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Calcium influx elevates mitochondrial oxidant stress (mOS) in dorsal motor nucleus of the vagus (DMV) neurons that are prone to Lewy body pathologies in presymptomatic Parkinson's disease (PD) patients. In experimental PD models, treatment with isradipine, the dihydropyridine with the highest affinity to Cav1.3 channels, prevents subthreshold calcium influx via Cav1.3 channels into midbrain dopamine neurons and protects them from mOS. In DMV neurons, isradipine is also effective in reducing mOS despite overwhelming evidence that subthreshold calcium influx is negligible compared with spike-triggered influx. To solve this conundrum we combined slice electrophysiology, two-photon laser scanning microscopy, mRNA profiling, and computational modeling. We find that the unusually depolarized subthreshold voltage trajectory of DMV neurons is positioned between the relatively hyperpolarized activation curve of Cav1.3 channels and that of other high-voltage activated (HVA) calcium channels, thus creating a functional segregation between Cav1.3 and HVA calcium channels. The HVA channels flux the bulk of calcium during spikes but can only influence pacemaking through their coupling to calcium-activated potassium currents. In contrast, Cav1.3 currents, which we show to be more than an order-of-magnitude smaller than the HVA calcium currents, are able to introduce sufficient inward current to speed up firing. However, Kv4 channels that are constitutively open in the subthreshold range guarantee slow pacemaking, despite the depolarizing action of Cav1.3 and other pacemaking currents. We propose that the efficacy of isradipine in preventing mOS in DMV neurons arises from its mixed effect on Cav1.3 channels and on HVA Cav1.2 channels., (Copyright © 2015 the American Physiological Society.)

The induction of corticostriatal long-term depression (LTD) in striatal spiny projection neurons (SPNs) requires coactivation of group I metabotropic glutamate receptors (mGluRs) and L-type Ca(2+) channels. This combination leads to the postsynaptic production of endocannabinoids that act presynaptically to reduce glutamate release. Although the necessity of coactivation is agreed upon, why it is necessary in physiologically meaningful settings is not. The studies described here attempt to answer this question by using two-photon laser scanning microscopy and patch-clamp electrophysiology to interrogate the dendritic synapses of SPNs in ex vivo brain slices from transgenic mice. These experiments revealed that postsynaptic action potentials induce robust ryanodine receptor (RYR)-dependent Ca(2+)-induced-Ca(2+) release (CICR) in SPN dendritic spines. Depolarization-induced opening of voltage-gated Ca(2+) channels was necessary for CICR. CICR was more robust in indirect pathway SPNs than in direct pathway SPNs, particularly in distal dendrites. Although it did not increase intracellular Ca(2+) concentration alone, group I mGluR activation enhanced CICR and slowed Ca(2+) clearance, extending the activity-evoked intraspine transient. The mGluR modulation of CICR was sensitive to antagonism of inositol trisphosphate receptors, RYRs, src kinase, and Cav1.3 L-type Ca(2+) channels. Uncaging glutamate at individual spines effectively activated mGluRs and facilitated CICR induced by back-propagating action potentials. Disrupting CICR by antagonizing RYRs prevented the induction of corticostriatal LTD with spike-timing protocols. In contrast, mGluRs had no effect on the induction of long-term potentiation. Taken together, these results make clearer how coactivation of mGluRs and L-type Ca(2+) channels promotes the induction of activity-dependent LTD in SPNs.

The propensity of a neuron to synchronize is captured by its infinitesimal phase response curve (iPRC). Determining whether an iPRC is biphasic, meaning that small depolarizing perturbations can actually delay the next spike, if delivered at appropriate phases, is a daunting experimental task because negative lobes in the iPRC (unlike positive ones) tend to be small and may be occluded by the normal discharge variability of a neuron. To circumvent this problem, iPRCs are commonly derived from numerical models of neurons. Here, we propose a novel and natural method to estimate the iPRC by direct estimation of its spectral modes. First, we show analytically that the spectral modes of the iPRC of an arbitrary oscillator are readily measured by applying weak harmonic perturbations. Next, applying this methodology to biophysical neuronal models, we show that a low-dimensional spectral reconstruction is sufficient to capture the structure of the iPRC. This structure was preserved reasonably well even with added physiological scale jitter in the neuronal models. To validate the methodology empirically, we applied it first to a low-noise electronic oscillator with a known design and then to cortical pyramidal neurons, recorded in whole cell configuration, that are known to possess a monophasic iPRC. Finally, using the methodology in conjunction with perforated-patch recordings from pallidal neurons, we show, in contrast to recent modeling studies, that these neurons have biphasic somatic iPRCs. Biphasic iPRCs would cause lateral somatically targeted pallidal inhibition to desynchronize pallidal neurons, providing a plausible explanation for their lack of synchrony in vivo.

Motor cortex is a key brain center involved in motor control in rodents and other mammals, but specific intracortical mechanisms at the microcircuit level are largely unknown. Neuronal expression of hyperpolarization-activated current (I(h)) is cell class specific throughout the nervous system, but in neocortex, where pyramidal neurons are classified in various ways, a systematic pattern of expression has not been identified. We tested whether I(h) is differentially expressed among projection classes of pyramidal neurons in mouse motor cortex. I(h) expression was high in corticospinal neurons and low in corticostriatal and corticocortical neurons, a pattern mirrored by mRNA levels for HCN1 and Trip8b subunits. Optical mapping experiments showed that I(h) attenuated glutamatergic responses evoked across the apical and basal dendritic arbors of corticospinal but not corticostriatal neurons. Due to I(h), corticospinal neurons resonated, with a broad peak at ∼4 Hz, and were selectively modulated by α-adrenergic stimulation. I(h) reduced the summation of short trains of artificial excitatory postsynaptic potentials (EPSPs) injected at the soma, and similar effects were observed for short trains of actual EPSPs evoked from layer 2/3 neurons. I(h) narrowed the coincidence detection window for EPSPs arriving from separate layer 2/3 inputs, indicating that the dampening effect of I(h) extended to spatially disperse inputs. To test the role of corticospinal I(h) in transforming EPSPs into action potentials, we transfected layer 2/3 pyramidal neurons with channelrhodopsin-2 and used rapid photostimulation across multiple sites to synaptically drive spiking activity in postsynaptic neurons. Blocking I(h) increased layer 2/3-driven spiking in corticospinal but not corticostriatal neurons. Our results imply that I(h)-dependent synaptic integration in corticospinal neurons constitutes an intracortical control mechanism, regulating the efficacy with which local activity in motor cortex is transferred to downstream circuits in the spinal cord. We speculate that modulation of I(h) in corticospinal neurons could provide a microcircuit-level mechanism involved in translating action planning into action execution.

Experience-dependent modification in the electrical properties of central neurons is a form of intrinsic plasticity that occurs during development and has been observed following behavioral learning. We report a novel form of intrinsic plasticity in hippocampal CA1 pyramidal neurons mediated by the KV7/KCNQ and CaV1/L-type Ca2+ channels. Enhancing Ca2+ influx with a conditioning spike train (30 Hz, 3 s) potentiated the KV7/KCNQ channel function and led to a long-lasting, activity-dependent increase in spike frequency adaptation-a gradual reduction in the firing frequency in response to sustained excitation. These effects were abolished by specific blockers for CaV1/L-type Ca2+ channels, KV7/KCNQ channels, and protein kinase A (PKA). Considering the widespread expression of these two channel types, the influence of Ca2+ influx and subsequent activation of PKA on KV7/KCNQ channels may represent a generalized principle in fine tuning the output of central neurons that promotes stability in firing-an example of homeostatic regulation of intrinsic membrane excitability.

The cholinergic innervation of the prefrontal cortex (PFC) plays a pivotal role in regulating executive functions. Muscarinic receptors activated by acetylcholine depolarize pyramidal neurons in the rodent PFC homologue, but the mechanisms mediating this modulation are controversial. To address this question, we studied the responses of layer V rat pre- and infralimbic cortex pyramidal neurons to muscarinic receptor stimulation. Consistent with previous findings, M(1) receptor stimulation produced a strong depolarization, leading to tonic firing. Voltage-clamp analysis revealed that M(1) activation reduced constitutively active inwardly rectifying (Kir2) K(+) channel currents. Blocking protein kinase C activation or depleting intracellular Ca(2+) stores did not affect the modulation. However, reversal of the modulation was prevented by the phosphoinositide kinase inhibitor, wortmanin, suggesting the modulation was mediated by depletions of membrane phosphatidylinositol-4,5-bisphosphate (PIP(2)). Reduction of Kir2 channel currents by M(1) receptor stimulation significantly increased the temporal summation of excitatory synaptic potentials (EPSPs) evoked by repetitive stimulation of layer I. This action was complimented by M(2/4) receptor mediated presynaptic inhibition of the same terminals. As a consequence of this dual modulation, the responses to a single, isolated afferent volley was reduced, but the response to a high-frequency afferent burst was potentiated.

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.

Single-unit activity in the neostriatum of awake monkeys shows a marked dependence on expected reward. Responses to visual cues differ when animals expect primary reinforcements, such as juice rewards, in comparison to secondary reinforcements, such as tones. The mechanism of this reward-dependent modulation has not been established experimentally. To assess the hypothesis that direct neuromodulatory effects of dopamine on spiny neurons can account for this modulation, we develop a computational model based on simplified representations of key ionic currents and their modulation by D1 dopamine receptor activation. This minimal model can be analyzed in detail. We find that D1-mediated increases of inward rectifying potassium and L-type calcium currents cause a bifurcation: the native up/down state behavior of the spiny neuron model becomes truly bistable, which modulates the peak firing rate and the duration of the up state and introduces a dependence of the response on the past state history. These generic consequences of dopamine neuromodulation through bistability can account for both reward-dependent enhancement and suppression of spiny neuron single-unit responses to visual cues. We validate the model by simulating responses to visual targets in a memory-guided saccade task; our results are in close agreement with the main features of the experimental data. Our model provides a conceptual framework for understanding the functional significance of the short-term neuromodulatory actions of dopamine on signal processing in the striatum.

There is growing evidence linking alterations in serotonergic signaling in the prefrontal cortex to the etiology of schizophrenia. Prefrontal pyramidal neurons are richly innervated by serotonergic fibers and express high levels of serotonergic 5-HT(2)-class receptors. It is unclear, however, how activation of these receptors modulates cellular activity. To help fill this gap, whole cell voltage-clamp and single-cell RT-PCR studies of acutely isolated layer V-VI prefrontal pyramidal neurons were undertaken. The vast majority (>80%) of these neurons had detectable levels of 5-HT(2A) or 5-HT(2C) receptor mRNA. Bath application of 5-HT(2) agonists inhibited voltage-dependent Ca(2+) channel currents. L-type Ca(2+) channels were a particularly prominent target of this signaling pathway. The L-type channel modulation was blocked by disruption of G(alphaq) signaling or by inhibition of phospholipase Cbeta. Antagonism of intracellular inositol trisphosphate signaling, chelation of intracellular Ca(2+), or depletion of intracellular Ca(2+) stores also blocked this modulation. Inhibition of the Ca(2+)-dependent phosphatase calcineurin prevented receptor-mediated modulation of L-type currents. Last, the 5-HT(2) receptor modulation was robustly expressed in neurons from Ca(v)1.3 knockout mice. These findings argue that 5-HT(2) receptors couple through G(alphaq) proteins to trigger a phospholipase Cbeta/inositol trisphosphate signaling cascade resulting in the mobilization of intracellular Ca(2+), activation of calcineurin, and inhibition of Ca(v)1.2 L-type Ca(2+) currents. This modulation and its blockade by atypical neuroleptics could have wide-ranging effects on synaptic integration and long-term gene expression in deep-layer prefrontal pyramidal neurons.

Whole cell recordings from acutely dissociated neocortical pyramidal neurons and striatal medium spiny neurons exhibited a calcium-channel current resistant to known blockers of L-, N-, and P/Q-type Ca(2+) channels. These R-type currents were characterized as high-voltage-activated (HVA) by their rapid deactivation kinetics, half-activation and half-inactivation voltages, and sensitivity to depolarized holding potentials. In both cell types, the R-type current activated at potentials relatively negative to other HVA currents in the same cell type and inactivated rapidly compared with the other HVA currents. The main difference between cell types was that R-type currents in neocortical pyramidal neurons inactivated at more negative potentials than R-type currents in medium spiny neurons. Ni(2+) sensitivity was not diagnostic for R-type currents in either cell type. Single-cell RT-PCR revealed that both cell types expressed the alpha1E mRNA, consistent with this subunit being associated with the R-type current.

Dopamine is a critical determinant of neostriatal function, but its impact on intrastriatal GABAergic signaling is poorly understood. The role of D(1) dopamine receptors in the regulation of postsynaptic GABA(A) receptors was characterized using whole cell voltage-clamp recordings in acutely isolated, rat neostriatal medium spiny neurons. Exogenous application of GABA evoked a rapidly desensitizing current that was blocked by bicuculline. Application of the D(1) dopamine receptor agonist SKF 81297 reduced GABA-evoked currents in most medium spiny neurons. The D(1) dopamine receptor antagonist SCH 23390 blocked the effect of SKF 81297. Membrane-permeant cAMP analogues mimicked the effect of D(1) dopamine receptor stimulation, whereas an inhibitor of protein kinase A (PKA; Rp-8-chloroadenosine 3',5' cyclic monophosphothioate) attenuated the response to D(1) dopamine receptor stimulation or cAMP analogues. Inhibitors of protein phosphatase 1/2A potentiated the modulation by cAMP analogues. Single-cell RT-PCR profiling revealed consistent expression of mRNA for the beta1 subunit of the GABA(A) receptor-a known substrate of PKA-in medium spiny neurons. Immunoprecipitation assays of radiolabeled proteins revealed that D(1) dopamine receptor stimulation increased phosphorylation of GABA(A) receptor beta1/beta3 subunits. The D(1) dopamine receptor-induced phosphorylation of beta1/beta3 subunits was attenuated significantly in neostriata from DARPP-32 mutants. Voltage-clamp recordings corroborated these results, revealing that the efficacy of the D(1) dopamine receptor modulation of GABA(A) currents was reduced in DARPP-32-deficient medium spiny neurons. These results argue that D(1) dopamine receptor stimulation in neostriatal medium spiny neurons reduces postsynaptic GABA(A) receptor currents by activating a PKA/DARPP-32/protein phosphatase 1 signaling cascade targeting GABA(A) receptor beta1 subunits.

Adenosine is a potent regulator of acetylcholine release in the striatum, yet the mechanisms mediating this regulation are largely undefined. To begin to fill this gap, adenosine receptor expression and coupling to voltage-dependent Ca(2+) channels were studied in cholinergic interneurons by combined whole cell voltage-clamp recording and single-cell reverse transcription-polymerase chain reaction. Cholinergic interneurons were identified by the presence of choline acetyltransferase mRNA. Nearly all of these interneurons (90%, n = 28) expressed detectable levels of A(1) adenosine receptor mRNA. A(2a) and A(2b) receptor mRNAs were less frequently detected. A(3) receptor mRNA was undetectable. Adenosine rapidly and reversibly reduced N-type Ca(2+) currents in cholinergic interneurons. The A(1) receptor antagonist 8-cyclopentyl-1, 3-dimethylxanthine completely blocked the effect of adenosine. The IC(50) of the A(1) receptor selective agonist 2-chloro-N6-cyclopentyladenosine was 45 nM, whereas it was near 30 microM for the A(2a) receptor agonist CGS-21680. Dialysis with GDPbetaS or brief exposure to the G protein (G(i/o)) alkylating agent N-ethylmaleimide also blocked the adenosine modulation. The reduction in N-type currents was partially reversed by depolarizing prepulses. A membrane-delimited pathway mediated the modulation, because it was not seen in cell-attached patches when agonist was applied to the bath. Activation of protein kinase C attenuated the adenosine modulation. Taken together, our results argue that activation of A(1) adenosine receptors in cholinergic interneurons reduces N-type Ca(2+) currents via a membrane-delimited, G(i/o) class G-protein pathway that is regulated by protein kinase C. These observations establish a cellular mechanism by which adenosine may serve to reduce acetylcholine release.

We used the whole cell patch-clamp technique and single-cell reverse transcription-polymerase chain reaction (RT-PCR) to study the muscarinic receptor-mediated modulation of calcium channel currents in both acutely isolated and cultured pyramidal neurons from rat sensorimotor cortex. Single-cell RT-PCR profiling for muscarinic receptor mRNAs revealed the expression of m1, m2, m3, and m4 subtypes in these cells. Muscarine reversibly reduced Ca2+ currents in a dose-dependent manner. The modulation was blocked by the muscarinic antagonist atropine. When the internal recording solution included 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N, N,N',N'-tetraacetic acid (EGTA) or 10 mM bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid (BAPTA), the modulation was rapid (tauonset approximately 1.2 s). Under conditions where intracellular calcium levels were less controlled (0.0-0.1 mM BAPTA), a slowly developing component of the modulation also was observed (tauonset approximately 17 s). Both fast and slow components also were observed in recordings with 10 mM EGTA or 20 mM BAPTA when Ca2+ was added to elevate internal [Ca2+] ( approximately 150 nM). The fast component was due to a reduction in both N- and P-type calcium currents, whereas the slow component involved L-type current. N-ethylmaleimide blocked the fast component but not the slow component of the modulation. Preincubation of cultured neurons with pertussis toxin (PTX) also greatly reduced the fast portion of the modulation. These results suggest a role for both PTX-sensitive G proteins as well as PTX-insensitive G proteins in the muscarinic modulation. The fast component of the modulation was reversed by strong depolarization, whereas the slow component was not. Reblock of the calcium channels by G proteins (at -90 mV) occurred with a median tau of 68 ms. We conclude that activation of muscarinic receptors results in modulation of N- and P-type channels by a rapid, voltage-dependent pathway and of L-type current by a slow, voltage-independent pathway.

1. Voltage-dependent facilitation of Ca2+ channels was studied in acutely isolated adult rat neostriatal neurons. Particular attention was paid to the facilitation of L-type channels. 2. In the absence of neuromodulators, the current-voltage relationship for whole cell Ba2+ currents was enhanced by a prepulse to +100 mV. The median enhancement at -20 mV was nearly 60%. The voltage dependence and kinetics of the processes underlying the facilitation were similar to those reported in other neurons. N-, P-, Q-, and L-type currents contributed to the observed facilitation. 3. Voltage-dependent facilitation of L-type currents was studied by subtracting nifedipine-insensitive currents from control currents. Although the kinetics were similar to those of the whole cell currents, the half-activation voltage for facilitation of L-type currents [half-activation voltage (Vh) = -0.6 mV, slope factors (Vc) = 11.8 mV, [n = 5] was significantly less depolarized than that of the pooled currents (Vh = 47.3 mV, Vc = 12.3 mV, n = 7). 4. Repetitive depolarization with spikelike waveforms was also able to induce facilitation of L-type currents, suggesting that facilitation was not simply a consequence of a modal shift in gating like that induced by Bay K 8644. 6. Combined whole cell recording and single-cell reverse transcription-polymerase chain reaction amplification revealed that neostriatal medium spiny neurons expressed detectable levels of either class C or class D L-type channel alpha 1, subunit mRNA. Both neurons expressing class C L-type channels and neurons expressing class D L-type channels exhibited voltage-dependent facilitation.

1. Depolarization-activated, calcium-independent potassium (K+) currents were studied with the use of whole cell voltage-clamp recording from neostriatal neurons acutely isolated from adult (> or = 4 wk old) rats. The whole cell K+ current was composed of transient and persistent components. The aims of the experiments were to isolate the persistent component and then to characterize its voltage dependence and kinetics. 2. Application of 10 mM 4-aminopyridine (4-AP) completely blocked the transient currents while reducing the persistent current by approximately 40% [50% inhibitory concentration (IC50), of blockable current = 125 microM]. The persistent K+ current also was reduced by tetraethylammonium (TEA). Two components to the TEA block were present, having IC50s of 125 microM (23% of the blockable current) and 5.9 mM (77% of the blockable current). Collectively, these results suggested that the persistent components of the total K+ current was pharmacologically heterogeneous. The properties of the 4-AP-resistant, persistent K+ current (IKrp) were subsequently studied. 3. The kinetics of activation and deactivation of IKrp were voltage dependent. Examination of the entire activation/deactivation time constant profile showed that it was bell shaped, with time constants being moderately rapid (tau approximately 50 ms) at membrane potentials corresponding to the resting potential of neostriatal cells (approximately -80 mV), becoming considerably longer (tau approximately 100 ms) at potentials near the cells' spike thresholds (approximately -45 mV), and decreasing to a minimum (tau approximately 5 ms) at potentials associated with the peak of the cells' action potentials (approximately +20 mV). The inactivation kinetics of IKrp also were voltage dependent. The time constants of inactivation varied between 1 and 8 s at potentials between -10 and +35 mV. 4. Unlike persistent K+ currents in many other cell types, IKrp activated at relatively hyperpolarized membrane potentials (approximately -70 mV). The Boltzmann function describing activation had a half-activation voltage of -13 mV and a slope factor of 12 mV. In addition, the Boltzmann function describing the voltage dependence of inactivation of IKrp had a relatively depolarized half-inactivation voltage of -55 and a large slope factor of 19 mV, indicating that this current was available over a broad range of membrane potentials (between -100 and -10 mV). 5. Neostriatal neurons recorded in vivo exhibit subthreshold shifts in membrane potential of variable duration (tens of ms to s) from a hyperpolarized resting state to a depolarized state that is limited in amplitude just below spike threshold. The voltage dependence of activation and inactivation of IKrp indicates that it will be available on depolarization from the hyperpolarized state. However, the slow activation rate of this current suggests that it will contribute little either to limiting the amplitude of the initial depolarization associated with entry into the depolarized state or to depolarizing episodes of short duration (e.g., < 50 ms). However, IKrp should limit the amplitude of membrane depolarizations associated with prolonged excursions into the depolarized state.

1. Large, projection-like neurons from the adult (> 3 wk post-natal) rat globus pallidus (GP) were acutely isolated and subjected to whole-cell voltage-clamp (n = 37). Ca2+ currents were isolated pharmacologically in cells with whole-cell capacitances of 15-34 pF. 2. With 5 mM Ba2+ as a charge carrier, whole-cell currents began to activate near -40 mV and peaked near 0 mV. Based on activation threshold and inactivation kinetics, currents appeared to be of the high-voltage-activated type. 3. Cd2+ blocked whole-cell currents with an IC50 near 2 microM. Currents activated at negative potentials were not relatively resistant to Cd2+, supporting the inference that low-voltage-activated currents were not prominent in these neurons. 4. The dihydropyridine, L-channel antagonist, nifedipine (5 microM), reduced peak current by 21 +/- 4% (SD) (n = 10). The dihydropyridine agonist, BayK 8644 (1-2 microM) enhanced peak current and slowed current deactivation (n = 4). 5. The N-channel antagonist, omega-conotoxin GVIA (omega-CgTx, 2 microM) blocked 25 +/- 7% of the peak whole-cell current (n = 10). The blocks produced by omega-CgTx and nifedipine were additive, blocking an average of 46 +/- 8% of the current (n = 10). 6. The current resistant to the selective N- and L-channel antagonists was partially blocked by the P-channel antagonist omega-agatoxin IVA (omega-AgTx, 100 nM). omega-AgTx blocked about one-half of the current not attributable to N- and L-type channels (22 +/- 5% of the total current, n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

1. We describe three outward K+ current components in acutely dissociated neurons from rat sensorimotor cortex on the basis of inactivation kinetics and voltage dependence. 2. The fast A current (IAf) was completely inactivated at -40 mV and half-inactivated at -52 mV. It activated [time to peak (TTP) 8 ms at -10 mV] and was inactivated (tau inact = 12 ms at -10 mV) rapidly. Recovery from inactivation had a time constant of approximately 80 ms at -100 mV. It was insensitive to tetraethyl ammonium (TEA) and dendrotoxin but was blocked by 4-aminopyridine (4-AP, IC50 = 1 mM). 3. The slowly inactivating current (IKS) was the largest current seen in acutely dissociated adult neurons. It was completely inactivated at -40 mV, half-inactivated at -98 mV, and was kinetically slower (TTP = 130 ms at -10 mV; tau inact = 293 ms at -10 mV) than the fast A current. Deactivation tails were fit with the sum of two exponentials with time constants of 2-10 and 15-40 ms. IKS recovered from inactivation with a time constant of approximately 1,200 ms at -100 mV. 4. There were two components that inactivated with even slower kinetics. The very slowly inactivating current (IKSS) was operationally defined as the current remaining after a 5-s hold at -40 mV. One component inactivated with a time constant of 1,927 ms at -10 mV. The other component showed no inactivation over a 5-s test command, but in 40- to 50-s steps to -10 mV, inactivated with a tau of approximately 20 s. The very slowly inactivating current activated with similar kinetics to IKS (TTP = 121 ms at -10 mV), and two deactivation tails, with kinetics similar to those after the -100 mV prepulse, were observed after holding at -40 mV. 5. Both IKS and IKSS were sensitive to TEA. Seventy-six percent (76%) of IKSS was blocked by 30 mM TEA. Two components to the TEA block were present for IKSS, with IC50s of 88 microM (67% of blockable current) and 7 mM (33%). Seventy percent (70%) of IKS was blocked by 30 mM TEA. For the IKS current, there were also two effective concentrations, with IC50s of 8 microM (21% of blockade current) and 3 mM (79%). 6. IKS and IKSS were also sensitive to 4-AP. Seventy-six percent (76%) of IKSS was blocked by 3-5 mM 4-AP. IKSS exhibited two components of 4-AP block.(ABSTRACT TRUNCATED AT 400 WORDS)

Data analyzed in this study were derived from the responses of 128 spinothalamic tract (STT) cells and 110 thalamic neurons recorded in 75 anesthetized monkeys. A k-means cluster analysis, a nonhierarchical clustering technique, was performed using the relative magnitudes of responses to a graded series of innocuous and noxious mechanical stimuli applied to the receptive field. For comparison, a parallel analysis was performed based on definitions of low-threshold (LT), wide dynamic range (WDR), and high-threshold (HT) cells used by our laboratory. For 128 STT cells, a classification scheme with three clusters was found statistically to be the best. This yielded groups of 22, 57, and 49 cells in clusters 1, 2, and 3, respectively. Cluster 1 cells were activated best by low-intensity mechanical stimuli, whereas cluster 3 cells were activated primarily by nociceptive stimuli. Cluster 2 cells had intermediate characteristics. When the classification scheme based on the cluster analysis was compared with the classification of the same neurons as LT, WDR, and HT cells, cluster 1 cells were divided into LT and WDR cells, whereas cluster 2 and 3 cells included WDR and HT cells. For 110 thalamic neurons, a classification scheme with five clusters was found statistically to be the best. Clusters 1-5 contained 25, 34, 17, 10, and 24 cells, respectively. Response characteristics of cells in each group indicated a gradual change in sensitivity to higher intensities of peripheral input from cluster 1 to 5. When this classification scheme was compared with the classification scheme previously used by our laboratory, cluster 1 cells belonged to the LT group, clusters 2 and 3 split into LT and WDR cells, and clusters 4 and 5 included WDR and HT cells. It is concluded that a classification scheme based on a cluster analysis of the responses of neurons to standardized stimuli may provide an objective and functionally meaningful way to categorize somatosensory neurons.

1. The spontaneous discharge of 30 spinothalamic tract (STT) neurons in the lumbosacral spinal cord of anesthetized monkeys was studied. Interval, correlation, and spectral analyses were performed. 2. Three patterns of discharge were found; these were referred to as the SP1, SP2, and SP3 patterns. 3. The SP1 group had moderately regular discharge trains that were devoid of short-interval spike bursts. 4. The SP2 group had spike trains dominated by short-interval bursts without evidence of low-frequency rhythmicity. 5. The SP3 group had spike trains with features of both the SP1 and SP2 groups. 6. Some correlations were found between the mean discharge rate and stimulus-response classes previously defined by our group (25). Correlations were deduced from a parent data set of 221 STT neurons. Type 1 neurons, which were driven primarily by tactile afferents, were found to have significantly lower mean rates than other "within-neuron" groups. On the other hand, type C neurons, which had strong input from afferents signalling pressure and noxious stimuli, were found to have significantly higher mean rates than all other "across-neuron" classes. 7. A weak relationship was found between the pattern of discharge and the within-neuron stimulus-response classification. Neurons with a largely tactile coding orientation (types 1 and 2) were most frequently of the SP1 and SP2 classes, whereas neurons with a more prominent nociceptive input (types 3 and 4) were most frequently of the SP2 and SP3 classes. No relationship was apparent between discharge pattern and the across-neuron classes.

The activity of 132 neurons in the caudal part of the ventral posterior lateral nucleus (VPLc) of the thalamus was recorded from 23 anesthetized monkeys. All single thalamic units that could be excited by electrical search stimuli applied to the contralateral sciatic nerve were investigated. Responses of these cells to mechanical, thermal, and electrical stimuli applied in the periphery indicated that at least half of the sampled cells were nociceptive. Based on responses to graded mechanical stimuli applied to the periphery, 110 of the sampled cells that received a predominant input from cutaneous receptive fields were classified. There were 56 low-threshold, 39 wide dynamic range, and 15 high-threshold cells. The same neurons were also classified into five mechanical types based on a cluster analysis: types 1-5 contained 25, 34, 17, 10, and 24 cells, respectively. The fact that about half the population of cells belonged to either the wide dynamic or the high threshold group (or mechanical types 3-5) suggested that a large population of VPLc neurons respond to mechanical nociceptive stimuli either exclusively or preferentially. Responses of 63 thalamic neurons were tested to noxious heat pulses applied to their cutaneous receptive fields with a contact thermostimulator. Of these, 47 cells were excited, whereas only 16 cells did not respond. The peripheral nerve that innervated the receptive field of each of 82 thalamic neurons was stimulated with graded strengths to activate A fibers only or both A and C fibers. All tested cells responded to peripheral A fiber volleys. In addition, 42 of these cells responded to peripheral C fiber volleys. The C fiber responses could be either short lasting (a few hundreds of milliseconds) or long lasting (up to several seconds). The recording sites of 80 cells were reconstructed. Of these, 78 were in the VPLc nucleus and the remaining two were in the reticular nucleus of the thalamus. No obvious relationship between the response characteristics and the locations of the cells within the VPLc nucleus was found. Sampled thalamic units had a variety of sources of input from the periphery, including both cutaneous and/or deep tissue receptive fields. The majority of the cells, however, had exclusively cutaneous receptive fields. The sizes of the cutaneous receptive fields were often very small, so that nearly half (41%) of the receptive fields of cells sampled occupied an area of skin smaller than half the foot.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Two hundred and twenty-one spinothalamic tract (STT) neurons in the lumbar spinal cord of anesthetized monkeys were studied. The majority of the recordings were in laminae IV-VI. Thirteen of these neurons were intracellularly injected with horseradish peroxidase and histologically reconstructed. 2. A standard series of four mechanical cutaneous stimuli, which ranged in intensity from innocuous brushing to tissue-damaging pinching, were used to test the mechanical responsiveness of STT neurons. The mean alterations in discharge rate produced by these test stimuli when delivered to a neuron's excitatory receptive field were used as response measures. 3. Univariate and bivariate analyses of these response measures failed to reveal natural groupings of STT neurons. To assess whether natural groupings dependent upon shared multivariate response patterns were present, a k-means cluster analysis of the responses was performed. 4. Because an assumption about the type of coding used by the STT system had to be made prior to clustering, two independent analyses were performed. One approach assumed a labeled line coding model; response magnitudes were determined within the context of the neuron under study (within-neuron analysis). The other approach assumed a population coding model; response magnitudes were determined within the context of the STT population (across-neuron analysis). 5. The within-neuron analysis suggested that the STT sample could be partitioned into four groups. The smallest group (n = 18, 8%) responded primarily to brushing but often had a convergent nociceptive input; this group was referred to as type I. A second group (n = 31, 14%) had strong responses to low-intensity stimuli, particularly pressure, and modestly larger responses to noxious stimuli; this group was referred to as type II. The clustering in these two groups was relatively weak, reflecting some heterogeneity in response pattern. 6. The largest within-neuron group (n = 108, 49%) was most responsive to noxious stimuli but had a saturating response function; because of their apparent role in coding intermediate intensity stimuli, this group was referred to as type III. The fourth group (n = 64, 29%) responded best to the most intense stimulus used; this group was referred to as type IV. 7. The across-neuron analysis also suggested that the STT sample could be partitioned into four groups. The largest group (n = 122, 55%) had relatively weak responses to all the cutaneous stimuli; this group was referred to as type A. 8. All of the remaining across-neuron groups had mean responses at or above the mean for all cutaneous stimuli.(ABSTRACT TRUNCATED AT 400 WORDS)

Extracellular recordings were made from 81 primate spinothalamic (STT) neurons in the L7-S1 segments of the spinal cord. The majority of the sample was recorded from within laminae IV-V. The responses of STT neurons to noxious thermal stimulation of glabrous and hairy skin were studied in an attempt to identify a neural substrate for the differences in thermal sensation evoked by noxious stimulation of these two types of skin. In addition, the responses to graded mechanical stimuli were examined for evidence of differential sensitivity. Thermal intensity-response functions were constructed from the alteration in the mean discharge rate produced by a 30-s thermal pulse of 43-55 degrees C. Generally, the functions derived from stimulation of both hairy and glabrous skin were either linear or positively accelerating. Deceleration in the response functions was occasionally observed above 53 degrees C. The population mean discharge rate derived from glabrous skin stimulation was significantly greater than that derived from hairy skin stimulation above 49 degrees C. Cluster analysis was used to assess whether the STT population could be partitioned into functionally relevant subgroups. No clustering was evident on the basis of the alteration in discharge rate during stimulation alone. Analysis of the alteration in mean discharge rate during and following thermal stimulation identified four groups; these were referred to as the Amnr, Bmnr, Cmnr, and Dmnr classes. The clustering was not dependent on differences in the responses evoked from hairy and glabrous skin. The mechanical and thermal sensitivities of each thermal class covaried. The capacity of the STT population to code the quality of noxious thermal stimuli, as judged by changes in the across-neuron discharge pattern, was assessed with a multidimensional scaling technique (MDS). The results suggest that the population discharge could be used to order stimuli correctly from 45 to 55 degrees C. Also, it was found that a substantial change in the population's discharge pattern occurred to stimuli between 47 and 49 degrees C when delivered to hairy skin. A similar alteration in the population's discharge pattern occurred to glabrous skin stimuli near 51 degrees C. These alterations in population behavior may underly the alterations in sensory quality in humans that occur in these temperature ranges when stimulating hairy and glabrous skin. The possible roles of the thermally and mechanically based classes in thermal intensity and quality coding were examined. Within the lower intensity ranges (less than 49-51 degrees C), the Cmnr and Dmnr classes appeared to be best suited to intensity coding.(ABSTRACT TRUNCATED AT 400 WORDS)

Fifty-two slowly adapting proprioceptive neurons in the cuneate nucleus of chloralose-anesthetized cats were studied. Recordings were made from 3 mm rostral to the obex to 5 mm caudal. The highest densities of proprioceptive neurons were found above and more than 3 mm caudal to the obex. Analysis of the spike trains produced with the forelimb held fixed revealed three basic periodic patterns. Neurons exhibiting these patterns were partitioned into three groups, referred to as the A, B, and C classes. Class A neurons (42%; 22/52) produced regular spike trains that were qualitatively similar to muscle spindle fibers. Interval distributions for this class were typically unimodal and slightly positively skewed. Adjacent intervals were frequently positively correlated. Spectral analysis suggested that 91% of class A spike trains had one to two periodic components. Class B neurons (21%; 11/52) had additional spikes interposed in their periodic discharge; these "interrupting" spikes did not significantly alter the timing of the dominant periodic discharge. Interval distributions were typically bimodal and adjacent intervals were negatively correlated. Spectral analysis suggested that two or more periodic components were present in their spike trains. Class C neurons (36%; 26/52) had spike trains with a basic rhymicity, but when this specific discharge was interrupted, the subsequent interval was near modal length; thus, they were "reset." Interval distributions were usually multimodal and adjacent intervals were frequently negatively correlated. Spectral analysis suggested that C spike trains usually had four or more periodic components. Estimates of information-carrying capacity of each class using a mean rate code and those of primary muscle spindle fibers suggested that a sizable information loss may occur in synaptic transmission. This potential loss was smaller for A-neurons (40%) than for B- (69%) or C-neurons (64%). Electrical stimulation of cutaneous structures influenced 55% (22/52) of the sample. All were members of the B and C classes. Responses were typically biphasic. The cutaneous receptive fields nearly always included a portion of the forepaw. No relationship was found between movement sensitivity and receptive field topography. Contralateral input was found in half (10/20) the neurons tested.

Extracellular recordings were made from 81 primate spinothalamic (STT) neurons in the L7-S1 segments of the spinal cord. The majority of the sample was recorded from within laminae IV-V. The temporal features of the responses to noxious thermal stimulation of glabrous and hairy skin were studied in an attempt to determine whether natural groupings of STT neurons could be identified on the basis of response time course alone and whether these groups were skin type dependent. The relationship between these groups and those based on static response features (37) was also explored in an attempt to define more fully their potential functional roles. In most STT neurons, the thermally evoked responses typically appeared to have two response components, particularly at stimulus temperatures above 49 degrees C. The first response phase typically peaked within 1-12 s of stimulus onset and then adapted. The second phase slowly rose to a maximum, typically 15-30 s following stimulus onset. The existence of natural groupings of STT neurons based upon the characteristics of these two response components was assessed with a k-means cluster analysis. On the basis of the onset and early peak latencies, two well-defined short and long latency neuronal clusters were found in the responses evoked from both glabrous and hairy skin; these were referred to as the SP1 and LP1 classes, respectively. The glabrous and hairy skin SP1 classes did not differ significantly in either onset or early peak latency for stimuli of 47-55 degrees C. However, the hairy skin LP1 class had significantly shorter onset latencies than the glabrous skin LP1 class for stimuli of 49-53 degrees C, as well as shorter peak latencies for stimuli of 49 and 51 degrees C. The SP1 class constituted 62% of the hairy skin subset, whereas the LP1 class constituted 57% of the glabrous skin subset. A cluster analysis of the late-peak latencies also revealed two subgroups. In the responses evoked from both glabrous and hairy skin, the longer latency classes (LP2) constituted more than 80% of the samples. With one exception, no dependence upon the type of skin that was stimulated was found in the latencies of either the LP2 class or the shorter latency SP2 class. Prior conditioning of the skin with a 30-s thermal pulse of 51-55 degrees C led to a suppression of the early response phase and an enhancement of the late phase in nearly all cases examined (n = 11). This pattern was independent of skin type.(ABSTRACT TRUNCATED AT 400 WORDS)

The intrinsic processes contributing to the three discharge patterns of proprioceptive cuneate neurons described by Surmeier and Towe were studied experimentally and with computer simulation. Examination of the alterations in excitability produced by antidromic activation suggested that a prolonged inhibition was a concomitant of discharge in proprioceptive cuneate neurons. Computer simulation was performed to test the possible roles of inhibitory hyperpolarizing processes in governing the observed discharge patterns. These simulations used two constant threshold models. The simplest model linearly integrated synaptic potentials until the spike threshold was reached. After the discharge, synaptic potentials that preceded the spike were ignored (i.e., the model was "reset"). The second model was similar to the first except that following a spike two hyperpolarizing processes were activated and preceding events continued to play a role in membrane potential. Simulation of class A spike trains that possessed positive correlations between nearby intervals was successful only with a resetting model. This suggested that class A neurons have fast, no-memory postspike conductance changes, which effectively shunt synaptic charge. Simulation of class B spike trains was possible with the nonresetting model. At least two periodic inputs, which evoked brief, relatively large EPSPs, were required. In addition, a prominent, fast, spike-dependent hyperpolarization and a small-amplitude, slow hyperpolarization were required. Simulation of class C spike trains was also possible with the nonresetting model. Several periodic inputs were required; one input had to evoke a slow suprathreshold EPSP. In contrast to class B simulations, class C spike train simulation required that a large-amplitude, slow hyperpolarization, as well as a brief hyperpolarization, following spike initiation. The results of class B and C simulations suggested that these two groups differed primarily in the amplitude of a slow, hyperpolarizing, postspike conductance. Some role may also be played by the time course of the driving EPSPs.