Calcium currents in retrogradely labeled pyramidal cells from rat sensorimotor cortex

Our previous studies of calcium (Ca(2+)) currents in cortical pyramidal cells revealed that the percentage contribution of each Ca(2+) current type to the whole cell Ca(2+) current varies from cell to cell. The extent to which these currents are modulated by neurotransmitters is also variable. This study was directed at testing the hypothesis that a major source of this variability is recording from multiple populations of pyramidal cells. We used the whole cell patch-clamp technique to record from dissociated corticocortical, corticostriatal, and corticotectal projecting pyramidal cells. There were significant differences between the three pyramidal cell types in the mean percentage of L-, P-, and N-type Ca(2+) currents. For both N- and P-type currents, the range of percentages expressed was small for corticostriatal and corticotectal cells as compared with cells which project to the corpus callosum or to the general population. The variance was significantly different between cell types for N- and P-type currents. These results suggest that an important source of the variability in the proportions of Ca(2+) current types present in neocortical pyramidal neurons is recording from multiple populations of pyramidal cells.     

Age-related functional changes of high-voltage-activated calcium channels in different neuronal subtypes of mouse striatum

By means of whole-cell patch-clamp recordings, we characterized the developmental profile of high-voltage-activated (HVA) calcium (Ca(2+)) channel subtypes in distinct neuronal populations of mouse striatum. Acutely dissociated medium spiny neurons (MSNs) and cholinergic interneurons (ChIs) were recorded from mice at five developmental stages: postnatal-days (PD) 14, 23, 40, 150 and 270. During ageing, total HVA Ca(2+) current recorded from both MSNs and ChIs was unchanged. However, the pharmacological analysis of the differential contribution of HVA Ca(2+) channel subtypes showed a significant rearrangement of each component. In both neuronal subtypes, a large fraction of the total HVA current recorded from PD14 mice was inhibited by the L-type HVA channel blocker nifedipine. This dihydropyridine-sensitive component accounted for nearly 50%, in MSNs, and 35%, in ChIs, of total current at PD14, but its contribution was down-regulated up to 20-25% at 9 months. Likewise, the N-type, omega-conotoxin GVIA-sensitive component decreased from 35% to 40% to about 25% in MSNs and 15% in ChIs. The P-type, omega-agatoxin-sensitive fraction did not show significant changes in both neuronal subtypes, whereas the Q-type, omega-conotoxin MVIIC-sensitive channels did show a significant up-regulation at 9 months. As compared with striatal neurons, we recorded pyramidal neurons dissociated from cortical layers IV-V and found no significant developmental change in the different components of HVA Ca(2+) currents. In conclusion, our data demonstrate a functional reconfiguration of HVA Ca(2+) channels in striatal but not cortical pyramidal neurons during mouse development. Such changes might have profound implications for physiological and pathophysiological processes of the striatum.     

Blocker-resistant Ca2+ currents in rat CA1 hippocampal pyramidal neurons

Ca(2+) currents resistant to organic Ca(2+) channel antagonists are present in different types of central neurons. Here, we describe the properties of such currents in CA1 neurons acutely dissociated from rat hippocampus. Blocker-resistant Ca(2+) currents were isolated by combined application of N-, P/Q- and L-type Ca(2+) current antagonists (omega-conotoxin GVIA 2 microM; omega-conotoxin MVIIC 3 microM; omega-agatoxin IVA 200 nM; nifedipine 10 microM) and constituted approximately 21% of the total Ba(2+) current.The blocker-resistant current showed properties similar to R-type currents in other cell types, i.e. voltages of half-maximal inactivation and activation of -76 and -17 mV, respectively, and strong inactivation during the test pulse. In addition, blocker-resistant Ca(2+) currents in CA1 neurons displayed a characteristically rapid deactivation. Application of mock action potentials revealed that charge transfer through blocker-resistant Ca(2+) channels is highly sensitive to action potential shape and changes in resting membrane voltage. Pharmacological experiments showed that these currents were highly sensitive to the divalent cation Ni(2+) (half-maximal block at 28 microM), but were relatively resistant to the spider toxin SNX-482 (8% and 52% block at 0.1 and 1 microM, respectively).In addition to the functional analysis, we examined the expression of pore-forming and accessory Ca(2+) channel subunits on the messenger RNA level in isolated CA1 neurons using quantitative real-time polymerase chain reaction. Of the pore-forming alpha subunits encoding high-threshold Ca(2+) channels, Ca(v)2.1, Ca(v)2.2 and Ca(v)2.3 messenger RNA levels were most prominent, corresponding to the high proportion of N-, P/Q- and R-type currents in these neurons.In summary, CA1 neurons display blocker-resistant Ca(2+) currents with distinctive biophysical and pharmacological properties similar to R-type currents in other neuron types, and express Ca(2+) channel messenger RNAs that give rise to R-type Ca(2+) currents in expression systems.     

Pharmacological and biophysical characterization of voltage-gated calcium currents in the endopiriform nucleus of the guinea pig

The endopiriform nucleus (EPN) is a well-defined structure that is located deeply in the piriform region at the border with the striatum and is characterized by dense intrinsic connections and prominent projections to piriform and limbic cortices. The EPN has been proposed to promote synchronization of large populations of neurons in the olfactory cortices via the activation of transient depolarizations possibly mediated by Ca(2+) spikes. It is known that principal cells in the EPN express both a low- and high-voltage-activated (HVA) Ca(2+) currents. We further characterized HVA conductances possibly related to Ca(2+)-spike generation in the EPN with a whole cell, patch-clamp study on neurons acutely dissociated from the EPN of the guinea pig. To study HVA currents in isolation, experiments were performed from a holding potential of -60 mV, using Ba(2+) as the permeant ion. Total Ba(2+) currents (I(Ba)) evoked by depolarizing square pulses peaked at 0/+10 mV and were completely abolished by 200 microM Cd(2+). The pharmacology of HVA I(Ba)s was analyzed by applying saturating concentrations of specific Ca(2+)-channel blockers. The L-type blocker nifedipine (10 microM; n = 11), the N-type-channel blocker omega-conotoxin GVIA (0.5 microM; n = 24), and the P/Q-type blocker omega-conotoxin MVIIC (1 microM; n = 16) abolished fractions of total I(Ba)s equal on average to 24.7 +/- 5.4%, 27.1 +/- 3.4%, and 22.2 +/- 2.4%, respectively (mean +/- SE). The simultaneous application of the three blockers reduced I(Ba) by 68.5 +/- 6.6% (n = 10). Nifedipine-sensitive currents and most N- and P/Q-type currents were slowly decaying, the average fractional persistence after 300 ms of steady depolarization being 0.77 +/- 0.02, 0.60 +/- 0.06, and 0.68 +/- 0.04, respectively. The residual, blocker-resistant (R-type) currents were consistently faster inactivating, with an average fractional persistence after 300 ms of 0.30 +/- 0.08. Fast-decaying R-type currents also displayed a more negative threshold of activation (by about 10 mV) than non-R-type HVA currents. These results demonstrate that EPN neurons express multiple pharmacological components of the HVA Ca(2+) currents and point to the existence of an R-type current with specific functional properties including fast inactivation kinetics and intermediate threshold of activation.     

Kv2 subunits underlie slowly inactivating potassium current in rat neocortical pyramidal neurons

We determined the expression of Kv2 channel subunits in rat somatosensory and motor cortex and tested for the contributions of Kv2 subunits to slowly inactivating K⁺ currents in supragranular pyramidal neurons. Single cell RT-PCR showed that virtually all pyramidal cells expressed Kv2.1 mRNA and ∼80% expressed Kv2.2 mRNA. Immunocytochemistry revealed striking differences in the distribution of Kv2.1 and Kv2.2 subunits. Kv2.1 subunits were clustered and located on somata and proximal dendrites of all pyramidal cells. Kv2.2 subunits were primarily distributed on large apical dendrites of a subset of pyramidal cells from deep layers. We used two methods for isolating currents through Kv2 channels after excluding contributions from Kv1 subunits: intracellular diffusion of Kv2.1 antibodies through the recording pipette and extracellular application of rStromatoxin-1 (ScTx). The Kv2.1 antibody specifically blocked the slowly inactivating K⁺ current by 25–50% (at 8 min), demonstrating that Kv2.1 subunits underlie much of this current in neocortical pyramidal neurons. ScTx (300 n m ) also inhibited ∼40% of the slowly inactivating K⁺ current. We observed occlusion between the actions of Kv2.1 antibody and ScTx. In addition, Kv2.1 antibody- and ScTx-sensitive currents demonstrated similar recovery from inactivation and voltage dependence and kinetics of activation and inactivation. These data indicate that both agents targeted the same channels. Considering the localization of Kv2.1 and 2.2 subunits, currents from truncated dissociated cells are probably dominated by Kv2.1 subunits. Compared with Kv2.1 currents in expression systems, the Kv2.1 current in neocortical pyramidal cells activated and inactivated at relatively negative potentials and was very sensitive to holding potential.     

Voltage-gated calcium channels in adult rat inferior colliculus neurons

The inferior colliculus (IC) plays a key role in the processing of auditory information and is thought to be an important site for genesis of wild running seizures that evolve into tonic-clonic seizures. IC neurons are known to have Ca(2+) channels but neither their types nor their pharmacological properties have been as yet characterized. Here, we report on biophysical and pharmacological properties of Ca(2+) channel currents in acutely dissociated neurons of adult rat IC, using electrophysiological and molecular techniques. Ca(2+) channels were activated by depolarizing pulses from a holding potential of -90 mV in 10 mV increments using 5 mM barium (Ba(2+)) as the charge carrier. Both low (T-type, VA) and high (HVA) threshold Ca(2+) channel currents that could be blocked by 50 microM cadmium, were recorded. Pharmacological dissection of HVA currents showed that nifedipine (10 microM, L-type channel blocker), omega-conotoxin GVIA (1 microM, N-type channel blocker), and omega-agatoxin TK (30 nM, P-type channel blocker) partially suppressed the current by 21%, 29% and 22%, respectively. Since at higher concentration (200 nM) omega-agatoxin TK also blocks Q-type channels, the data suggest that Q-type Ca(2+) channels carry approximately 16% of HVA current. The fraction of current (approximately 12%) resistant to the above blockers, which was blocked by 30 microM nickel and inactivated with tau of 15-50 ms, was considered as R-type Ca(2+) channel current. Consistent with the pharmacological evidences, Western blot analysis using selective Ca(2+) channel antibodies showed that IC neurons express Ca(2+) channel alpha(1A), alpha(1B), alpha(1C), alpha(1D), and alpha(1E) subunits. We conclude that IC neurons express functionally all members of HVA Ca(2+) channels, but only a subset of these neurons appear to have developed functional LVA channels.     

Kv1.2-containing K+ channels regulate subthreshold excitability of striatal medium spiny neurons

A slowly inactivating, low-threshold K(+) current has been implicated in the regulation of state transitions and repetitive activity in striatal medium spiny neurons. However, the molecular identity of the channels underlying this current and their biophysical properties remain to be clearly determined. Because previous work had suggested this current arose from Kv1 family channels, high-affinity toxins for this family were tested for their ability to block whole cell K(+) currents activated by depolarization of acutely isolated neurons. alpha-Dendrotoxin, which blocks channels containing Kv1.1, Kv1.2, or Kv1.6 subunits, decreased currents evoked by depolarization. Three other Kv1 family toxins that lack a high affinity for Kv1.2 subunits, r-agitoxin-2, dendrotoxin-K, and r-margatoxin, failed to significantly reduce currents, implicating channels with Kv1.2 subunits. RT-PCR results confirmed the expression of Kv1.2 mRNA in identified medium spiny neurons. Currents attributable to Kv1.2 channels activated rapidly, inactivated slowly, and recovered from inactivation slowly. In the subthreshold range (ca. -60 mV), these currents accounted for as much as 50% of the depolarization-activated K(+) current. Moreover, their rapid activation and relatively slow deactivation suggested that they contribute to spike afterpotentials regulating repetitive discharge. This inference was confirmed in current-clamp recordings from medium spiny neurons in the slice preparation where Kv1.2 blockade reduced first-spike latency and increased discharge frequency evoked from hyperpolarized membrane potentials resembling the "down-state" found in vivo. These studies establish a clear functional role for somato-dendritic Kv1.2 channels in the regulation of state transitions and repetitive discharge in striatal medium spiny neurons.     

Characteristics of calcium currents in rat geniculate ganglion neurons

Geniculate ganglion (GG) cell bodies of chorda tympani (CT), greater superficial petrosal (GSP), and posterior auricular (PA) nerves transmit orofacial sensory information to the rostral nucleus of the solitary tract (rNST). We used whole cell recording to study the characteristics of the Ca(2+) channels in isolated Fluorogold-labeled GG neurons that innervate different peripheral receptive fields. PA neurons were significantly larger than CT and GSP neurons, and CT neurons could be further subdivided based on soma diameter. Although all GG neurons possess both low voltage-activated (LVA) "T-type" and high voltage-activated (HVA) Ca(2+) currents, CT, GSP, and PA neurons have distinctly different Ca(2+) current expression patterns. Of GG neurons that express T-type currents, the CT and GSP neurons had moderate and PA neurons had larger amplitude T-type currents. HVA Ca(2+) currents in the GG neurons were separated into several groups using specific Ca(2+) channel blockers. Sequential applications of L, N, and P/Q-type channel antagonists inhibited portions of Ca(2+) current in all CT, GSP, and PA neurons to a different extent in each neuron group. No difference was observed in the percentage of L- and N-type Ca(2+) currents reduced by the antagonists in CT, GSP, and PA neurons. Action potentials in GG neurons are followed by a Ca(2+) current initiated after depolarization (ADP) that may influence intrinsic firing patterns. These results show that based on Ca(2+) channel expression the GG contains a heterogeneous population of sensory neurons possibly related to the type of sensory information they relay to the rNST.     

Correlation of physiologically and morphologically identified neuronal types in human association cortex in vitro.

1. We examined whether the three physiologically defined neuron types described for rodent neocortex were also evident in human association cortex studied in an in vitro brain slice preparation. We also examined the relationship between physiological and morphological cell type in human neocortical neurons. In particular, we tested whether burst-firing neurons were numerous in regions of human cortex that are susceptible to seizures. 2. Although we sampled regular-spiking and fast-spiking neurons, we observed no true burst-firing neurons, as defined for rodent cortex. We did find neurons that displayed a voltage-dependent shift in firing behavior. Because this behavior was due, in large part, to a low-threshold calcium conductance, we called these cells low-threshold spike (LTS) neurons. 3. Regular-spiking neurons and LTS neurons only differed in the voltage dependence of firing behavior and the first few interspike intervals (ISIs) of repetitive firing in response to small current injections (from hyperpolarized membrane potentials). Because of the general similarities between the two types, we consider the LTS cells to be a subgroup of regular-spiking cells. 4. All biocytin-filled regular-spiking neurons were spiny and pyramidal and found in layers II-VI. The lone filled fast-spiking cell was aspiny and nonpyramidal (layer V). The LTS neurons were morphologically heterogeneous. We found 80% of LTS neurons to be spiny and pyramidal, but 20% were aspiny nonpyramidal cells. LTS neurons were located in layers II-VI. 5. In conclusion, human association cortex contains two of three physiological cell types described in rodent cortex: regular spiking and fast spiking. These physiological types corresponded to spiny, pyramidal, and aspiny, nonpyramidal cells, respectively. We sampled no intrinsic burst-firing neurons in human association cortex. LTS neurons exhibited voltage-dependent changes in firing behavior and were morphologically heterogeneous: most LTS cells were spiny and pyramidal, but two cells were found to be aspiny and nonpyramidal. It is not clear whether the absence of burst-firing neurons or the morphological heterogeneity of LTS neurons are due to species differences or differences in cortical areas.     

Pharmacological and biophysical properties of Ca2+ channels and subtype distributions in human adrenal chromaffin cells

In this study, we explored the pharmacological and biophysical properties of voltage-activated Ca(2+) channels in human chromaffin cells using the perforated-patch configuration of the patch-clamp technique. According to their pharmacological sensitivity to Ca(2+) channel blockers, cells could be sorted into two groups of similar size showing the predominance of either N- or P/Q-type Ca(2+) channels. R-type Ca(2+) channels, blocked by 77% with 20 muM Cd(2+) and not affected by 50 muM Ni(2+), were detected for the first time in human chromaffin cells. Immunocytochemical experiments revealed an even distribution of alpha (1E) Ca(2+) channels in these cells. With regard to their biophysical properties, L- and R-type channels were activated at membrane potentials that were 15-20 mV more negative than P/Q- and N-type channels. Activation time constants showed no variation with voltage for the L-type channels, decreased with increasing potentials for the R- and P/Q-type channels, and displayed a bell shape with a maximum at 0 mV for the N-type channels. R-type channels were also the most inactivated channels. We thus show here that human chromaffin cells possess all the Ca(2+) channel types described in neurons, L, N, P/Q, and R channels, but the relative contributions of N and P/Q channels differ among cells. Given that N- and P/Q-type Ca(2+) channel types can be differentially modulated, these findings suggest the possibility of cell-specific regulation in human chromaffin cells.     

Activation of metabotropic glutamate receptors inhibits high-voltage-gated calcium channel currents of chicken nucleus magnocellularis neurons

Using whole cell patch-clamp recordings, we pharmacologically characterized the voltage-gated Ca2+ channel (VGCC) currents of chicken nucleus magnocellularis (NM) neurons using barium as the charge carrier. NM neurons possessed both low- and high-voltage-activated Ca2+ channel currents (HVA I(Ba2+)). The N-type channel blocker (omega-conotoxin-GVIA) inhibited more than half of the total HVA I(Ba2+), whereas blockers of L- and P/Q-type channels each inhibited a small fraction of the current. Metabotropic glutamate receptor (mGluR)-mediated modulation of the HVA I(Ba2+) was examined by bath application of glutamate (100 microM), which inhibited the HVA I(Ba2+) by an average of 16%. The inhibitory effect was dose dependent and was partially blocked by omega-conotoxin-GVIA, indicating that mGluRs modulate N and other type HVA I(Ba2+). The nonspecific mGluR agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarbosylic acid (1S,3R-ACPD), mimicked the inhibitory effect of glutamate on HVA I(Ba2+). Group I-III mGluR agonists showed inhibition of the HVA current with the most potent being the group III agonist L(+)-2-amino-4-phosphonobutyric acid. 1S,3R-ACPD (200 microM) had no effect on K+ or Na+ currents. The firing properties of NM neurons were also not altered by 1S,3R-ACPD. We propose that the inhibition of VGCC currents by mGluRs limits depolarization-induced Ca2+ entry into these highly active NM neurons and regulates their Ca2+ homeostasis.     

Effects of spike parameters and neuromodulators on action potential waveform-induced calcium entry into pyramidal neurons

Neocortical pyramidal neurons express several different calcium channel types. Previous studies with square voltage steps have found modest biophysical differences between these calcium channel types as well as differences in their modulation by transmitters. We used acutely dissociated neocortical pyramidal neurons to test whether this diversity extends to different activation by physiological stimuli. We conclude that 1) peak amplitude, latency to peak, and the total charge entry for the Ca(2+) channel current is dependent on the shape of the mock action potential waveforms (APWs). 2) The percent contribution of the five high-voltage-activated currents to the whole cell current was not altered by using an APW as opposed to a voltage step to elicit the current. 3) The identity of the charge carrier affects the amplitude and decay of the whole cell current. With Ca(2+), there was a greater contribution of T-type current to the whole cell current. 4) Total Ba(2+) charge entry is linearly dependent on the number of spikes in the stimulating waveform and relatively insensitive to spike frequency. 5) Current decay was greatest with Ca(2+) as the charge carrier and with minimal internal chelation. 6) Voltage-dependent neurotransmitter-mediated modulations can be reversed by multiple spikes. The extent of the reversal is dependent on the number of spikes in the stimulating waveform. Thus the neuronal activity pattern can determine the effectiveness of voltage-dependent and -independent modulatory pathways in neocortical pyramidal neurons.     

Voltage-gated calcium currents in axotomized adult rat cutaneous afferent neurons

The effect of sciatic nerve injury on the somatic expression of voltage-gated calcium currents in adult rat cutaneous afferent dorsal root ganglion (DRG) neurons identified via retrograde Fluoro-gold labeling was studied using whole cell patch-clamp techniques. Two weeks after a unilateral ligation and transection of the sciatic nerve, the L(4)-L(5) DRG were dissociated and barium currents were recorded from cells 3-10 h later. Cutaneous afferents (35-50 microm diam) were classified as type 1 (possessing only high-voltage-activated currents; HVA) or type 2 (having both high- and low-voltage-activated currents). Axotomy did not change the percentage of neurons exhibiting a type 2 phenotype or the properties of low-threshold T-type current found in type 2 neurons. However, in type 1 neurons the peak density of HVA current available at a holding potential of -60 mV was reduced in axotomized neurons (83.9 +/- 5.6 pA/pF, n = 53) as compared with control cells (108.7 +/- 6.9 pA/pF, n = 58, P < 0.01, unpaired t-test). A similar reduction was observed at more negative holding potentials, suggesting differences in steady-state inactivation are not responsible for the effect. Separation of the type 1 cells into different size classes indicates that the reduction in voltage-gated barium current occurs selectively in the larger (capacitance >80 pF) cutaneous afferents (control: 112.4 +/- 10.6 pA/pF, n = 30; ligated: 72.6 +/- 5.0 pA/pF, n = 36; P < 0.001); no change was observed in cells with capacitances of 45-80 pF. Isolation of the N- and P?Q-type components of the HVA current in the large neurons using omega-conotoxin GVIA and omega-agatoxin TK suggests a selective reduction in N-type barium current after nerve injury, as the density of omega-CgTx GVIA-sensitive current decreased from 56.9 +/- 6.6 pA/pF in control cells (n = 13) to 31.3 +/- 4.6 pA/pF in the ligated group (n = 12; P < 0.005). The HVA barium current of large cutaneous afferents also demonstrates a depolarizing shift in the voltage dependence of inactivation after axotomy. Injured type 1 cells exhibited faster inactivation kinetics than control neurons, although the rate of recovery from inactivation was similar in the two groups. The present results indicate that nerve injury leads to a reorganization of the HVA calcium current properties in a subset of cutaneous afferent neurons.     

Pharmacological characterization of voltage-dependent calcium currents in rat hippocampal neurons.

The kinetics and pharmacology of voltage-dependent calcium (Ca) currents in primary cultures of hippocampal neurons were studied using the whole cell clamp technique. The low voltage-activated (LVA) Ca current was activated at -50 mV and completely inactivated within 100 ms. This current was insensitive to omega-conotoxin (omega-CgTx) and to the calcium agonist Bay K 8644. The high-voltage-activated (HVA) Ca current was activated at -20 mV and inactivated incompletely during pulses of 200 ms duration. The snail toxin omega-CgTx revealed two pharmacological components of the HVA Ca current, one irreversibly blocked and the other insensitive to the toxin. Bay K 8644 had a clear agonistic action mainly on the omega-CgTx insensitive component of the HVA Ca current.     

Voltage-gated sodium channels shape subthreshold EPSPs in layer 5 pyramidal neurons from rat prefrontal cortex

The role of voltage-dependent channels in shaping subthreshold excitatory postsynaptic potentials (EPSPs) in neocortical layer 5 pyramidal neurons from rat medial prefrontal cortex (PFC) was investigated using patch-clamp recordings from visually identified neurons in brain slices. Small-amplitude EPSPs evoked by stimulation of superficial layers were not affected by the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonopentanoic acid but were abolished by the AMPA receptor antagonist 6-cyano-7-nitroquinoxalene-2,3-dione, suggesting that they were primarily mediated by AMPA receptors. AMPA receptor-mediated EPSPs (AMPA-EPSPs) evoked in the apical dendrites were markedly enhanced, or increased in peak and duration, at depolarized holding potentials. Enhancement of AMPA-EPSPs was reduced by loading the cells with lidocaine N-ethylbromide (QX-314) and by local application of the Na(+) channel blocker tetrodotoxin (TTX) to the soma but not to the middle/proximal apical dendrite. In contrast, blockade of Ca(2+) channels by co-application of Cd(2+) and Ni(2+) to the soma or apical dendrite did not affect the AMPA-EPSPs. Like single EPSPs, EPSP trains were shaped by Na(+) but not Ca(2+) channels. EPSPs simulated by injecting synaptic-like current into proximal/middle apical dendrite (simEPSPs) were enhanced at depolarized holding potentials similarly to AMPA-EPSPs. Extensive blockade of Ca(2+) channels by bath application of the Cd(2+) and Ni(2+) mixture had no effects on simEPSPs, whereas bath-applied TTX removed the depolarization-dependent EPSP amplification. Inhibition of K(+) currents by 4-aminopyridine (4-AP) and TEA increased the TTX-sensitive EPSP amplification. Moreover, strong inhibition of K(+) currents by high concentrations of 4-AP and TEA revealed a contribution of Ca(2+) channels to EPSPs that, however, seemed to be dependent on Na(+) channel activation. Our results indicate that in layer 5 pyramidal neurons from PFC, Na(+), and K(+) voltage-gated channels shape EPSPs within the voltage range that is subthreshold for somatic action potentials.     

Repeated cocaine administration suppresses HVA-Ca2+ potentials and enhances activity of K+ channels in rat nucleus accumbens neurons

The nucleus accumbens (NAc) is an important forebrain area involved in sensitization, withdrawal effects, and self-administration of cocaine. However, little is known about cocaine-induced alterations in the neuronal excitability and whole cell neuroplasticity in this region that may affect behaviors. Our recent investigations have demonstrated that repeated cocaine administration decreases voltage-sensitive sodium and calcium currents (VSSCs and VSCCs, respectively) in freshly dissociated NAc neurons of rats. In this study, current-clamp recordings were performed in slice preparations to determine the effects of chronic cocaine on evoked Ca(2+) potentials and voltage-sensitive K(+) currents in NAc neurons. Repeated cocaine administration with 3-4 days of withdrawal caused significant alterations in Ca(2+) potentials, including suppression of Ca(2+)-mediated spikes, increase in the intracellular injected current intensity required for generation of Ca(2+) potentials (rheobase), reduced duration of Ca(2+) plateau potentials, and abolishment of secondary Ca(2+) potentials associated with the primary Ca(2+) plateau potential. Application of nickel (Ni(2+)), which blocks low-voltage activated T-type Ca(2+) channels, had no impact on evoked Ca(2+) plateau potentials in NAc neurons, indicating that these Ca(2+) potentials are high-voltage activated (HVA). In addition, repeated cocaine pretreatment also hyperpolarized the resting membrane potential, increased the amplitude of afterhyperpolarization in Ca(2+) spikes, and enhanced the outward rectification observed during membrane depolarization. These findings indicate that repeated cocaine administration not only suppressed HVA-Ca(2+) potentials but also significantly enhanced the activity of various K(+) channels in NAc neurons. They also demonstrate an integrative role of whole cell neuroplasticity during cocaine withdrawal, by which the subthreshold membrane excitability of NAc neurons is significantly decreased.     

High-voltage-activated calcium current in developing neurons is insensitive to nifedipine

We have analyzed the effect of nifedipine on the macroscopic high-threshold, voltage-activated (HVA) calcium current in four cell types: postnatal rat Purkinje and dorsal root ganglion (DRG) neurons, embryonic chick DRG neurons, and adult cat ventricular myocytes. As is consistent with previous reports, nifedipine reduced HVA current in myocytes in a voltage-sensitive manner. Analysis of nifedipine actions on neurons, however, was compromised by slow inactivation of the current at holding potentials between –80mV and –40 mV. The slow inactivation was voltage-dependent, irreversible after 5 min, and contributed to rundown of the current. At –40 mV, slow inactivation displayed two time constants: 12±8 s and 7±4 min. When slow inactivation was taken into account, we found no evidence for a nifedipine-sensitive component of the HVA current in these neurons. Consistent with previous studies, DRG neurons were reduced irreversibly by -conotoxin, whereas cardiac and Purkinje cells were unaffected. Our biophysical and pharmacological results are consistent with two types of neuronal HVA currents (N type and P type) in developing neurons that are distinct from cardiac HVA currents (L type).     

Electrophysiological and morphological characterization of neurons within neocortical ectopias

Focal developmental abnormalities in neocortex, including ectopic collections of neurons in layer I (ectopias), have been associated with behavioral and neurological deficits. In this study, we used infrared differential interference contrast microscopy and whole cell patch-clamp to complete the first characterization of neurons within and surrounding neocortical ectopias. Current-clamp recordings revealed that neurons within ectopias display multiple types of action potential firing patterns, and biocytin labeling indicated that approximately 20% of the cells in neocortical ectopias can be classified as nonpyramidal cells and the rest as atypically oriented pyramidal cells. All cells had spontaneous excitatory (glutamatergic) and inhibitory (GABAergic) postsynaptic currents. Exhibitory postsynaptic currents consisted of both N-methyl-D-aspartate (NMDA) receptor-mediated and AMPA/kainate (A/K) receptor-mediated currents. The NMDA receptor-mediated component had decay time constants of 15.35 +/- 2.2 (SE) ms, while the A/K component had faster decay kinetics of 7.6 +/- 1.7 ms at -20 mV. GABA(A) receptor-mediated synaptic currents in ectopic cells reversed at potentials near the Cl- equilibrium potential and had decay kinetics of 16.65 +/- 1.3 ms at 0 mV. Furthermore we show that cells within ectopias receive direct excitatory and inhibitory input from adjacent normatopic cortex and can display a form of epileptiform activity.     

Cannabinoids depress inhibitory synaptic inputs received by layer 2/3 pyramidal neurons of the neocortex

Using whole cell voltage-clamp recordings we investigated the effects of a synthetic cannabinoid (WIN55,212-2) on inhibitory inputs received by layer 2/3 pyramidal neurons in slices of the mouse auditory cortex. Activation of the type 1 cannabinoid receptor (CB1R) with WIN55,212-2 reliably reduced the amplitude of GABAergic inhibitory postsynaptic currents evoked by extracellular stimulation within layer 2/3. The suppression of this inhibition was blocked and reversed by the highly selective CB1R antagonist AM251, confirming a CB1R-mediated inhibition. Pairing evoked inhibitory postsynaptic currents (IPSCs) at short interstimulus intervals while applying WIN55,212-2 resulted in an increase in paired-pulse facilitation suggesting that the probability of GABA release was reduced. A presynaptic site of cannabinoid action was verified by an observed decrease in the frequency with no change in the amplitude or kinetics of action potential-independent postsynaptic currents (mIPSCs). When Cd(2+) was added or Ca(2+) was omitted from the recording solution, the remaining fraction of Ca(2+)-independent mIPSCs did not respond to WIN55,212-2. These data suggest that cannabinoids are capable of suppressing the inhibition of neocortical pyramidal neurons by depressing Ca(2+)-dependent GABA release from local interneurons.