Propylparaben reduces the excitability of hippocampal neurons by blocking sodium channels

Propylparaben (PPB) is an antimicrobial preservative widely used in food, cosmetics, and pharmaceutics. Virtual screening methodologies predicted anticonvulsant activity of PPB that was confirmed in vivo. Thus, we explored the effects of PPB on the excitability of hippocampal neurons by using standard patch clamp techniques. Bath perfusion of PPB reduced the fast-inactivating sodium current (I_Na) amplitude, causing a hyperpolarizing shift in the inactivation curve of the I_Na, and markedly delayed the sodium channel recovery from the inactivation state. Also, PPB effectively suppressed the riluzole-sensitive, persistent sodium current (I_NaP). PPB perfusion also modified the action potential kinetics, and higher concentrations of PPB suppressed the spike activity. Nevertheless, the modulatory effects of PPB did not occur when PPB was internally applied by whole-cell dialysis. These results indicate that PPB reduces the excitability of CA1 pyramidal neurons by modulating voltage-dependent sodium channels. The mechanistic basis of this effect is a marked delay in the recovery from inactivation state of the voltage-sensitive sodium channels. Our results indicate that similar to local anesthetics and anticonvulsant drugs that act on sodium channels, PPB acts in a use-dependent manner.     

INaP underlies intrinsic spiking and rhythm generation in networks of cultured rat spinal cord neurons

We have shown previously that rhythm generation in disinhibited spinal networks is based on intrinsic spiking, network recruitment and a network refractory period following the bursts. This refractory period is based mainly on electrogenic Na/K pump activity. In the present work, we have investigated the role of the persistent sodium current (INaP) in the generation of bursting using patch-clamp and multielectrode array recordings. We detected INaP exclusively in the intrinsic spiking cells. The blockade of INaP by riluzole suppressed the bursting by silencing the intrinsic spiking cells and suppressing network recruitment. The blockade of the persistent sodium current produced a hyperpolarization of the membrane potential of the intrinsic spiking cells, but had no effect on non-spiking cells. We also investigated the involvement of the hyperpolarization-activated cationic current (I(h)) in the rhythmic activity. The bath application of ZD7288, a specific I(h) antagonist, slowed down the rate of the bursts by increasing the interburst intervals. I(h) was present in approximately 70% of the cells, both in the intrinsic spiking cells as well as in the non-spiking cells. We also found both kinds of cells in which I(h) was not detected. In summary, in disinhibited spinal cord cultures, a persistent sodium current underlies intrinsic spiking, which, via recurrent excitation, generates the bursting activity. The hyperpolarization-activated cationic current contributes to intrinsic spiking and modulates the burst frequency.     

Intrinsic membrane potential oscillations in hippocampal neurons in vitro

Membrane potential oscillations (MPOs) of 2-10 Hz and up to 6 mV were found in almost all stable hippocampal CA1 and CA3 neurons in the in vitro slice preparation. MPOs were prominent for pyramidal cells but less pronounced in putative interneurons. MPOs were activated at threshold depolarizations that evoked a spike and the frequency of the MPOs increased with the level of depolarization. MPOs were distinct from and seemed to regulate spiking, with a spike often riding near the top of a depolarizing MPO wave. Analysis of the periodicity of the oscillations indicate that the period of MPOs did not depend on the afterhyperpolarization (AHP) following a single spike. MPOs persisted in low (0-0.1 mM) Ca2+ medium, with or without Cd2+ (0.2 mM), when synaptic transmission was blocked. Choline-substituted low-Na+ (0-26 mM) medium, 3 microM tetrodotoxin (TTX) or intracellular injection of QX-314 reduced or abolished the fast Na(+)-spike and reduced inward anomalous rectification. About 40% of CA1 neurons had no MPOs after Na+ currents were blocked, suggesting that these MPOs were Na(+)-dependent. In about 60% of the cells, a large depolarization activated Ca(2+)-dependent MPOs and slow spikes. MPOs were not critically affected by extracellular Ba2+ or Cs2+, or by 0.2 mM 4-aminopyridine, with or without 2 mM tetraethylammonium (TEA). However, in 5-10 mM TEA medium, MPOs were mostly replaced by 0.2-3 Hz spontaneous bursts of wide-duration spikes followed by large AHPs. Low Ca2+, Cd2+ medium greatly reduced the spike width but not the spike-bursts. In conclusion, each cycle of an MPO in normal medium probably consists of a depolarization phase mediated by Na+ currents, possibly mixed with Ca2+ currents activated at a higher depolarization. The repolarization/hyperpolarization phase may be mediated by Na+/Ca2+ current inactivation and partly by TEA-sensitive, possibly the delayed rectifier, K+ currents. The presence of prominent intrinsic, low-threshold MPOs in all hippocampal pyramidal neurons suggests that MPOs may play an important role in information processing in the hippocampus.     

Extracellular fields influence transmembrane potentials and synchronization of hippocampal neuronal activity

The influence of extracellular fields on the transmembrane potential (TMP) of CA1 pyramidal neurons was investigated following both ortho- and antidromic stimulation in the in vitro hippocampal slice preparation. A short latency negative deflection on the intracellular potential coincided with the falling phase of the extracellular population spike. Subtraction of extracellular field potentials from ground referenced intracellular records revealed a sharp depolarizing wave of the TMP superimposed upon the underlying synaptic potential. This graded depolarization was capable of discharging CA1 cells and displayed a parallel shift in latency and amplitude with the extracellular population spike. A similar depolarizing wave was associated with the antidromically evoked population spike which persisted following blockade of synaptic activity. Finally, multiple population spike activity similar to that observed during epileptiform discharge was associated with repetitive depolarizing waves of the TMP. These data suggest that extracellular field potentials can ephaptically discharge CA1 neurons and may play a role in recruitment and synchronization of neuronal activity in the hippocampus.     

Ion currents and spiking properties of identified subtypes of locust octopaminergic dorsal unpaired median neurons

Efferent dorsal unpaired median (DUM) neurons are key elements of an insect neuromodulatory system. In locusts, subpopulations of DUM neurons mediate octopaminergic modulation at specific targets depending on their activity during different behaviours. This study investigates whether in addition to synaptic inputs, activity in DUM neurons depends on intrinsic membrane properties. Intracellular in situ recordings and whole-cell patch-clamp recordings from freshly isolated somata characterize somatic voltage signals and the underlying ion currents of individual subtypes of DUM neurons identified beforehand by a vital retrograde tracing technique. Na(+), Ca(2+), K(+) currents and a hyperpolarization-activated (I(h)) current are described in detail for their (in-)activation properties and subtype-specific current densities. In addition, a Ca(2+)-dependent K(+) current is demonstrated by its sensitivity to cadmium and charybdotoxin. This complex current composition determines somatic excitability similar in all subtypes of DUM neurons. Both Na(+) and Ca(2+) currents generate overshooting somatic action potentials. Repolarizing K(+) currents, in particular transient, subthreshold-activating A-currents, regulate the firing frequency and cause delayed excitation by shunting depolarizing input. An opposing hyperpolarization-activated (I(h)) current contributes to the resting membrane potential and induces rebound activity after prolonged inhibition phases. A quantitative analysis reveals subtype-specific differences in current densities with more inhibitory I(K) but less depolarizing I(Na) and I(h) - at least in DUM3 neurons promoting a reliable suppression of their activity as observed during behaviour. In contrast, DUM neurons that are easily activated during behaviour (DUM3,4,5 and DUMETi) express less I(K) and a pronounced depolarizing I(h) promoting excitability.     

Efficacy of antiseizure medication in a mouse model of HCN1 developmental and epileptic encephalopathy

Acquisition of drug-sensitivity profiles is challenging in rare epilepsies. Anecdotal evidence suggests that antiseizure medications that block sodium channels as their primary mechanism of action exacerbate seizures in HCN1 developmental and epileptic encephalopathies (DEEs), whereas sodium valproate is effective for some patients. The Hcn1 M294L heterozygous knock-in (Hcn1^M294L) mouse carries the homologue of the recurrent gain-of-function HCN1 M305L pathogenic variant and recapitulates the seizure and some behavioral phenotypes observed in patients. We used this mouse model to study drug efficacy in HCN1 DEE. Hcn1^M294L mice display epileptiform spiking on electrocorticography (ECoG), which we used as a quantifiable measure of drug effect. Phenytoin, lamotrigine, and retigabine significantly increased ECoG spike frequency, with lamotrigine and retigabine triggering seizures in a subset of the mice tested. In addition, there was a strong trend for carbamazepine to increase spiking. In contrast, levetiracetam, diazepam, sodium valproate, and ethosuximide all significantly reduced ECoG spike frequency. Drugs that reduced spiking did not cause any consistent ECoG spectral changes, whereas drugs that increased spiking all increased power in the slower delta and/or theta bands. These data provide a framework on which to build our understanding of gain-of-function HCN1 DEE pharmacosensitivity in the clinical setting.     

Action of serotonin on the hyperpolarization-activated cation current (Ih) in rat CA1 hippocampal neurons

We studied the effects of serotonin (5-HT) on hippocampal CA1 pyramidal neurons. In current-clamp mode, 5-HT induced a hyperpolarization and reduction of excitability due to the opening of inward rectifier K+ channels, followed by a late depolarization and partial restoration of excitability. These two components could be dissociated, as in the presence of BaCl2 to block K+ channels, 5-HT induced a depolarization accompanied by a reduction of membrane resistance, whereas in the presence of ZD 7288 [4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride], a selective blocker of the hyperpolarization-activated cation current (Ih), 5-HT only hyperpolarized neurons. We then studied the action of 5-HT on Ih in voltage-clamp conditions. 5-HT increased Ih at -90 mV by 29.1 +/- 2.9% and decreased the time constant of activation by 20.1 +/- 1.7% (n = 16), suggesting a shift in the voltage dependence of the current towards more positive potentials; however, the fully activated current measured at -140 mV also increased (by 14.1 +/- 1.7%, n = 14); this increase was blocked by ZD 7288, implying an effect of 5-HT on the maximal conductance of Ih. Both the shift of activation curve and the increase in maximal conductance were confirmed by data obtained with ramp protocols. Perfusion with the membrane-permeable analogue of cAMP, 8-bromoadenosine 3'5'-cyclic monophosphate (8-Br-cAMP), increased Ih both at -90 and -140 mV, although the changes induced were smaller than those due to 5-HT. Our data indicate that 5-HT modulates Ih by shifting its activation curve to more positive voltages and by increasing its maximal conductance, and that this action is likely to contribute to the 5-HT modulation of excitability of CA1 cells.     

Ionic currents underlying spontaneous action potentials in isolated cerebellar Purkinje neurons.

Acutely dissociated cell bodies of mouse Purkinje neurons spontaneously fired action potentials at approximately 50 Hz (25 degrees C). To directly measure the ionic currents underlying spontaneous activity, we voltage-clamped the cells using prerecorded spontaneous action potentials (spike trains) as voltage commands and used ionic substitution and selective blockers to isolate individual currents. The largest current flowing during the interspike interval was tetrodotoxin-sensitive sodium current (approximately -50 pA between -65 and -60 mV). Although the neurons had large voltage-dependent calcium currents, the net current blocked by cobalt substitution for calcium was outward at all times during spike trains. Thus, the electrical effect of calcium current is apparently dominated by rapidly activated calcium-dependent potassium currents. Under current clamp, all cells continued firing spontaneously (though approximately 30% more slowly) after block of T-type calcium current by mibefradil, and most cells continued to fire after block of all calcium current by cobalt substitution. Although the neurons possessed hyperpolarization-activated cation current (Ih), little current flowed during spike trains, and block by 1 mM cesium had no effect on firing frequency. The outward potassium currents underlying the repolarization of the spikes were completely blocked by 1 mM TEA. These currents deactivated quickly (<1 msec) after each spike. We conclude that the spontaneous firing of Purkinje neuron cell bodies depends mainly on tetrodotoxin-sensitive sodium current flowing between spikes. The high firing rate is promoted by large potassium currents that repolarize the cell rapidly and deactivate quickly, thus preventing strong hyperpolarization and restoring a high input resistance for subsequent depolarization.     

In vivo modulation of post-spike excitability in vasopressin cells by kappa-opioid receptor activation

An endogenous kappa-opioid agonist reduces the duration of phasic bursts in vasopressin cells. Non-synaptic post-spike depolarizing after-potentials underlie activity during bursts by increasing post-spike excitability and kappa-receptor activation reduces depolarizing after-potential amplitude in vitro. To investigate the effects of kappa-opioids on post-spike excitability in vivo, we analysed extracellular recordings of the spontaneous activity of identified supraoptic nucleus vasopressin cells in urethane-anaesthetized rats infused with Ringer's solution (n = 17) or the kappa-agonist, U50,488H (2.5 microg/h at 0.5 microl/h; n = 23), into the supraoptic nucleus over 5 days. We plotted the mean hazard function for the interspike interval distributions as a measure of the post-spike excitability of these cells. Following each spike, the probability of another spike firing in vasopressin cells recorded from U50,488H infused nuclei was markedly reduced compared to Ringer's treated vasopressin cells. To determine whether U50,488H could reduce post-spike excitability in cells that displayed spontaneous phasic activity, we infused U50,488H (50 microg/h at 1 microl/h, i.c.v.), for 1-12 h while recording vasopressin cell activity. Nine of 10 vasopressin cells were silenced by i.c.v. U50,488H 15 +/- 5 min into the infusion. Six cells exhibited spontaneous phasic activity before U50,488H infusion and recordings from three of these phasic cells were maintained until activity recovered; during U50,488H infusion, the activity of these three cells was irregular. Generation of the mean hazard function before and during U50,488H infusion revealed a reduction in post-spike excitability during U50,488H infusion. Thus, kappa-receptor activation reduces post-spike excitability in vivo; this may reflect inhibition of depolarizing after-potentials and may thus underlie the reduction in burst duration of vasopressin cells caused by an endogenous kappa-agonist in vivo.     

Suppression by topiramate of epileptiform burst discharges in hippocampal CA3 neurons of spontaneously epileptic rat in vitro

Topiramate, a novel antiepileptic drug, inhibits the seizures of spontaneously epileptic rat (SER), a double mutant (zi/zi, tm/tm) which exhibits both tonic convulsion and absence-like seizures from the age of 8-weeks. Hippocampal CA3 pyramidal neurons in SER show a long-lasting depolarization shift with accompanying repetitive firing when a single electrostimulation is delivered to the mossy fibers in vitro. The effects of topiramate on the excitability of CA3 pyramidal neurons in SER were examined to elucidate the mechanism underlying the antiepileptic action. Intracellular recordings were performed in 23 hippocampal slice preparations of 16 SER aged 8–17 weeks. Topiramate (10–100 μM) dose-dependently inhibited the depolarizing shifts with repetitive firing induced by mossy fiber stimulation without affecting the first spike and resting membrane potentials in hippocampal CA3 neurons of SER. Higher dose of topiramate (100 μM) sometimes inhibited the first spike, and decreased excitatory postsynaptic potentials in the SER CA3 neurons. However, topiramate up to 100 μM did not affect the single action potential elicited by the stimulation in the hippocampal CA3 neurons of age-matched Wistar rat devoid of the seizure. Application of topiramate (100 μM) did not significantly affect the firing induced by depolarizing pulse applied in the CA3 neurons of the SER. In addition, topiramate (100 μM) had no effects on the Ca²⁺ spike induced by intracellularly applied depolarizing pulse in the presence of tetrodotoxin and tetraethylammonium. In contrast, a dose-dependent inhibition of depolarization and repetitive firing induced by bath application of glutamate in CA3 pyramidal neurons was obtained with topiramate (10–100 μM). Furthermore, topiramate (100 μM) decreased the number of miniature postsynaptic potential of CA3 pyramidal neurons of SER. In patch clamp whole cell recording using acutely dissociated hippocampal CA3 neurons from SER aged 8-weeks and age-matched normal Wistar rats, there were no remarkable effects on voltage dependent Ca²⁺ current with topiramate up to 300 μM in either animal; the current was completely blocked by Cd²⁺ at a concentration of 1 mM. These findings suggest that topiramate inhibits release of glutamate from the nerve terminals and/or abnormal firing of the CA3 pyramidal neurons of SER by mainly blocking glutamate receptors in the neurons.     

Postnatal development of temporal integration,spike timing and spike threshold regulation by a dendrotoxin‐sensitive K+ current in rat CA1 hippocampal cells

Spike timing and network synchronization are important for plasticity, development and maturation of brain circuits. Spike delays and timing can be strongly modulated by a low‐threshold, slowly inactivating, voltage‐gated potassium current called D‐current (I_D). I_D can delay the onset of spiking, cause temporal integration of multiple inputs, and regulate spike threshold and network synchrony. Recent data indicate that I_D can also undergo activity‐dependent, homeostatic regulation. Therefore, we have studied the postnatal development of I_D‐dependent mechanisms in CA1 pyramidal cells in hippocampal slices from young rats (P7–27), using somatic whole‐cell recordings. At P21–27, these neurons showed long spike delays and pronounced temporal integration in response to a series of brief depolarizing current pulses or a single long pulse, whereas younger cells (P7–20) showed shorter discharge delays and weak temporal integration, although the spike threshold became increasingly negative with maturation. Application of α‐dendrotoxin (α‐DTX), which blocks I_D, reduced the spiking latency and temporal integration most strongly in mature cells, while shifting the spike threshold most strongly in a depolarizing direction in these cells. Voltage‐clamp analysis revealed an α‐DTX‐sensitive outward current (I_D) that increased in amplitude during development. In contrast to P21–23, I_D in the youngest group (P7–9) showed smaller peri‐threshold amplitude. This may explain why long discharge delays and robust temporal integration only appear later, 3 weeks postnatally. We conclude that I_D properties and I_D‐dependent functions develop postnatally in rat CA1 pyramidal cells, and I_D may modulate network activity and plasticity through its effects on synaptic integration, spike threshold, timing and synchrony.     

The effects of QX-314 on medullary respiratory neurones

The synaptic and current-evoked responses of respiratory neurones located in the nucleus of the tractus solitarius, the para- and retroambigual regions and the nucleus ambiguus, were examined after voltage-dependent sodium currents were blocked by intracellular application of the quaternary lidocaine derivative QX-314. (1) QX-314 abolished orthodromically and antidromically evoked action potential discharge. Only antidromic action potentials recovered during negative DC current injection. (2) QX-314 did not alter the amplitude or duration of small and short excitatory and inhibitory postsynaptic potentials evoked by vagus or superior laryngeal nerve stimulation. Larger and longer waves of spontaneous membrane depolarizations, however, were slightly diminished. (3) The repetitive discharge evoked by depolarizing current pulses was blocked by QX-314. Positive current pulses produced less membrane depolarization than under control and often evoked only a single action potential at the beginning of the pulse, indicating that QX-314 interferes with the processes responsible for repetitive firing. (4) When fast spike discharges were completely blocked, positive current pulses occasionally evoked depolarizing 'spikes' and potentials which were followed by a hyperpolarization. We conclude that a noninactivating sodium inward current and calcium currents contribute to the electroresponsiveness of respiratory neurones.     

Sodium depletion in the periaxonal space of the squid axon treated with pyrethroids

Pyrethriods are known to increase the steady-state sodium current during a step depolarization and to increase and prolong the tail sodium current associated with a step repolarization of the membrane. The pyrethroid-induced tail sodium current of squid axons developed as a function of the duration of the conditioning depolarizing pulse. However, with further lengthening the conditioning pulse duration, it decreased, further increased, or remained constant depending on the direction of sodium current during the depolarization, irrespective of the membrane potential per se. The depletion or accumulation of sodium in the periaxonal space during a 200-ms conditioning depolarizing pulse in the axon internally treated with pronase, pyrethroids, or both, was demonstrated by measurements of the changes in sodium reversal potential. Thus the observed changes in tail current amplitude as a function of the conditioning pulse duration are explicable in terms of changes in sodium concentration in the periaxonal space without assuming inactivation of the pyrethroid-modified channel.     

Sequential acquisition of cacophony calcium currents,sodium channels and voltage‐dependent potassium currents affects spike shape and dendrite growth during postembryonic maturation of an identified Drosophila motoneuron

During metamorphosis the CNS undergoes profound changes to accommodate the switch from larval to adult behaviors. In Drosophila and other holometabolous insects, adult neurons differentiate either from respecified larval neurons, newly born neurons, or are born embryonically but remain developmentally arrested until differentiation during pupal life. This study addresses the latter in the identified Drosophila flight motoneuron 5. In situ patch‐clamp recordings, intracellular dye fills and immunocytochemistry address the interplay between dendritic shape, excitability and ionic current development. During pupal life, changes in excitability and spike shape correspond to a stereotyped, progressive appearance of voltage‐gated ion channels. High‐voltage‐activated calcium current is the first current to appear at pupal stage P4, prior to the onset of dendrite growth. This is followed by voltage‐gated sodium as well as transient potassium channel expression, when first dendrites grow, and sodium‐dependent action potentials can be evoked by somatic current injection. Sustained potassium current appears later than transient potassium current. During the early stages of rapid dendritic growth, sodium‐dependent action potentials are broadened by a calcium component. Narrowing of spike shape coincides with sequential increases in transient and sustained potassium currents during stages when dendritic growth ceases. Targeted RNAi knockdown of pupal calcium current significantly reduces dendritic growth. These data indicate that the stereotyped sequential acquisition of different voltage‐gated ion channels affects spike shape and excitability such that activity‐dependent calcium influx serves as a partner of genetic programs during critical stages of motoneuron dendrite growth.     

Enhancement of persistent Na+ current by sea anemone toxin (ATX II) exerts dual action on hippocampal excitability

We used anemone toxin II (ATX II) to study how a selective enhancement of persistent Na+ current (INaP) would affect the excitability of CA1 pyramidal neurons in the hippocampal slice. In whole-cell recordings from CA1 cell somata, local application of ATX II (10 microM) into the stratum pyramidale invariably depolarized the neurons and produced sustained burst discharges with depolarizing plateau potentials of variable amplitude and length. However, the strong excitatory action of ATX II, observed on the single cell level, was not mirrored in field potential recordings from the same hippocampal subfield. The amplitude of the electrically evoked population spike declined, reflecting the decreased availability of fast Na+ channels, and the intracellulary recorded burst discharges were not detected by the field electrode. The lacking synchronization of cellular bursting activity was seen during both local and bath application of ATX II, suggesting that the toxin, in addition to promoting burst discharges of individual neurons, simultaneously dampens network excitability. In fact, ATX II reduced afferent fibre volleys (reflecting axonal excitability) and field excitatory postsynaptic potentials (EPSPs) in a similar fashion. As the expression of different Na+ channel subtypes appears to be compartmentalized within hippocampal neurons, we propose that point mutations leading to pathologically enhanced INaP might exert quite opposite effects, depending on the type and location of the Na+ channel affected. Whereas alterations of somatodendritic Na+ channels would give rise to bursting activity, alterations of axonal Na+ channels would primarily decrease network excitability.     

Repeated morphine exposure decreased the nucleus accumbens excitability during short-term withdrawal

It is well known that the nucleus accumbens plays an important role in drug reinforcing effect and relapse. However, the cellular neuroadaptations that take place in accumbens neurons after repeated drug exposure are still not well understood, especially for opioids. Here, we examined how nucleus accumbens neuronal excitability becomes affected in rats exposed to morphine using whole-cell patch-clamp recordings. Medium spiny neurons recorded from brain slices of repeated morphine treated rats exhibited a significant decrease in the intrinsic excitability after 3-4 days withdrawal, compared to that of neurons from saline treated animals, which was indicated by the increase of current to evoke the first spike and the decrease of spike number induced by depolarizing current steps in the morphine group. Moreover, the excitability decrease was accompanied by related membrane property changes, such as resting membrane potential hyperpolarization, input resistance, and membrane time constant decrease, inward rectification increase, and action potential duration decrease. Taken together, repeated morphine exposure and short-term withdrawal may reduce nucleus accumbens activity and output by modulating intrinsic membrane properties of its output neurons, which could be an important neuroadaptation process that mediates morphine addictive effect.     

The Effects of Dopamine on the Subthreshold Electrophysiological Responses of Rat Prefrontal Cortex Neurons In Vitro

The rat prefrontal cortex is densely innervated by dopaminergic fibres originating in the mesencephalic ventral tegmental area, and dopamine application in vivo has an inhibitory effect. We have studied the effects of dopamine on the persistent sodium current that is present in prefrontal cortex neurons and on the subthreshold electrophysiological responses generated by that current: a slow depolarization and a fast oscillatory activity. Experiments were made in coronal slices of rat frontal cortex (300–400 pm thickness) and intracellular recordings from regularly spiking cells were obtained with 3 M potassium acetate-filled glass microelectrodes (80–150 MΩ). Dopamine was applied dissolved in the extracellular medium and, in current-clamp recordings, reversibly inhibited the slow subthreshold depolarization. Dopamine was ineffective when applied after tetrodotoxin (1 μM) had blocked the action potentials. This inhibition was dose-dependent in the range of 0.1–10 μM. Dopamine, applied at 10 μM, decreased the steady-state firing frequency and also inhibited the subthreshold fast oscillatory activity. The currents activated in the subthreshold range were recorded with the single-electrode voltage-clamp technique and a clear persistent, tetrodotoxin-sensitive component was isolated. This component was inhibited by 50% in a reversible way by 20 μM dopamine. These results show that dopamine increases the threshold for spike firing and suggest a mechanism for the inhibitory action of this neurotransmitter in the prefrontal cortex.     

Analysis of distinct short and prolonged components in rebound spiking of deep cerebellar nucleus neurons

Deep cerebellar nucleus (DCN) neurons show pronounced post-hyperpolarization rebound burst behavior, which may contribute significantly to responses to strong inhibitory inputs from cerebellar cortical Purkinje cells. Thus, rebound behavior could importantly shape the output from the cerebellum. We used whole-cell recordings in brain slices to characterize DCN rebound properties and their dependence on hyperpolarization duration and depth. We found that DCN rebounds showed distinct fast and prolonged components, with different stimulus dependence and different underlying currents. The initial depolarization leading into rebound spiking was carried by hyperpolarization-activated cyclic nucleotide-gated current, and variable expression of this current could lead to a control of rebound latency. The ensuing fast rebound burst was due to T-type calcium current, as previously described. It was highly variable between cells in strength, and could be expressed fully after short periods of hyperpolarization. In contrast, a subsequent prolonged rebound component required longer and deeper periods of hyperpolarization before it was fully established. We found using voltage-clamp and dynamic-clamp analyses that a slowly inactivating persistent sodium current fits the conductance underlying this prolonged rebound component, resulting in spike rate increases over several seconds. Overall, our results demonstrate that multiphasic DCN rebound properties could be elicited differentially by different levels of Purkinje cell activation, and thus create a rich repertoire of potential rebound dynamics in the cerebellar control of motor timing.     

Differential excitability and voltage-dependent Ca2+ signalling in two types of medial entorhinal cortex layer V neurons

The entorhinal cortex (EC) is a key structure in memory formation, relaying sensory information to the hippocampal formation and processed information to the neocortex. EC neurons in the deep layers modulate the transfer of sensory information by the superficial layers and the dentate gyrus, and form the output to the neocortex. Here we characterize two types of EC layer V neurons by their fluorescence morphology, electrophysiology and intracellular Ca2+ signalling using intracellular recording and Ca2+ imaging. Pyramidal neurons show, in response to depolarizing current pulses, regular firing with strong adaptation and a fast and medium afterhyperpolarization (AHP) which are separated by a depolarizing notch and, with hyperpolarizing current injection, a transient sag. Multipolar cells respond to depolarization with delayed firing with very weak adaptation and have no depolarizing notch between fast and medium AHP and no sag with hyperpolarization. The delayed firing was blocked by 30 micro m 4-aminopyridine, indicating mediation by the D-type potassium current. Subthreshold depolarization evoked membrane potential oscillations of 2-5 Hz in both cell types and an increase in [Ca2+]i of 37 nm in pyramidal and 59 nm in multipolar neurons. Repetitive firing at 10 Hz for 30 s increased [Ca2+]i in pyramidal and multipolar neurons by 194 and 295 nm, respectively. Differential temporal firing and Ca2+ signalling suggest specific information processing and synaptic memory storage possibilities in these two layer V cell types of the EC.     

Effects of vigabatrin on epileptiform abnormal discharges in hippocampal CA3 neurons of spontaneously epileptic rats (SER)

Vigabatrin, a γ-amino butyric acid (GABA) transaminase inhibitor, is known to inhibit partial epilepsy in humans. The spontaneously epileptic rat (SER), a double mutant (zi/zi, tm/tm), exhibits both tonic convulsion and absence-like seizures from the age of 8 weeks. Hippocampal CA3 pyramidal neurons in SER show a long-lasting depolarization shift with accompanying repetitive firing when a single stimulus is delivered to the mossy fibers in slice preparations. The effects of vigabatrin on the abnormal excitability of hippocampal CA3 pyramidal neurons in SER were examined to elucidate the mechanism underlying the antiepileptic action of the drug. Intracellular recordings were performed in 24 hippocampal slice preparations of 20 SER aged 8–17 weeks old. Bath application of vigabatrin (1 mM) inhibited the depolarizing shifts with repetitive firing induced by mossy fiber stimulation in 15 min without affecting the first spike and resting membrane potentials in hippocampal CA3 neurons of SER. A higher dose of vigabatrin (10 mM) sometimes inhibited the first spike. However, vigabatrin at doses up to 10 mM did not significantly affect the single action potential elicited by stimulation of the mossy fibers in the hippocampal CA3 neurons of age-matched Wistar rats. In addition, application of vigabatrin (10 mM) did not significantly affect the firing induced by depolarizing pulse applied in the CA3 neurons of the SER, nor the miniature excitatory postsynaptic potential (mEPSP) recorded in the CA3 neurons of SER. The inhibitory effect of vigabatrin (1 mM) on the mossy fiber stimulation-induced depolarization shift with repetitive firing was blocked by concomitant application of bicuculline (10 μM), a GABA_A receptor antagonist. These findings strongly suggested that GABA increased by inhibition of GABA transaminase with vigabatrin inhibits abnormal excitation of hippocampal CA3 neurons of SER via GABA_A receptors, although the possibility that the drug acted directly on the GABA_A receptors of CA3 neurons could not be completely excluded.