An allosteric gating model recapitulates the biophysical properties of IK,L expressed in mouse vestibular type I hair cells.

KEY POINTS: Vestibular type I and type II hair cells and their afferent fibres send information to the brain regarding the position and movement of the head. The characteristic feature of type I hair cells is the expression of a low-voltage-activated outward rectifying K+ current, IK,L , whose biophysical properties and molecular identity are still largely unknown. In vitro, the afferent nerve calyx surrounding type I hair cells causes unstable intercellular K+ concentrations, altering the biophysical properties of IK,L . We found that in the absence of the calyx, IK,L in type I hair cells exhibited unique biophysical activation properties, which were faithfully reproduced by an allosteric channel gating scheme. These results form the basis for a molecular and pharmacological identification of IK,L . ABSTRACT: Type I and type II hair cells are the sensory receptors of the mammalian vestibular epithelia. Type I hair cells are characterized by their basolateral membrane being enveloped in a single large afferent nerve terminal, named the calyx, and by the expression of a low-voltage-activated outward rectifying K+ current, IK,L . The biophysical properties and molecular profile of IK,L are still largely unknown. By using the patch-clamp whole-cell technique, we examined the voltage- and time-dependent properties of IK,L in type I hair cells of the mouse semicircular canal. We found that the biophysical properties of IK,L were affected by an unstable K+ equilibrium potential (Veq K+ ). Both the outward and inward K+ currents shifted Veq K+ consistent with K+ accumulation or depletion, respectively, in the extracellular space, which we attributed to a residual calyx attached to the basolateral membrane of the hair cells. We therefore optimized the hair cell dissociation protocol in order to isolate mature type I hair cells without their calyx. In these cells, the uncontaminated IK,L showed a half-activation at -79.6 mV and a steep voltage dependence (2.8 mV). IK,L also showed complex activation and deactivation kinetics, which we faithfully reproduced by an allosteric channel gating scheme where the channel is able to open from all (five) closed states. The 'early' open states substantially contribute to IK,L activation at negative voltages. This study provides the first complete description of the 'native' biophysical properties of IK,L in adult mouse vestibular type I hair cells.

Long-term effect of neonatal inhibition of APP gamma-secretase on hippocampal development in the Ts65Dn mouse model of Down syndrome.

Neurogenesis impairment is considered a major determinant of the intellectual disability that characterizes Down syndrome (DS), a genetic condition caused by triplication of chromosome 21. Previous evidence obtained in the Ts65Dn mouse model of DS showed that the triplicated gene APP (amyloid precursor protein) is critically involved in neurogenesis alterations. In particular, excessive levels of AICD (amyloid precursor protein intracellular domain) resulting from APP cleavage by gamma-secretase increase the transcription of Ptch1, a Sonic Hedgehog (Shh) receptor that keeps the mitogenic Shh pathway repressed. Previous evidence showed that neonatal treatment with ELND006, an inhibitor of gamma-secretase, reinstates the Shh pathway and fully restores neurogenesis in Ts65Dn pups. In the framework of potential therapies for DS, it is extremely important to establish whether the positive effects of early intervention are retained after treatment cessation. Therefore, the goal of the current study was to establish whether early treatment with ELND006 leaves an enduring trace in the brain of Ts65Dn mice. Ts65Dn and euploid pups were treated with ELND006 in the postnatal period P3-P15 and the outcome of treatment was examined at ~one month after treatment cessation. We found that in treated Ts65Dn mice the pool of proliferating cells in the hippocampal dentate gyrus (DG) and total number of granule neurons were still restored as was the number of pre- and postsynaptic terminals in the stratum lucidum of CA3, the site of termination of the mossy fibers from the DG. Accordingly, patch-clamp recording from field CA3 showed functional normalization of the input to CA3. Unlike in field CA3, the number of pre- and postsynaptic terminals in the DG of treated Ts65Dn mice was no longer fully restored. The finding that many of the positive effects of neonatal treatment were retained after treatment cessation provides proof of principle demonstration of the efficacy of early inhibition of gamma-secretase for the improvement of brain development in DS.

Synthesis and biological evaluation of a new class of benzothiazines as neuroprotective agents.

Neurodegenerative diseases are disorders related to the degeneration of central neurons that gradually lead to various, severe alterations of cognitive and/or motor functions. Currently, for no such diseases does any pharmacological treatment exist able to arrest its progression. Riluzole (1) is a small molecule able to interfere with multiple cellular and molecular mechanisms of neurodegeneration, and is the only approved treatment of amyotrophic lateral sclerosis (ALS), the progression of which proved to significantly slow, thus increasing somewhat average survival. Here we report the synthesis of differently functionalized 4H-3,1-benzothiazine (5-6) and 2H-1,4-benzothiazine (7) series as superior homologues of 1. Biological evaluation demonstrated that amidine 4H-3,1-benzothiazine derivatives 5b-d can reduce glutamate and LDH release in the oxygen/glucose deprivation and reperfusion model (OGD/R) applied to brain slices with a higher potency than 1. Moreover the mentioned compounds significantly reduce glutamate- and 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in neuroblastoma cells. In addition, the same compounds limit ROS formation in both neuronal preparations. Finally, 5c proved effective in inhibiting neuronal voltage-dependent Na+ and Ca2+-channels, showing a profile comparable with that of 1.

Two distinct types of depolarizing afterpotentials are differentially expressed in stellate and pyramidal-like neurons of entorhinal-cortex layer II.

Two types of principal neurons, stellate cells and pyramidal-like cells, are found in medial entorhinal-cortex (mEC) layer II, and are believed to represent two distinct channels of information processing and transmission in the entorhinal cortex-hippocampus network. In this study, we found that depolarizing afterpotentials (DAPs) that follow single action potentials (APs) evoked from various levels of holding membrane voltage (Vh ) show distinct properties in the two cells types. In both, an evident DAP followed the AP at near-threshold Vh levels, and was accompanied by an enhancement of excitability and spike-timing precision. This DAP was sensitive to voltage-gated Na(+)-channel block with TTx, but not to partial removal of extracellular Ca(2+). Application of 5-µM anandamide, which inhibited the resurgent and persistent Na(+) -current components in a relatively selective way, significantly reduced the amplitude of this particular DAP while exerting poor effects on the foregoing AP. In the presence of background hyperpolarization, DAPs showed an opposite behavior in the two cell types, as in stellate cells they became even more prominent, whereas in pyramidal-like cells their amplitude was markedly reduced. The DAP observed in stellate cells under this condition was strongly inhibited by partial extracellular-Ca(2+) removal, and was sensitive to the low-voltage-activated Ca(2+)-channel blocker, NNC55-0396. This Ca(2+) dependence was not observed in the residual DAP evoked in pyramidal-like cells from likewise negative Vh levels. These results demonstrate that two distinct mechanism of DAP generation operate in mEC layer-II neurons, one Na(+)-dependent and active at near-threshold Vh levels in both stellate and-pyramidal-like cells, the other Ca(2+)-dependent and only expressed by stellate cells in the presence of background membrane hyperpolarization.

Elementary properties of Ca(2+) channels and their influence on multivesicular release and phase-locking at auditory hair cell ribbon synapses.

Voltage-gated calcium (Cav1.3) channels in mammalian inner hair cells (IHCs) open in response to sound and the resulting Ca(2+) entry triggers the release of the neurotransmitter glutamate onto afferent terminals. At low to mid sound frequencies cell depolarization follows the sound sinusoid and pulses of transmitter release from the hair cell generate excitatory postsynaptic currents (EPSCs) in the afferent fiber that translate into a phase-locked pattern of action potential activity. The present article summarizes our current understanding on the elementary properties of single IHC Ca(2+) channels, and how these could have functional implications for certain, poorly understood, features of synaptic transmission at auditory hair cell ribbon synapses.

Fine Tuning of CaV1.3 Ca2+ channel properties in adult inner hair cells positioned in the most sensitive region of the Gerbil Cochlea.

Hearing relies on faithful signal transmission by cochlear inner hair cells (IHCs) onto auditory fibres over a wide frequency and intensity range. Exocytosis at IHC ribbon synapses is triggered by Ca(2+) inflow through Ca(V)1.3 (L-type) Ca(2+) channels. We investigated the macroscopic (whole-cell) and elementary (cell-attached) properties of Ca(2+) currents in IHCs positioned at the middle turn (frequency ∼ 2 kHz) of the adult gerbil cochlea, which is their most sensitive hearing region. Using near physiological recordings conditions (body temperature and a Na(+) based extracellular solution), we found that the macroscopic Ca(2+) current activates and deactivates very rapidly (time constant below 1 ms) and inactivates slowly and only partially. Single-channel recordings showed an elementary conductance of 15 pS, a sub-ms latency to first opening, and a very low steady-state open probability (Po: 0.024 in response to 500-ms depolarizing steps at ∼-18 mV). The value of Po was significantly larger (0.06) in the first 40 ms of membrane depolarization, which corresponds to the time when most Ca(2+) channel openings occurred clustered in bursts (mean burst duration: 19 ms). Both the Po and the mean burst duration were smaller than those previously reported in high-frequency basal IHCs. Finally, we found that middle turn IHCs are likely to express about 4 times more Ca(2+) channels per ribbon than basal cells. We propose that middle-turn IHCs finely-tune Ca(V)1.3 Ca(2+) channel gating in order to provide reliable information upon timing and intensity of lower-frequency sounds.

Burst activity and ultrafast activation kinetics of CaV1.3 Ca²âº channels support presynaptic activity in adult gerbil hair cell ribbon synapses.

Auditory information transfer to afferent neurons relies on precise triggering of neurotransmitter release at the inner hair cell (IHC) ribbon synapses by Ca²âº entry through CaV1.3 Ca²âº channels. Despite the crucial role of CaV1.3 Ca²âº channels in governing synaptic vesicle fusion, their elementary properties in adult mammals remain unknown. Using near-physiological recording conditions we investigated Ca²âº channel activity in adult gerbil IHCs. We found that Ca²âº channels are partially active at the IHC resting membrane potential (-60 mV). At -20 mV, the large majority (>70%) of Ca²âº channel first openings occurred with an estimated delay of about 50 µs in physiological conditions, with a mean open time of 0.5 ms. Similar to other ribbon synapses, Ca²âº channels in IHCs showed a low mean open probability (0.21 at -20 mV), but this increased significantly (up to 0.91) when Ca²âº channel activity switched to a bursting modality. We propose that IHC Ca²âº channels are sufficiently rapid to transmit fast signals of sound onset and support phase-locking. Short-latency Ca²âº channel opening coupled to multivesicular release would ensure precise and reliable signal transmission at the IHC ribbon synapse.

Pharmacotherapy with fluoxetine restores functional connectivity from the dentate gyrus to field CA3 in the Ts65Dn mouse model of down syndrome.

Down syndrome (DS) is a high-incidence genetic pathology characterized by severe impairment of cognitive functions, including declarative memory. Impairment of hippocampus-dependent long-term memory in DS appears to be related to anatomo-functional alterations of the hippocampal trisynaptic circuit formed by the dentate gyrus (DG) granule cells - CA3 pyramidal neurons - CA1 pyramidal neurons. No therapies exist to improve cognitive disability in individuals with DS. In previous studies we demonstrated that pharmacotherapy with fluoxetine restores neurogenesis, granule cell number and dendritic morphology in the DG of the Ts65Dn mouse model of DS. The goal of the current study was to establish whether treatment rescues the impairment of synaptic connectivity between the DG and CA3 that characterizes the trisomic condition. Euploid and Ts65Dn mice were treated with fluoxetine during the first two postnatal weeks and examined 45-60 days after treatment cessation. Untreated Ts65Dn mice had a hypotrophyc mossy fiber bundle, fewer synaptic contacts, fewer glutamatergic contacts, and fewer dendritic spines in the stratum lucidum of CA3, the terminal field of the granule cell projections. Electrophysiological recordings from CA3 pyramidal neurons showed that in Ts65Dn mice the frequency of both mEPSCs and mIPSCs was reduced, indicating an overall impairment of excitatory and inhibitory inputs to CA3 pyramidal neurons. In treated Ts65Dn mice all these aberrant features were fully normalized, indicating that fluoxetine can rescue functional connectivity between the DG and CA3. The positive effects of fluoxetine on the DG-CA3 system suggest that early treatment with this drug could be a suitable therapy, possibly usable in humans, to restore the physiology of the hippocampal networks and, hence, memory functions.

Distinct developmental patterns in the expression of transient, persistent, and resurgent Na+ currents in entorhinal cortex layer-II neurons.

Sub- and near-threshold voltage-dependent Na+ currents (VDSCs) are of major importance in determining the electrical properties of medial entorhinal cortex (mEC) layer-II neurons. Developmental changes in the ability of mEC layer-II stellate cells (SCs) to generate Na+ -dependent, subthreshold electrical events have been reported between P14 and P18. In this study we examined the modifications occurring in the various components of VDSCs during postnatal development of mEC SCs. The transient, resurgent, and persistent Na+ currents (I(NaT), I(NaR), and I(NaP), respectively) showed distinct patterns of developmental expression in the time window considered (P5 to P24-27). All three currents prominently and steeply increased in absolute amplitude and conductance from P5 to at least P16. However, capacitive charge accumulation, an index of membrane surface area, also markedly increased in the same time window, and in the case of I(NaT) the specific conductance per unit of accumulated capacitive charge remained relatively constant. By contrast, specific I(NaR) and I(NaP) conductances showed a significant tendency to increase, especially from P5 to P18. Neither I(NaR) nor I(NaP) represented a constant fraction of the total Na+ current at all developmental ages. Indeed, detectable levels of I(NaR) and I(NaP) were present in only ~20% and ~70%, respectively, of the cells on P5, and were observed in all cells only from P10 onwards. Moreover, the average I(NaR)-to-I(NaT) conductance ratio increased steadily from ~0.004 (P5) up to a plateau level of ~0.05 (P22+), whereas the I(NaP)-to-I(NaT) conductance ratio increased only from ~0.009 on P5 to ~0.02 on P22+. The relative increase in conductance ratio from P5 to P22 was significantly greater for I(NaR) than for I(NaP), indicating that I(NaR) expression starts later than that of I(NaP). These findings show that in mEC layer-II SCs the single functional components of the VDSC are regulated differentially from each other as far as their developmental expression is concerned.

Differential effects of Zn2+ on activation, deactivation, and inactivation kinetics in neuronal voltage-gated Na+ channels.

Whole-cell, patch-clamp recordings were carried out in acutely dissociated neurons from entorhinal cortex (EC) layer II to study the effects of Zn(2+) on Na(+) current kinetics and voltage dependence. In the presence of 200 µM extracellular Cd(2+) to abolish voltage-dependent Ca(2+) currents, and 100 mM extracellular Na(+), 1 mM Zn(2+) inhibited the transient Na(+) current, I (NaT), only to a modest degree (~17% on average). A more pronounced inhibition (~36%) was induced by Zn(2+) when extracellular Na(+) was lowered to 40 mM. Zn(2+) also proved to modify I (NaT) voltage-dependent and kinetic properties in multiple ways. Zn(2+) (1 mM) shifted the voltage dependence of I (NaT) activation and that of I (NaT) onset speed in the positive direction by ~5 mV. The voltage dependence of I (NaT) steady-state inactivation and that of I (NaT) inactivation kinetics were markedly less affected by Zn(2+). By contrast, I (NaT) deactivation speed was prominently accelerated, and its voltage dependence was shifted by a significantly greater amount (~8 mV on average) than that of I (NaT) activation. In addition, the kinetics of I (NaT) recovery from inactivation were significantly slowed by Zn(2+). Zn(2+) inhibition of I (NaT) showed no signs of voltage dependence over the explored membrane-voltage window, indicating that the above effects cannot be explained by voltage dependence of Zn(2+)-induced channel-pore block. These findings suggest that the multiple, voltage-dependent state transitions that the Na(+) channel undergoes through its activation path are differentially sensitive to the gating-modifying effects of Zn(2+), thus resulting in differential modifications of the macroscopic current's activation, inactivation, and deactivation. Computer modeling provided support to this hypothesis.

Persistent Nav1.6 current at axon initial segments tunes spike timing of cerebellar granule cells.

Cerebellar granule (CG) cells generate high-frequency action potentials that have been proposed to depend on the unique properties of their voltage-gated ion channels. To address the in vivo function of Nav1.6 channels in developing and mature CG cells, we combined the study of the developmental expression of Nav subunits with recording of acute cerebellar slices from young and adult granule-specific Scn8a KO mice. Nav1.2 accumulated rapidly at early-formed axon initial segments (AISs). In contrast, Nav1.6 was absent at early postnatal stages but accumulated at AISs of CG cells from P21 to P40. By P40-P65, both Nav1.6 and Nav1.2 co-localized at CG cell AISs. By comparing Na(+) currents in mature CG cells (P66-P74) from wild-type and CG-specific Scn8a KO mice, we found that transient and resurgent Na(+) currents were not modified in the absence of Nav1.6 whereas persistent Na(+) current was strongly reduced. Action potentials in conditional Scn8a KO CG cells showed no alteration in threshold and overshoot, but had a faster repolarization phase and larger post-spike hyperpolarization. In addition, although Scn8a KO CG cells kept their ability to fire action potentials at very high frequency, they displayed increased interspike-interval variability and firing irregularity in response to sustained depolarization. We conclude that Nav1.6 channels at axon initial segments contribute to persistent Na(+) current and ensure a high degree of temporal precision in repetitive firing of CG cells.

Elementary properties of CaV1.3 Ca(2+) channels expressed in mouse cochlear inner hair cells.

Mammalian cochlear inner hair cells (IHCs) are specialized to process developmental signals during immature stages and sound stimuli in adult animals. These signals are conveyed onto auditory afferent nerve fibres. Neurotransmitter release at IHC ribbon synapses is controlled by L-type Ca(V)1.3 Ca(2+) channels, the biophysics of which are still unknown in native mammalian cells. We have investigated the localization and elementary properties of Ca(2+) channels in immature mouse IHCs under near-physiological recording conditions. Ca(V)1.3 Ca(2+) channels at the cell pre-synaptic site co-localize with about half of the total number of ribbons present in immature IHCs. These channels activated at about 70 mV, showed a relatively short first latency and weak inactivation, which would allow IHCs to generate and accurately encode spontaneous Ca(2+) action potential activity characteristic of these immature cells. The Ca(V)1.3 Ca(2+) channels showed a very low open probability (about 0.15 at 20 mV: near the peak of an action potential). Comparison of elementary and macroscopic Ca(2+) currents indicated that very few Ca(2+) channels are associated with each docked vesicle at IHC ribbon synapses. Finally, we found that the open probability of Ca(2+) channels, but not their opening time, was voltage dependent. This finding provides a possible correlation between presynaptic Ca(2+) channel properties and the characteristic frequency/amplitude of EPSCs in auditory afferent fibres.

Synthesis and biological evaluation of amidine, guanidine, and thiourea derivatives of 2-amino(6-trifluoromethoxy)benzothiazole as neuroprotective agents potentially useful in brain diseases.

A series of amidine, thiourea, and guanidine derivatives of 2-amino-6-(trifluoromethoxy)benzothiazole termed 2, 3, and 4, respectively, and structurally related to riluzole, a neuroprotective drug in many animal models of brain disease, have been synthesized. The biological activity of compounds 2a-e, 3a-f, and 4a,b was preliminarily tested by means of an in vitro protocol of ischemia/reperfusion injury. The results demonstrated that 2c and 3a-d significantly attenuated neuronal injury. Selected for testing of their antioxidant properties, compounds 3a-d were shown to be endowed with a direct ROS scavenging activity. Compounds 3b and 3d were also evaluated for their activity on voltage-dependent Na(+) and Ca(2+) currents in neurons from rat piriform cortex. At 50 microM, compound 3b inhibited the transient Na(+) current to a much smaller extent than riluzole, whereas 3d was almost completely ineffective.

Axonal Na+ channels ensure fast spike activation and back-propagation in cerebellar granule cells.

In most neurons, Na+ channels in the axon are complemented by others localized in the soma and dendrites to ensure spike back-propagation. However, cerebellar granule cells are neurons with simplified architecture in which the dendrites are short and unbranched and a single thin ascending axon travels toward the molecular layer before bifurcating into parallel fibers. Here we show that in cerebellar granule cells, Na+ channels are enriched in the axon, especially in the hillock, but almost absent from soma and dendrites. The impact of this channel distribution on neuronal electroresponsiveness was investigated by multi-compartmental modeling. Numerical simulations indicated that granule cells have a compact electrotonic structure allowing excitatory postsynaptic potentials to diffuse with little attenuation from dendrites to axon. The spike arose almost simultaneously along the whole axonal ascending branch and invaded the hillock the activation of which promoted spike back-propagation with marginal delay (<200 micros) and attenuation (<20 mV) into the somato-dendritic compartment. These properties allow granule cells to perform sub-millisecond coincidence detection of pre- and postsynaptic activity and to rapidly activate Purkinje cells contacted by the axonal ascending branch.

Analysis of resurgent sodium-current expression in rat parahippocampal cortices and hippocampal formation.

The resurgent Na(+) current (I(NaR)) is a component of neuronal voltage-dependent Na(+) currents that is activated by repolarization and is believed to result from an atypical path of Na(+)-channel recovery from inactivation. So far, I(NaR) has only been identified in a small number of central neuronal populations in the cerebellum, diencephalon, and brainstem. The possible presence and roles of I(NaR) in neurons of the cerebral cortex and temporal-lobe memory system are still uncharacterized. In this study whole-cell, patch-clamp experiments were carried out in acute rat brain slices to investigate I(NaR) expression and properties in several neuronal populations of the parahippocampal region and hippocampal formation. Specifically, we examined pyramidal neurons of perirhinal cortex areas 36 and 35 (layers II and V); neurons of superficial and deep layers of medial entorhinal cortex (mEC); dentate gyrus (DG) granule cells; and pyramidal cells of the CA3 and CA1 hippocampal fields. I(NaR) was found to be thoroughly expressed in parahippocampal cortices. The most consistent and prominent I(NaR) expression was observed in mEC layer-II cells. A vast majority of areas 36 and 35 neurons (both in layers II and V) and mEC layer-III and -V neurons were also endowed with I(NaR), although at lower amplitude levels. I(NaR) was expressed by approximately 60% of DG granule cells and approximately 35% of CA1 pyramidal cells of the ventral hippocampus, whereas it was never observed in CA3 neurons (both in the ventral and dorsal hippocampus) and CA1 neurons of the dorsal hippocampus. The biophysical properties of I(NaR) were very similar in all of the neuronal types in which the current was observed, with a peak in the current-voltage relationship at -35/-40 mV. Our results show that the parahippocampal region and part of the hippocampal formation are sites of major I(NaR) expression, and provide a new basis for further studies on the molecular correlates of I(NaR).

Resurgent Na+ current in pyramidal neurones of rat perirhinal cortex: axonal location of channels and contribution to depolarizing drive during repetitive firing.

The perirhinal cortex (PRC) is a supra-modal cortical area that collects and integrates information originating from uni- and multi-modal neocortical regions and directed to the hippocampus. The mechanisms that underlie the specific excitable properties of the different PRC neuronal types are still largely unknown, and their elucidation may be important in understanding the integrative functions of PRC. In this study we investigated the expression and properties of resurgent Na(+) current (I(NaR)) in pyramidal neurones of rat PRC area 35 (layer II). Patch-clamp experiments in acute PRC slices were first carried out. A measurable I(NaR) was expressed by a large majority of neurones (31 out of 35 cells). I(NaR) appeared as an inward, slowly decaying current elicited upon step repolarization after depolarizations sufficient to induce nearly complete inactivation of the transient Na(+) current (I(NaT)). I(NaR) had a peak amplitude of approximately 2.5% that of I(NaT), and showed the typical biophysical properties also observed in other neuronal types (i.e. cerebellar Purkinje and granule cells), including a bell-shaped current-voltage relationship with a peak at approximately -40 mV, and a characteristic acceleration of activation and decay speed at potentials negative to -45 mV. Current-clamp experiments were then carried out in which repetitive action-potential discharge at various frequencies was induced with depolarizing current injection. The voltage signals thus obtained were then used as command waveforms for voltage-clamp recordings. These experiments showed that a Na(+) current identifiable as I(NaR) activates in the early interspike phase even at relatively high firing frequencies (20 Hz), thereby contributing to the depolarizing drive and possibly enhancing repetitive discharge. In acutely dissociated area 35 layer II neurones, as well as in nucleated patches from the same neurones, I(NaR) was never observed, despite the presence of typical I(NaT)s. Since in both preparations neuronal processes are lost, we carried out experiments of focal tetrodotoxin (TTX) application in slices to verify whether the channels responsible for I(NaR) are located in compartment(s) different from the soma. We found that TTX preferentially inhibited I(NaR) when applied close to the site of axon emergence from soma, whereas application to the apical pole of the soma had a significantly smaller effect on I(NaR). Our results indicate that in area 35 pyramidal cells I(NaR) is largely generated in the axon initial segment, where it may participate in setting the coding properties of these neurones.

High-voltage-activated Ca2+ currents show similar patterns of expression in stellate and pyramidal cells from rat entorhinal cortex layer II.

High-voltage-activated (HVA) Ca2+ currents were studied in acutely isolated neurons from rat entorhinal cortex (EC) layer II. Stellate and pyramidal cells, the two main neuronal types of this structure, were visually identified based on morphological criteria. HVA currents were recorded by applying the whole-cell, patch-clamp technique, using 5-mM Ba2+ as the charge carrier. In both neuronal types, the amplitude of total HVA Ba2+ currents (IBas) showed a significant tendency to increase with postnatal age in the time window considered [postnatal day 15 (P15) to P28-29]. At P20-P29, when IBa expression reached stable levels, IBa density per unit of membrane area was not different in stellate versus pyramidal cells. The same was also observed when Ca2+, instead of Ba2+, was used as the charge carrier. The pharmacological current subtypes composing total HVA currents were characterized using selective blockers. Again, no significant differences were found between stellate and pyramidal cells with respect to the total-current fractions attributable to specific pharmacological Ca2+ channel subtypes. In both cell types, approximately 52-55% of total IBas was abolished by the L-type channel blocker, nifedipine (10 microM), approximately 23-30% by the N-type channel blocker, omega-conotoxin GVIA (1 microM), approximately 22-24% by the P/Q-type channel blocker, omega-agatoxin IVA (100 nM), and approximately 11-13% remained unblocked (R-type current) after simultaneous application of L-, N-, and P/Q-type channel blockers. The Cav 2.3 (alpha1E) channel blocker, SNX-482 (100 nM), abolished approximately 57-62% of total R-type current. We conclude that HVA Ca2+ currents are expressed according to similar patterns in the somata and proximal dendrites of stellate and pyramidal cells of rat EC layer II.

Kinetic and functional analysis of transient, persistent and resurgent sodium currents in rat cerebellar granule cells in situ: an electrophysiological and modelling study.

Cerebellar neurones show complex and differentiated mechanisms of action potential generation that have been proposed to depend on peculiar properties of their voltage-dependent Na+ currents. In this study we analysed voltage-dependent Na(+) currents of rat cerebellar granule cells (GCs) by performing whole-cell, patch-clamp experiments in acute rat cerebellar slices. A transient Na+ current (I(NaT)) was always present and had the properties of a typical fast-activating/inactivating Na+ current. In addition to I(NaT), robust persistent (I(NaP)) and resurgent (I(NaR)) Na+ currents were observed. I(NaP) peaked at approximately -40 mV, showed half-maximal activation at approximately -55 mV, and its maximal amplitude was about 1.5% of that of I(NaT). I(NaR) was elicited by repolarizing pulses applied following step depolarizations able to activate/inactivate I(NaT), and showed voltage- and time-dependent activation and voltage-dependent decay kinetics. The conductance underlying I(NaR) showed a bell-shaped voltage dependence, with peak at -35 mV. A significant correlation was found between GC I(NaR) and I(NaT) peak amplitudes; however, GCs expressing I(NaT) of similar size showed marked variability in terms of I(NaR) amplitude, and in a fraction of cells I(NaR) was undetectable. I(NaT), I(NaP) and I(NaR) could be accounted for by a 13-state kinetic scheme comprising closed, open, inactivated and blocked states. Current-clamp experiments carried out to identify possible functional correlates of I(NaP) and/or I(NaR) revealed that in GCs single action potentials were followed by depolarizing afterpotentials (DAPs). In a majority of cells, DAPs showed properties consistent with I(NaR) playing a role in their generation. Computer modelling showed that I(NaR) promotes DAP generation and enhances high-frequency firing, whereas I(NaP) boosts near-threshold firing activity. Our findings suggest that special properties of voltage-dependent Na+ currents provides GCs with mechanisms suitable for shaping activity patterns, with potentially important consequences for cerebellar information transfer and computation.

Multiple conductance substates in pharmacologically untreated Na(+) channels generating persistent openings in rat entorhinal cortex neurons.

Na(+)-channel activity recorded in cell-attached patches from entorhinal cortex neurons in the absence of gating-modifying drugs was examined to determine the possible occurrence of substate openings. Brief sojourns to subconductance levels were occasionally observed within prolonged ("persistent") burst openings. Subconductance occurrence and amplitude were determined following two distinct, complementary approaches: (1) direct visual inspection and (2) automated detection by application of a method that exploits the current variance of fixed-width tracing segments to sort amplitude estimations. The two approaches led to comparable results. At least six subconductance levels in addition to the full open state were revealed, with amplitudes that were approximately 20%, 30%, 40%, 50%, 60% and 75% that of full openings. The global probability of subconductance opening occurrence within a burst as well as the probability of observing one particular subconductance level within a burst showed no clear dependence upon membrane potential in the -40 to +10 mV range. Open- and closed-time distributions of substate openings could either be similar to those observed in burst full openings or show distinct patterns. Low-amplitude late openings were also observed in isolation, separately from full-size openings. These openings corresponded to conductance levels very similar to those of the substates observed within full-size burst openings; therefore, they were interpreted as isolated subconductance openings. Early, transient openings responsible for the fast-inactivating whole-cell Na(+)-current component also manifested distinct conductance levels, the two most prominent of which were in an approximate 75:100 amplitude ratio. Interestingly, the 75% conductance level observed among early openings occurred much more frequently than in "persistent" burst openings. We conclude that pharmacologically untreated Na(+) channels from native neurons generate substate openings that may influence differently the multiple gating modes displayed by these channels.

Ionic mechanisms in the generation of subthreshold oscillations and action potential clustering in entorhinal layer II stellate neurons.

A multicompartmental biophysical model of entorhinal cortex layer II stellate cells was developed to analyze the ionic basis of physiological properties, such as subthreshold membrane potential oscillations, action potential clustering, and the medium afterhyperpolarization. In particular, the simulation illustrates the interaction of the persistent sodium current (I(Nap)) and the hyperpolarization activated inward current (Ih) in the generation of subthreshold membrane potential oscillations. The potential role of Ih in contributing to the medium hyperpolarization (mAHP) and rebound spiking was studied. The role of Ih and the slow calcium-activated potassium current Ikappa(AHP) in action potential clustering was also studied. Representations of Ih and I(Nap) were developed with parameters based on voltage-clamp data from whole-cell patch and single channel recordings of stellate cells (Dickson et al., J Neurophysiol 83:2562-2579, 2000; Magistretti and Alonso, J Gen Physiol 114:491-509, 1999; Magistretti et al., J Physiol 521:629-636, 1999a; J Neurosci 19:7334-7341, 1999b). These currents interacted to generate robust subthreshold membrane potentials with amplitude and frequency corresponding to data observed in the whole cell patch recordings. The model was also able to account for effects of pharmacological manipulations, including blockade of Ih with ZD7288, partial blockade with cesium, and the influence of barium on oscillations. In a model with a wider range of currents, the transition from oscillations to single spiking, to spike clustering, and finally tonic firing could be replicated. In agreement with experiment, blockade of calcium channels in the model strongly reduced clustering. In the voltage interval during which no data are available, the model predicts that the slow component of Ih does not follow the fast component down to very short time constants. The model also predicts that the fast component of Ih is responsible for the involvement in the generation of subthreshold oscillations, and the slow component dominates in the generation of spike clusters.