Neurokinins inhibit low threshold inactivating K currents in capsaicin responsive DRG neurons

Neurokinins (NK) released from terminals of dorsal root ganglion (DRG) neurons may control firing of these neurons by an autofeedback mechanism. In this study we used patch clamp recording techniques to determine if NKs alter excitability of rat L4-S3 DRG neurons by modulating K⁺ currents. In capsaicin (CAPS)-responsive phasic neurons substance P (SP) lowered action potential (AP) threshold and increased the number of APs elicited by depolarizing current pulses. SP and a selective NK₂ agonist, [βAla⁸]-neurokinin A (4–10) also inhibited low threshold inactivating K⁺ currents isolated by blocking non-inactivating currents with a combination of high TEA, (−) verapamil and nifedipine. Currents recorded under these conditions were heteropodatoxin-sensitive (Kv4 blocker) and α-dendrotoxin-insensitive (Kv1.1 and Kv1.2 blocker). SP and NKA elicited a > 10 mV positive shift of the voltage dependence of activation of the low threshold currents. This effect was absent in CAPS-unresponsive neurons. The effect of SP or NKA on K⁺ currents in CAPS-responsive phasic neurons was fully reversed by an NK₂ receptor antagonist (MEN10376) but only partially reversed by a PKC inhibitor (bisindolylmaleimide). An NK₁ selective agonist ([Sar⁹, Met¹¹]-substance P) or direct activation of PKC with phorbol 12,13-dibutyrate, did not change firing in CAPS-responsive neurons, but did inhibit various types of K⁺ currents that activated over a wide range of voltages. These data suggest that the excitability of CAPS-responsive phasic afferent neurons is increased by activation of NK₂ receptors and that this is due in part to inhibition and a positive voltage shift in the activation of heteropodatoxin-sensitive Kv4 channels.     

Excitatory actions of substance P in the rat lateral posterior nucleus

The lateral posterior nucleus (LP) receives inputs from both neocortex and superior colliculus (SC), and is involved with integration and processing of higher‐level visual information. Relay neurons in LP contain tachykinin receptors and are innervated by substance P (SP)‐containing SC neurons and by layer V neurons of the visual cortex. In this study, we investigated the actions of SP on LP relay neurons using whole‐cell recording techniques. SP produced a graded depolarizing response in LP neurons along the rostro‐caudal extent of the lateral subdivision of LP nuclei (LPl), with a significantly larger response in rostral LPl neurons compared with caudal LPl neurons. In rostral LPl, SP (5–2000 nm ) depolarized nearly all relay neurons tested (> 98%) in a concentration‐dependent manner. Voltage‐clamp experiments revealed that SP produced an inward current associated with a decreased conductance. The inward current was mediated primarily by neurokinin receptor (NK)₁ tachykinin receptors, although significantly smaller inward currents were produced by specific NK₂ and NK₃ receptor agonists. The selective NK₁ receptor antagonist RP67580 attenuated the SP‐mediated response by 71.5% and was significantly larger than the attenuation of the SP response obtained by NK₂ and NK₃ receptor antagonists, GR159897 and SB222200, respectively. The SP‐mediated response showed voltage characteristics consistent with a K⁺ conductance, and was attenuated by Cs⁺, a K⁺ channel blocker. Our data suggest that SP may modulate visual information that is being processed and integrated in the LPl with inputs from collicular sources.     

Differential contributions of Ca2+‐activated K+ channels and Na+/K+‐ATPases to the generation of the slow afterhyperpolarization in CA1 pyramidal cells

In many types of CNS neurons, repetitive spiking produces a slow afterhyperpolarization (sAHP), providing sustained, intrinsically generated negative feedback to neuronal excitation. Changes in the sAHP have been implicated in learning behaviors, in cognitive decline in aging, and in epileptogenesis. Despite its importance in brain function, the mechanisms generating the sAHP are still controversial. Here we have addressed the roles of M‐type K⁺ current (I_M), Ca²⁺‐gated K⁺ currents (I_Ca(K)'s) and Na⁺/K⁺‐ATPases (NKAs) current to sAHP generation in adult rat CA1 pyramidal cells maintained at near‐physiological temperature (35 °C). No evidence for I_M contribution to the sAHP was found in these neurons. Both I_Ca(K)'s and NKA current contributed to sAHP generation, the latter being the predominant generator of the sAHP, particularly when evoked with short trains of spikes. Of the different NKA isoenzymes, α₁‐NKA played the key role, endowing the sAHP a steep voltage‐dependence. Thus normal and pathological changes in α₁‐NKA expression or function may affect cognitive processes by modulating the inhibitory efficacy of the sAHP.     

Effect of charybdotoxin and leiurotoxon I on potassium currents in bullfrog sympathetic ganglion and hippocampal neurons

The effects of charybdotoxin and leiurotoxin I were examined on several classes of K⁺ currents in bullfrog sympathetic ganglion and hippocampal CA1 pyramidal neurons. Highly purified preparations of charybdotoxin selectively blocked a large voltage- and Ca²⁺-dependent K⁺ current (I_c) responsible for action potential repolarization (IC₅₀ = 6 nM) while leiurotoxin I selectively blocked a small Ca²⁺-dependent K⁺ conductance (I_AHP) responsible for the slow afterhyperpolarization following an action potential (IC₅₀ = 7.5 nM) in bullfrog sympathetic ganglion neurons. Neither of the toxins had a significant effects on other K⁺ currents (M-current [I_M], A-current [I_A] and the delayed rectifier [I_KD] present in these cells. Leiurotoxin I at a concentration of 20 nM had no detectable effect on currents in hippocampal CA1 pyramidal neurons. This lack of effect on I_AHP in central neurons suggests that the channels underlying slow AHPs in those neurons are pharmacologically distinct from analogous channels in peripheral neurons.     

Characterization of substance P release from the intermediate area of rat thoracic spinal cord

Substance P (SP) nerve terminals innervate the intermediolateral cell column (IML) of the thoracic spinal cord, where SP coexists with serotonin (5-HT), neurokinin A (NKA) and thyrotropin-releasing hormone (TRH). Neither the depolarization-induced release of SP nor the presence of other neurochemicals in the regulation of SP release has been directly studied in this system. In the present study, basal and K⁺-stimulated release of SP from the microdissected intermediate area (including the IML, intercalated nucleus and central autonomic nucleus) of the rat thoracic spinal cord, and the regulation of SP release by presynaptic autoreceptors and by coexisting neurochemicals (5-HT, NKA and TRH) were studied using an in vitro superfusion system. Potassium evoked a concentration- and extracellular Ca²⁺-dependent release of SP. In rats pretreated with the serotoninergic neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), both SP content and the absolute amount of SP released were decreased. However, the fraction of the remaining tissue content of SP released by K⁺ depolarization was not changed subsequent to 5,7-DHT treatment. Moreover, 5-HT, 5-HT_1B agonists (CGS-12066B and RU 24969) and a 5-HT₃ agonist (2-methyl-5-HT) did not alter the K⁺-evoked release of SP. These data demonstrate that SP is released from the intermediate area of the rat thoracic spinal cord and some of the SP released comes from serotoninergic nerve terminals. Although 5-HT coexists with SP in the IML, neither endogenous 5-HT nor 5-HT receptor ligands appear to regulate the release of SP. Other colocalized neuropeptides (NKA and TRH) are not involved in the regulation of SP release because neither NKA, a NK₂ agonist (GR 64349) nor a TRH analog (MK-771) changed the K⁺-evoked release of SP. A neurokinin-1 (NK₁) antagonist (GR 82334) dose-dependently (10^-9-10^-7 M) increased the K⁺ stimulated release of SP. These data suggest the presence of presynaptic inhibitory NK₁ autoreceptors. Whereas, NK₁ agonists, [GR 73632 (10^-9-10^-6 M) and [Sar⁹, Met (O₂)^1]] SP (10^-8-10^-6 M)], increased the basal and K⁺-stimulated release of SP, the excitatory effects of GR 73632 were not blocked by the NK₁ antagonist. Moreover, GR 73632 increased the efflus of SP to a greater extent in the absence of peptidase inhibitors. Thus, the effect of NK₁ agonists on the release of SP may be related to an inhibition of peptide degradation rather than activation of NK₁ autoreceptors. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Effects of coexisting neurochemicals on the release of serotonin from the intermediate area of rat thoracic spinal cord

Serotonin (5-HT), substance P (SP), neurokinin A (NKA), and thyrotropin-releasing hormone (TRH) coexist in the nerve terminals of the intermediolateral cell column (IML) of the thoracic spinal cord. The Ca2⁺-dependent release of 5-HT from the microdissected intermediate area (including the IML) of the rat thoracic spinal cord, and the 5-HT_1B autoreceptor regulator of 5-HT release, were previously demonstrated. In this paper, the effects of SP, NKA, TRH, and/or their analogs on the release of [³H]5-HT from the intermediate area were investigated using an in vitro superfusion system. Both SP (the endogenous ligand for neurokinin-1 (NK₁) receptor) and an NK₁, agonist (GR 73632) significantly increased the basal release of [³3H]5-HT. SP and GR 73632 did not change the K⁺-stimulated release of [³H]5-HT. The effect of the NK₁ agonist on the basal release of [³H]5-HT was dose-dependent, was reduced by an NK₁ antagonist (GR 82334), and was not dependent on extracellular Ca²⁺. Neither NKA, an NK₂, agonist (GR 64349), nor a TRH analog (MK-771) altered the basal or stimulated release of [³H]5-HT. These data suggest that basal release of 5-HT from the intermediate area of the rat thoracic spinal cord is regulated by SP (acting through an NK₁ receptor), but not by NKA or TRH. These results provide evidence for the role of SP as a modulator of serotoninergic neurons in the intermediate area of the thoracic spinal cord, and may help to clarify the role of coexisting neurochemicals in the spinal regulation of the sympathetic nervous system. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

HERG- and IRK-like Inward Rectifier Currents are Sequentially Expressed During Neuronal Development of Neural Crest Cells and their Derivatives

Quail neural crest cells were cultured in a differentiative medium to study the inward K⁺ channel profile in neuronal precursors at various stages of maturation. Between 12 and 24 h of culture, neural crest-derived neurons displayed, in addition to the previously described outward depolarization-activated K⁺ currents, an inward current enhanced in high K⁺ medium. A biophysical and pharmacological analysis led us to conclude that this inward K⁺ current is identical to that previously demonstrated in mouse and human neuroblastoma cell lines (I_IR). This current (quail I_IR or ql_lR), which is active at membrane potentials positive to -35 mV, was blocked by Cs⁺ and by class Ill antiarrhythmic drugs, thus resembling the K⁺ current encoded by the human ether-a-go-go-related gene (HERG). At later stages of incubation (>48 h), neural crest-derived neurons underwent morphological and biochemical differentiation and expressed fast Na⁺ currents. At this stage the cells lost qll_R, displaying instead a classical inward rectifier K⁺ (IRK) current (quail I_IRK= qI_IRK). This substitution was reflected in the resting potential (V_REST), which became hyperpolarized by >20 mV compared with the 24 h cells. Neurons were also harvested from peripheral ganglia and other derivatives originating from the migration of neural crest cells, viz. ciliary ganglia, dorsal root ganglia, adrenal medulla and sympathetic chain ganglia. After brief culture following harvesting from young embryos, ganglionic neurons always expressed qi_lR. On the other hand, when ganglia were explanted from older embryos (7–12 days), briefly cultured neurons displayed the IRK-like current. Again, in all the above derivatives the ql_lR substitution by ql_lRK was accompanied by a 20 mV hyperpolarization of V_REST. Together, these data indicate that the V_REST of normal neuronal precursors is sequentially regulated by HERG- and IRK-like currents, suggesting that HERG-like channels mark an immature and transient stage of neuronal differentiation, probably the same stage frozen in neuroblastomas by neoplastic transformation.     

Inhibition of M-type K current by linopirdine, a neurotransmitter-release enhancer, in NG108-15 neuronal cells and rat cerebral neurons in culture

The effect of linopirdine, a neurotransmitter-release enhancer, on the M-type K⁺-current, I_K(M), was examined in NGPM1-27 cells, mouse neuroblastoma×rat glioma NG108-15 cells transformed to express m1-muscarinic acetylcholine (ACh) receptors, using the nystatin-perforated patch-recording mode under voltage-clamp conditions. The application of linopirdine induced the inward current associated with an inhibition of I_K(M), which mimics an excitatory part of the ACh-induced responses in NGPM1-27 cells. The affinity of linopirdine for the inhibition of I_K(M) was 24.7 μM in NGPM1-27 cells. In the presence of linopirdine, ACh failed to evoke a further inward current, but ACh still elicited an outward current, thus suggesting that the Ca²⁺-dependent K⁺ current is rather insensitive to linopirdine. Linopirdine also inhibited another voltage-gated potassium current (I_K(V)) at the concentration of 72.3 μM. Finally, the inhibitory effect of linopirdine on I_K(M) was confirmed in pyramidal neurons acutely dissociated from the rat cerebral cortex at 35.8 μM. The results suggest that linopirdine is thus considered to be an inhibitor of some type of K⁺ channels in both NGPM1-27 cells and the rat cerebral neurons.     

Similarities of SP-, NKA- and NKB-induced currents in rat dorsal root ganglion neurons

Tachykinin (TK; including substance P (SP), neurokinin A (NKA) and neurokinin B (NKB))-induced currents (I(TK)s) were studied in freshly isolated rat dorsal root ganglion (DRG) neurons using whole-cell patch clamp recording and repatch techniques. All the three I(TK)s manifested features of fast activating kinetics, such as short latency and fast tau(on) and tau(off), and very slow desensitization. The concentration-response relationships for TKs show: (1). compared with the concentration-response curve for NKA, the curve for NKB shifted upwards, while that for SP shifted downwards; (2). the EC(50) values for NKB-, NKA- and SP-activated currents were very close to each other. The I-V curves for the three TKs were basically linear and arrayed in the order of NKB>NKA>SP; the reversal potentials for the three I(TK)s were all around +15 mV. Replacement of NaCl in the external solution by equimolar N-methyl-D-glucamine (NMDG) attenuated both NKA- and NKB-activated currents markedly, as it was the case with SP-activated current caused by the opening of Na(+) preferring non-selective cation channels observed in our previous work. All the three TKs proved to inhibit coexistent GABA(A) receptor-mediated current (I(GABA)); this effect was removed by intracellular dialysis of GDP-beta-S or H-7. However, these drugs did not block the SP-, NKA- and NKB-activated currents at all, which indicated that I(TK)s were G-protein independent. In short, the responses of rat DRG neurons to SP, NKA and NKB were similar in essence, although the amplitudes of currents induced by the same concentration of the three TKs were different. Taking the results of this study and our previous studies together, we hypothesized that SP, NKA and NKB may induce inward currents through undiscovered channels that are associated with tachykinins receptors (NK1R, NK2R, NK3R, which have already been cloned), but independent of G-protein coupling and remains to be further investigated.     

Electrophysiological properties and their modulation by norepinephrine in the ambiguus neurons of the guinea pig

Electrophysiological properties of guinea pig ambiguus (AMB) neurons were studied in a brainstem slice preparation. During subthreshold depolarization AMB neurons displayed an early slow depolarization and a late outward rectification both of which were blocked by replacing Ca²⁺ with Co²⁺ in the extracellular solution. AMB neurons showed hyperpolarizing inward rectification which was blocked by extracellular Cs⁺ and is likely caused by the activation of Ih. In 58% (n = 49) of AMB neurons spike firing was restricted to the early phase of a long-lasting depolarizing current injection (phasic firing). The remaining AMB neurons showed repetitive firing throughout the depolarization (tonic firing). A Ca²⁺-mediated K⁺ current (I_K(Ca)) caused an afterhyperpolarization that followed both single and repetitive spike firing. I_K(Ca) also controlled the firing pattern in both types of firing, especially in the phasic firing. Norepinephrine (NE) blocked both the hyperpolarizing inward rectification and the Ca²⁺-dependent AHP. These effects of NE were antagonized by propranolol. It is proposed that the blockade of I_K(Ca) and Ih contribute to the improvement of the ‘signal-to-noise ratio’ by NE in AMB neurons.     

Opioid peptides modulate GABAA receptor responses in neurons of bullfrog dorsal root ganglia

Effects of enkephalin and selective opioid-receptor agonists on GABA-induced current were examined in dissociated neurons of bullfrog dorsal root ganglia (DRG) by using whole-cell patch-clamp method. Leucine-(Leu)-enkephalin and methionine-(Met)-enkephalin depressed GABA_A receptor-mediated currents. DPDPE, DAMGO and dynorphin-A (Dyn-A) also depressed the inward current produced by GABA: the order of agonist potency was DPDPE ≥ DAMGO> Dyn-A. Naloxone blocked the inhibitory effects of ekephalins and other opioid agonists on the GABA current. Naltrindole (NTI), a δ-receptor antagonist, prevented the DPDPE-induced depression of the GABA current. β-Funaltrexamine (β-FNA), a μ-receptor antagonist, reduced the DAMGO-induced depression of GABA currents. Nor-binaltorphimine (nor-BNI), a κ-receptor antagonist, reduced the effects of Dyn-A in depressing the GABA current The results suggest that enkephalin down-regulates GABA_A receptor function through mainly δ- and μ-opioid receptors in bullfrog DRG neurons.     

Endogenous voltage-gated potassium channels in human embryonic kidney (HEK293) cells

Endogenous voltage-gated potassium currents were investigated in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells using whole-cell voltage clamp recording. Depolarizing voltage steps from −70 mV triggered an outwardly rectified current in nontransfected HEK293 cells. This current had an amplitude of 296 pA at +40 mV and a current density of 19.2 pA/pF. The outward current was eliminated by replacing internal K⁺ with Cs⁺ and suppressed by the K⁺ channel blockers tetraethylammonium and 4-aminopyridine. Raising external K⁺ attenuated the outward current and shifted the reversal potential towards positive potentials as predicted by the Nernst equation. The current had a fast activation phase but inactivated slowly. These features implicate delayed rectifier (I_K)-like channels as mediators of the observed current, which was comparable in size to I_K currents in many other cells. A small native inward rectifier current but no transient outward current I_A, the M current I_M, or Ca²⁺-dependent K⁺ currents were detected in HEK293 cells. In contrast to these findings in HEK293 cells, little or no I_K-like current was detected in CHO cells. The difference in endogenous voltage-activated currents in HEK293 and CHO cells suggest that CHO cell lines are a preferred system for exogenous K⁺ channel expression. J. Neurosci. Res. 52:612–617, 1998. © 1998 Wiley-Liss, Inc.     

Cationic modulation of 5-HT2 and 5-HT3 receptors in rat sensory neurons: the role of K, Ca and Mg

The effects of changes in external K⁺, Ca²⁺, and Mg²⁺ concentrations on 5-HT₂- and 5-HT₃ receptor-mediated depolarizations of the resting membrane potential in rat dorsal root ganglion (DRG) cells was studied. In cells exhibiting a 5-HT₂-mediated response, 5-HT and α-methyl 5-HT depolarized the resting membrane potential (RMP) and increased the slope of the current–voltage (I/V) relationship. The equilibrium potential (E_r) for the depolarization was linearly related to the logarithm of the [K⁺]_o, indicating the depolarization resulted from a decrease in resting K⁺ conductance. In a subpopulation of large-diameter acutely dissociated DRG neurons recorded from using the whole-cell patch-clamp configuration, 5-HT produced an inward shift in the current required to hold cells at −60 mV. This inward shift in holding current was associated with a reduction in membrane conductance and reversed near E_k. This data suggests that the 5-HT₂ receptor-mediated depolarization and increase in R_in seen in intact DRG preparation is produced by blockade of an outward K⁺ leak current. Increases in [K⁺]_o reduced the increase in R_in and depolarization induced by 5-HT with 50% inhibition of the depolarization occurring at 8.3 mM of [K⁺]_o. Half-normal Ca²⁺ (1.2 mM) produced a downward shift of the 5-HT concentration–response curve, reducing the maximal response by 40%, with minimal effect on the half-maximal response. Mg²⁺ ions did not affect this 5-HT response. In cells exhibiting a 5-HT₃ receptor response, 5-HT and 2-methyl-5-HT produced depolarization with decreased R_in. The E_r for this depolarizing response (−30.2±1.8 mV) became less negative (−11.5 mV) in 10 mM [K⁺]_o with minimal effect on the amplitude of the depolarization. In Na⁺-free superfusate, the 5-HT-induced depolarization was converted to hyperpolarization. This indicated the 5-HT₃ response increased a mixed Na⁺/K⁺ conductance. Elevated Ca²⁺ or Mg²⁺ markedly reduced the 5-HT₃ response. Incubation with 3.5 mM Ca²⁺ shifted the 5-HT concentration–response curve downward and to the right, decreasing the maximal response by 49% and increasing the EC₅₀ by 10-fold. Elevated Mg²⁺ produced similar effects. In cells where both 5-HT₂- and 5-HT₃-mediated responses could be demonstrated, the elevation of K⁺ or the reduction of Ca²⁺ converted a 5-HT₂ response to a 5-HT₃ response. The above data suggest that elevation of [K⁺]_o or reduction of [Ca²⁺]_o produced by rapid firing rates of sensory neurons will favor the expression of 5-HT₃ responses over 5-HT₂ responses.     

Interaction between Low Voltage-activated Currents in Reticular Thalamic Neurons in a Rat Model of Absence Epilepsy

A transient potassium (K⁺) outward current (I_A) contributes to the distinctive patterns of low-threshold spike firing observed in various classes of thalamic neurons through a functional interaction with a calcium (Ca²⁺)-mediated inward current (I_T). The present study was undertaken to investigate the properties of transient K⁺ currents and their interaction with I_T in neurons of the reticular thalamic nucleus, and to compare these properties in reticular thalamic nucleus neurons from a rat model of absence epilepsy, designated the Genetic Absence Epilepsy Rat from Strasbourg (GAERS), with those from a Non-epileptic Control strain (NEC). This comparative approach appeared to be particularly important in view of the recent finding of a selective increase in I_T in reticular thalamic nucleus neurons from GAERS. Neurons were acutely isolated from the reticular thalamic nucleus through enzymatic procedures, and identified by morphological and immunocytochemical criteria. Ionic currents were analysed using whole-cell patch-clamp techniques. Transient K⁺ currents in reticular thalamic nucleus neurons with properties indicative of I_A activated at ～?55 mV (with half-activation at ?27 and ?33 mV in NEC and GAERS respectively), declined rapidly with a voltage-dependent time constant (τ= 4 ms at +45 mV), were 50% steady-state-inactivated at ?81 and ?86 mV in the two strains of rats respectively, and rapidly recovered from inactivation with a monoexponential time course (τ= 31 and 37 ms respectively). No significant differences in I_A properties or densities were found between reticular thalamic nucleus neurons from GAERS and NEC rats. Analysis of the interaction between I_A and I_T indicated a shift in the balance between the two opposing membrane conductances towards the generation of a low-voltage-activated inward current in reticular thalamic nucleus neurons from GAERS compared with NEC, and a lack of I_A to functionally compensate for this shift, which in turn may contribute to pathological forms of low-threshold spike firing characterizing spike-and-wave discharges.     

Membrane properties of identified mesencephalic dopamine neurons in primary dissociated cell culture

Dopamine (DA)-containing neurons in primary dissociated cell cultures derived from the embryonic mouse mesencephalon (day E13) were studied by histochemical and electrophysiological techniques. DA neurons exhibited two distinct morphologies, fusiform and multipolar, tended to reside in groups and organize dendrited into common fascicles. While these neurons expressed the cell-surface marker acetylcholinesterase, the presence of this enzyme could not be used to identify DA neurons unequivocally, since it was also observed in nondopaminergic cells. Neurons were therefore identified as DA by their distinct morphology, and this identification was validated with a double-labeling procedure that entailed the intracellular deposition of a fluorescent dye (Lucifer yellow or ethidium bromide), followed by processing for tyrosine hydroxylase immunocytochemistry. DA neurons identified in this manner were observed to have resting membrane potentials between ? 50 and ? 75 m V, input resistances of 50–360 Mω, and membrane time constants of 4.1–14.1 msec. Forty-seven percent of these cells displayed spontaneous activity that was irregular in nature and often contained bursts (burst length was between two and six action potentials). The DA neurons displayed a variety of ionic conductances, including (1) a Na⁺ conductance (g_Na) that underlies the action potential, (2) Ca²⁺ conductances (g_Ca) that mediate the nonsomatic low- and high-threshold spikes observed, and (3) at least three K⁺ conductances (g_k). Voltage-clamp analysis revealed several distinct transmembrane ionic currents, including (1) a large, rapidly inactivating tetrodotoxin-sensitive inward Na⁺ current (_Na), (2) a 4-aminopyridine-sensitive, transient early outward K⁺ current that required a conditioning hyperpolarization of the membrane to be activated by a subsequent depolarization (A-current, I_A), (3) a slowly developing inward current that was seen only after a conditioning hyperpolarization of the membrane and that was dependent on the presence of external Ca²⁺ ions (I_Ca), and (4) a late-onset, noninactivating K⁺current. Between 25% and 54% of the late-onset K⁺ current was Ca²⁺ -dependent and was not affected by tetraethylammonium ions. This current was termed I_AHP. The remaining current was not sensitive to changes in the extracellular Ca²⁺ concentration but was blocked by external tetraethylammonium ions. This current was termed I_K. The direct pressure application of DA (1–200 μM) onto the soma dose-dependently hyperpolarized these neurons; this effect was potentiated by the presence of the catecholamine reuptake blocker cocaine hydrochloride (10–200 μM). Under voltage-clamp conditions, DA was observed to increase I_K, significantly and had little effect on I_AHP. Thus, DA neurons in monolayer cultures were shown to have many of the electrophysiological properties routinely observed for these cells in vivo indicating that this preparation may serve as a useful model system for the further study of the molecular biology of these catecholamine-containing neurons. © Wiley-Liss, Inc.     

Ionic properties of IK,n in outer hair cells of guinea pig cochlea

The ionic properties of voltage-dependent K⁺ current activated at the resting membrane potential (I_K,n) of outer hair cells (OHC) isolated from the guinea pig cochlea were studied using a patch-clamp technique in a whole-cell recording mode. The reversal potential of I_K,n indicated a high selectivity for K⁺, and the relative permeability ratios for various monovalent cations were K⁺ : Rb⁺ : NH⁺₄ = 1 : 1.21 : 0.13. Decrease in extracellular Cl⁻¹ inhibited the I_K,n. I_K,n was blocked by Cs⁺ and Ba²⁺, although the inhibitory manner of Cs⁺ and Ba²⁺ were voltage-dependent and voltage-independent, respectively. By the use of puff-application method, the local application of Ba²⁺ to basolateral surface of OHC shifted the holding current level in an inward direction, whereas the application to apex and hair showed little change. Indicating that the I_K,n channels preferentially locate at the basolateral region of cell membrane.     

Release of endogenous opioids during a chronic IBD model suppresses the excitability of colonic DRG neurons

Background Endogenous opioids are implicated in pain‐regulation in chronic inflammatory bowel disease (IBD). We sought to examine whether endogenous opioids suppress the excitability of colonic nociceptive dorsal root ganglia (DRG) neurons during chronic IBD, and if so, whether modulation of underlying voltage‐gated K⁺ currents was involved. Methods The effects of chronic dextran sulfate sodium (DSS) colitis on afferent signaling in mice was studied using patch clamp recordings. Colonic DRG neurons were identified using Fast Blue retrograde labeling and recordings obtained from small DRG neurons (<40 pF). Key Results In current‐clamp recordings, the rheobase of neurons was increased 47% (P < 0.01) and action potential discharge at twice rheobase decreased 23% (P < 0.05) following incubation in colonic supernatants from chronic DSS mice. β‐endorphin increased 14‐fold, and tissue opioid immunoreactivity and expression in CD4+ cells observed by flow cytometry increased in chronic DSS colons. Incubation of naïve neurons in the μ‐opioid receptor agonist D‐Ala², N‐ MePhe⁴, Gly‐ol (DAMGO) (10 nM) partially recapitulated the effects of supernatants from DSS mice on rheobase. Supernatant effects were blocked by the μ‐opioid receptor antagonist naloxone. In voltage clamp, chronic DSS supernatants and DAMGO increased I_A K⁺ currents. Conclusions & Inferences The release of endogenous opioids during chronic inflammation in mice suppresses the excitability of nociceptive DRG neurons. Targeting immune cells may provide a novel means of modulating IBD pain.     

Modulation of action potential during the late slow excitatory postsynaptic potential in bullfrog sympathetic ganglia

The spike peak and after-hyperpolarization of the action potential of bullfrog sympathetic ganglion cells were depressed during the late slow excitatory postsynaptic potential (EPSP). These changes in the action potential were mimicked by luteinizing hormone-releasing hormone (LH-RH), a neurotransmitter candidate for the late show EPSP. LH-RH (5 μM) suppressed the voltage-dependent K⁺ currents, both the delayed rectifier K⁺ current (I_K1) and the M current (I_K2). It is suggested that the depression of the after-hyperpolarization of the action potential during the late slow EPSP may be due to suppression of I_K1 and I_K2.     

Tachykinin Actions on Deep Dorsal Horn Neurons In Wm: An Electrophysiological and Morphological Study in the Immature Rat

To assess whether functional neurokinin receptors exist in the deep dorsal horn of the rat, the actions of the selective neurokinin-1 receptor (NK1R) agonist [Sar⁹,Met(O₂)¹¹]substance P [Sar⁹,Met(O₂)¹¹]]SP), the neurokinin-2 receptor (NK2R) agonists [β-Ala⁸]NKA_4-10 and GR64349 and the neurokinin-3 receptor (NK3R) agonist senktide were examined intracellularly in vitro. [Sar⁹,Met(O₂)¹¹]]SP (1–4 μM) and senktide (1-2 μM) elicited slow depolarizations (40 mV) associated with increased synaptic activity and cell firing. [β-Ala⁸]NKA_4-10 (10-20 μM) and GR64349 (0.25-10 μM) caused small depolarizations (<2.0 mV) and no firing. Neurons were categorized as either ‘tonic’ or ‘phasic’ depending on their firing response to direct current step depolarizations. Tonic neurons, which, unlike phasic neurons, display no spike firing accommodation, generated a significantly larger depolarization to the NK1R and NK3R agonists. The putative contribution of these receptors to primary afferent-mediated synaptic transmission was assessed by testing the NKIR antagonist GR82334 (1 μM), the NK2R antagonist MEN10,376 (1 μM) and the NK3R antagonist [Trp⁷,β-Ala⁸]NKA4_-10 (1 μM) against the dorsal root-evoked excitatory postsynaptic potential (DR-EPSP). GR82334 and [Trp⁷,β-Ala⁸]NKA_4-10 significantly reduced (P ≤ 0.05) the duration but not the amplitude of the DR-EPSP. MEN10,376 (1 μM) had no effect on DR-EPSP amplitude or duration. Morphological detail was obtained for seven biocytin-filled deep dorsal horn neurons tested with [Sar⁹,Met(O₂)¹¹]SP. Five neurons responded to the NKIR agonist, and two of these had dorsally directed dendrites into the substantia gelatinosa. The other three [Sar⁹,Met(O₂)¹¹]SP-sensitive neurons had dendrites within deeper laminae. These data support the existence of functional NK1Rs and NK3Rs in the deep dorsal horn which may be involved in mediating sensory afferent inputs from nociceptors.     

Effects of Acetazolamide on Transient K+ Currents and Action Potentials in Nodose Ganglion Neurons of Adult Rats

The aim of the present study was to determine whether acetazolamide (AZ) contributes to the inhibition of the fast inactivating transient K⁺ current (I_A) in adult rat nodose ganglion (NG) neurons. We have previously shown that pretreatment with either AZ or 4‐AP attenuated or blocked the CO₂‐induced inhibition of slowly adapting pulmonary stretch receptors in in vivo experiments. The patch‐clamp experiments were performed by using the isolated NG neurons. In addition to this, the RT‐PCR of mRNA and the expression of voltage‐gated K⁺ (Kv) 1.4, Kv 4.1, Kv 4.2, and Kv 4.3 channel proteins from nodose ganglia were examined. We used NG neurons sensitive to the 1 mM AZ application. The application of 1 mM AZ inhibited the I_A by approximately 27% and the additional application of 4‐AP (1 mM) further inhibited I_A by 48%. The application of 0.1 μM α‐dendrotoxin (α‐DTX), a slow inactivating transient K⁺ current (I_D) blocker, inhibited the baseline I_A by approximately 27%, and the additional application of 1 mM AZ further decreased the I_A by 51%. In current clamp experiments, AZ application (1 mM) increased the number of action potentials due to the decreased duration of the depolarizing phase of action potentials and/or due to a reduction in the resting membrane potential. Four voltage‐gated K⁺ channel proteins were present, and most (80–90%) of the four Kv channels immunoreactive neurons showed the co‐expression of carbonic anhydrase‐II (CA‐II) immunoreactivity. These results indicate that the application of AZ causes the reduction in I_A via the inhibition of four voltage‐gated K⁺ channel (Kv) proteins without affecting I_D.