 microM. Moreover, the rapidly decaying component of NMDA-evoked whole cell currents was predominant in young cultured neurons, and its contribution to the total current was reduced in old cultured neurons.(ABSTRACT TRUNCATED AT 250 WORDS)