Voltage clamp analysis of ethanol effects on pacemaker currents ofAplysia neurons

The effect of 4% ethanol on pacemaker currents of Aplysia neurons was studied under voltage clamp. In normal seawater the n-shape in the I-V disappeared and outward current increased. Ion substitution and drug blocking experiments determined that leakage current and the slow inward calcium current were decreased and that the outward currents, IA and IK, were increased. This knowledge can be used to explain ethanol effects on spontaneous firing patterns.     

Voltage clamp analysis of lamprey neurons — role of N-methyl-d-aspartate receptors in fictive locomotion

Spinal neurons in the lamprey have been subjected to a voltage clamp analysis of the excitatory currents generated during fictive locomotion with particular reference to the phasic activation of voltage dependent N-methyl-D-aspartate (NMDA) receptors. Voltage-clamped neurons observed during NMDA-induced fictive swimming show excitatory and inhibitory synaptic currents in phase with the ipsilateral and contralateral ventral root discharges, respectively. The excitatory synaptic currents showed a marked voltage dependence suggesting that potential sensitive conductances such as the NMDA ionophore are involved in the synaptic events underlying rhythmic locomotor activity. The effect of NMDA receptor activation during application of tetrodotoxin has also been analyzed during NMDA-induced pacemaker-like oscillations. Such NMDA-induced oscillations are essentially abolished during the voltage clamp. In the presence of NMDA current voltage plots reveal a negative slope conductance in the potential range of the inherent oscillations. The addition of tetraethyl ammonium (TEA) to NMDA solution enhanced a net steady state inward current by more than 10-fold due to a partial block of the outward currents. A kinetic analysis was done with a frequency domain technique using a white noise stimulus to linearly perturb the membrane potential over a wide range of frequencies. The analysis revealed that the induced negative conductance leads to a response which is nearly 180 degrees out of phase with the stimulus at low frequencies. This is an unstable condition which leads to the depolarizing phase of the induced oscillations.     

Uncoupling of GABA-benzodiazepine receptors in chick cerebral cortical neurons requires co-activation of both receptor sites

Primary cultures of chick cerebral cortical neurons were exposed to 1 microM flurazepam in vitro, and the effect of flurazepam on GABA-activated membrane current (IGABA) was examined using whole-cell voltage-clamp recording. Exposure of chick cerebral cortical neurons to flurazepam alone for 3-10 days resulted in a significant decrease in the degree of potentiation of IGABA elicited by 0.5 microM flurazepam. When GABA or nipecotic acid (a GABA uptake blocker) were added with flurazepam during chronic drug exposure, neuronal responses to GABA were significantly less sensitive to modulation by 0.5 microM flurazepam compared to flurazepam treatment alone. Furthermore, this effect was significantly reduced by co-administration of the GABAA receptor antagonist bicuculline. These results suggest that tolerance to benzodiazepines in vitro requires activation of both the GABA and the benzodiazepine binding sites on the GABAA receptor-channel complex.     

Suppression of taurine response in acutely dissociated substantia nigra neurons by intracellular cyclic AMP

The modulatory effect of intracellular cyclic AMP on the taurine response was investigated in acutely dissociated rat substantia nigra neurons in patch clamp configurations. Taurine acts mainly on the glycine receptor. An intracellular application of cyclic AMP (5×10⁻⁴ M) inhibited the response to a high concentration of taurine (10⁻³M) by about 50%, but did not affect the response to a low concentration of taurine (10⁻⁴ M). This inhibition was blocked somewhat by N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide (H-8) (10⁻⁶M), suggesting that the inhibition of taurine response might be partly mediated by an activation of protein kinase A.     

Nodes of ranvier above a neuroma in the rat sciatic nerve: Voltage clamp analysis and electron microscopy

Neuroma formation was induced in adult rat sciatic nerves and the animals were allowed to survive for 1-10 months. In 10 animals single large myelinated fibres from the nerve segment above the neuroma were subjected to voltage clamp analysis. Six animals were fixed by glutaraldehyde perfusion and nodes of Ranvier or large myelinated fibres above the neuroma were examined in the electron microscope (EM). Most fibres exhibited normal action potentials, but a few had a reduced excitability and small action potentials. Some fibres had increased membrane time constant and leak conductance and a markedly increased membrane capacitance. Most of the examined nodes of Ranvier exhibited abnormally large delayed K currents, which could be blocked with 4-aminopyridine (4-AP). The Na current was normal. In the EM most large cross-cut myelinated axons were markedly atrophic, particularly after long survival times. Evaginations from the paranodal region of these axons penetrated between the terminating paranodal myelin lamellae. The nodal axolemmal undercoating could be very prominent and in some cases the nodal axon was irregular. These findings show that large myelinated peripheral nerve fibres, which are chronically disconnected from their peripheral targets, exhibit specific structural and functional abnormalities of the nodes of Ranvier.     

Effects of insulin-like growth factor 1 on voltage-gated ion channels in cultured rat hippocampal neurons

Insulin-like growth factor 1 (IGF-1) has important functions in the brain, including metabolic, neurotrophic, neuromodulatory, and neuroendocrine actions, and it is also prevents amyloid beta-induced death of hippocampal neurons. However, its functions on the voltage-gated ion channels in hippocampus remain uncertain. In the present study, we investigated the effects of IGF-1 on voltage-gated potassium, sodium, and calcium channels in the cultured rat hippocampal neurons using the whole-cell patch clamp recordings. Following incubation with different doses of IGF-1 for 24 h, a block of the peak transient A-type K+ currents amplitude (IC50: 4.425 ng/ml, Hill coefficient: 0.621) was observed. In addition, after the application of IGF-1, the amplitude of high-voltage activated Ca2+ currents significantly increased but activation kinetics did not significantly alter (V1/2: -33.45 +/- 1.32 mV, k = 6.16 +/- 1.05) compared to control conditions (V1/2: -33.19 +/- 2.28 mV, k = 7.26 +/- 1.71). However, the amplitude of Na+, K+, and low-voltage activated Ca2+ currents was not affected by the application of IGF-1. These data suggest that IGF-1 inhibits transient A-type K+ currents and enhances high-voltage-activated Ca2+ currents, but has no effects on Na+ and low-voltage-activated Ca2+ currents.     

An improved method for patch clamp recording and calcium imaging of neurons in the intact dorsal root ganglion in rats

The properties of dorsal root ganglion (DRG) neurons have been mostly investigated in culture of dissociated cells, and it is uncertain whether these cells maintain the electrophysiological properties of the intact DRG neurons. Few attempts have been made to record from DRG neurons in the intact ganglion using the patch clamp technique. In this study, rat DRGs were dissected and incubated for at least 1h at 37 degrees C in collagenase (10mg/ml). We used oblique epi-illumination to visualize DRG neurons and perform patch clamp recordings. All DRG neurons exhibited strong delayed rectifier potassium current and a high threshold for spike generation (-15 mV) that rendered the cells very weakly excitable, generating only one action potential upon strong current injection (>300 pA). It is therefore possible that cultured DRG neurons, commonly used in studies of pain processing, may be hyperexcitable because they acquired "neuropathic" properties due to the injury induced by their dissociation. Electrical stimulation of the attached root produced an antidromic spike in the soma that could be blocked by intracellular hyperpolarization or high frequency stimulation. Imaging intracellular calcium concentration with Oregon Green BAPTA-1 indicates that antidromic stimulation caused a long-lasting increase in intracellular calcium concentration mostly near the cell membrane. This study describes a simple approach to examine the electrophysiological and pharmacological properties and intracellular calcium signaling in DRG neurons in the intact ganglion where the effects of somatic spike invasion can be studied as well.     

Voltage-dependent calcium channel abnormalities in hippocampal CA3 neurons of spontaneously epileptic rats

PURPOSE: Hippocampal CA3 neurons of spontaneously epileptic rats (SER; zi/zi, tm/tm), which show both absence-like seizures and tonic convulsions, exhibit a long-lasting depolarization shift with repetitive firing with a single stimulation of mossy fibers. Therefore a whole-cell patch-clamp study using temporarily dissociated hippocampal CA3 neurons from SER was performed to elucidate whether such abnormal excitability was due to abnormalities in voltage-dependent Ca(2+) channels (VDCCs). METHODS: Hippocampal CA3 neurons were temporarily dissociated with enzymatic and mechanical treatments. In a voltage-clamp mode with whole-cell recording, depolarizing step pulses were applied to induce Ca(2+) currents in the presence of tetrodotoxin and tetraethylammonium. RESULTS: The threshold level of the Ca(2+) current induced by depolarizing pulses was found to be lower in hippocampal CA3 neurons of SER compared with those of control Wistar rats. In addition, the Ca(2+) current peak amplitude was greater, and decay of the current was weaker in CA3 neurons of SER than in those of normal Wistar rats. CONCLUSIONS: These findings suggest that enhancements of Ca(2+) influx into hippocampal CA3 neurons due to the easier activation properties of VDCCs, as well as a decrease in decay, are involved in SER epileptic seizures.     

Glycine-activated chloride currents of neurons freshly isolated from the ventral tegmental area of rats

Properties of whole-cell glycine currents (I_Gly) of ventral tegmental area (VTA) neurons from 3- to 7-day old Sprague–Dawley rats were investigated with the patch-clamp technique. Ninety-three percent of the 126 neurons examined produced I_Gly in response to glycine. For 70% of these neurons, I_Gly did not decay in response to a threshold concentration of glycine (1–5 μM). At elevated glycine concentrations, I_Gly consistently decayed from a peak to a steady state (SS). I_Gly increased in amplitude sigmoidally as a function of the concentration of agonist with an EC₅₀ of 32 μM. Strychnine (STR), when co-applied with glycine after a prepulse of STR, suppressed both the peak and SS I_Gly noncompetitively. In the absence of a prepulse, STR had a smaller effect on peak I_Gly while increasing its decay rate; the SS amplitude decreased. These STR effects were concentration dependent with an IC₅₀ of 31 nM and 184 nM STR for the peak and SS I_Gly, with prepulse, respectively, and 732 nM and 193 nM for the peak and SS I_Gly, respectively, without prepulse. Picrotoxin (PTX) co-applied with glycine suppressed both the peak and the SS I_Gly with an IC₅₀ of 25 μM. In contrast to STR, 1 min preincubation with PTX had no effect on I_Gly. Thus, PTX acts on the open channel. The inhibitory effects of both STR and PTX on I_Gly did not depend on the membrane potential.     

Activation of protein kinase C induces long-term changes of postsynaptic currents in neocortical neurons

Intracellularly injected phorbol 12,13-dibutyrate (PdiB), a phorbol ester that activates protein kinase C (PKC), altered the postsynaptic responses of neurons of the motor cortex of cats. PdiB increased the amplitudes and durations of EPSPs and decreased the amplitudes and durations of IPSPs elicited by stimulation of the ventrolateral (VL) thalamus or the pyramidal tract (PT). The changes lasted for 50 min or longer. Corresponding changes in peak excitatory and inhibitory postsynaptic currents (EPSCs, IPSCs) were measured directly with the single electrode voltage clamp technique. Quantitative analysis of EPSCs in response to VL thalamic stimulation and IPSCs in response to PT stimulation made in a subgroup of fast PT cells suggested that PdiB acted within the injected neuron rather than presynaptically to alter the synaptic currents. No consistent changes in resting membrane parameters that would account for these modifications were found. Control injections of a phorbol ester that did not activate PKC failed to induce changes in synaptic responses or resting membrane properties. These observations suggest that activation of PKC, in vivo, can induce long-lasting changes in synaptic responses of neocortical neurons by direct modification of postsynaptic ion channel conductivities.     

Corticosteroid-mediated modulation of carbachol responsiveness in CA1 pyramidal neurons: A voltage clamp analysis

Pyramidal neurons in the rat hippocampal CA1 area contain mineralocorticoid (MRs) and glucocorticoid (GRs) receptors for corticosterone. Previous current clamp experiments showed that depolarizations evoked by carbachol(1–3 μM) depend on relative MR/GR occupation: carbachol responses are small with predominant MR-activation and larger when both receptor types are occupied. Multiple K-conductances underlie the carbachol-induced depolarization. In the present study we used the single electrode voltage clamp technique to examine which K-conductances modulated by carbachol are sensitive to corticosteroid treatment in vitro. We observed that 1 μM carbachol significantly reduced the I_{K, Leak} while the I_M was hardly affected; carbachol effects on the I_{K, Leak} were significantly reduced under conditions of predominant MR activation compared to simultaneous activation of MRs and GRs. With a higher (10 μM) carbachol dose, steroid modulation of the I_{K, Leak} showed a similar tendency. The amplitude of the I_M was largely reduced by 10 μM carbachol but appeared to be not affected by steroid treatment. We conclude that the previously described suppression of the carbachol-induced depolarization with predominant activation MRs is caused by an attenuation of the carbachol action on the I_{K, Leak}. © 1995 Wiley-Liss, Inc.     

Development of the fast, transient outward K current in embryonic sympathetic neurones

Voltage-activated outward potassium (K+) currents in developing sympathetic neurones, dissociated from the rat superior cervical ganglion (SCG), were studied using the whole-cell patch clamp recording technique. In voltage-clamped neonatal SCG cells, two voltage-dependent K+ currents were measured: the fast, transient K+ current, IA; and, the slower activating, non-inactivating delayed rectifier, IK. Only IK, however, appeared to be present in SCG neurones isolated from early embryonic (E14.5-16.5) rat pups; IA was not observed in these cells. When these embryonic neurones were maintained in cell culture, IA developed over a time course (approximately 4-6 days) similar to that seen in vivo. IA, therefore, which appears to facilitate the fast repolarization phase of the action potential in rat SCG neurones, is the last voltage-activated current to develop in these cells.     

An analysis of cholinoceptive neurons in the hippocampal formation by direct microinfusion

Microinfusions of cholinergic agents were made in various sites in the dorsal hippocampal formation of urethane anaesthetized rats. Infusions of eserine or carbachol elicited hippocampal theta activity when made in areas containing high levels of cholinergic markers: the stratum oriens and radiatum of the CA1 and CA3, the stratum moleculare and stratum granulosum of the dentate gyrus and the infragranular region of the hilus. Subsequent infusions of atropine sulfate antagonized the theta activity. Control infusions of equal volumes of saline in active sites were without effect. Infusions of eserine or carbachol in the vicinity of the hippocampal fissure, the stratum lacunosum/moleculare of the CA1 or CA3, in the deep regions of the hilus, and in the lateral ventricle and overlying neocortex, were also without effect. Furthermore, in active sites, the latency to onset of theta and subsequent theta frequency, were both directly related to the total amount of carbachol infused. Thus, areas in which theta could be elicited with a cholinergic agonist (carbachol), or an anticholinesterase (eserine) and antagonized with atropine, were found to correspond well to areas previously found to contain a high density of cholinoceptive neurons, using autoradiographic and immunohistochemical techniques. These results provide further support for the involvement of acetylcholine as a neurotransmitter in the generation of type 2 theta in the hippocampal formation.     

Electrophysiological properties of the rat area postrema neurons displaying both the transient outward current and the hyperpolarization-activated inward current

We found coexistence of the transient outward potassium current (I(TO)) and the hyperpolarization-activated inward current (I(H)) in 26 of 82 area postrema neurons tested using the whole-cell patch-clamp technique in rat brain slices. Cells displaying both the I(TO) and the I(H) typically showed "voltage sag" and "rebound potentials" in response to hyperpolarizing current injection and repetitive firing with strong adaptation was seen with depolarizing current injection. When cells were held at membrane potentials more negative than the resting level (e.g., -85mV), the afterhyperpolarization was enhanced. Voltage clamp recordings were performed to examine the characteristics of I(TO) and I(H) in and the contributions of these currents to the electroresponsiveness of area postrema cells. We show, in this study, the voltage-dependent properties of I(H) and I(TO), and how these currents modulate the intrinsic membrane properties of area postrema cells. We discuss the functional significance of the specific subset of area postrema neurons whose cells have both I(H) and I(TO) channels.     

Stimulation of a sodium influx by cAMP in Helix neurons

Brief pressure injections of aqueous solutions of cAMP in identified neurons of Helix pomatia caused depolarizations which lasted for tens of seconds. In voltage-clamped neurons an inward current of similar duration was induced which saturated at 10 μA/cm² cell surface. In the range of negative membrane potentials with little voltage-dependent activation, this current was not accompanied by a change in membrane conductance. The inward current was not produced by injection of ATP, ADP, adenosine, inosine or cGMP. cAMP derivatives produced longer-lasting effects. Prolongation of the inward current was also observed after inhibition of the phosphodiesterase by IBMX. Drugs which block active transport had no effect on the response to cAMP injection. The inward current depended on extracellular sodium, and was maximal when all other mono- and divalent cations were replaced by Na⁺. The cAMP-induced current was accompanied by a transient increase in [Na⁺]_i, but there was no change in [Cl⁻]_i. Li⁺ could largely substitute for Na⁺; Ca²⁺ was less effective. Addition of Mg²⁺ or Ca²⁺ to solutions containing a high Na⁺-concentration inhibited the response. Internal acidification with HCl reversibly enhanced the inward current. These data indicate that the depolarizing effect of cAMP can be accounted for by an inward movement of Na-ions, and that the effect is augmented by H⁺-ions.     

Excitation of locus coeruleus neurons by vasoactive intestinal peptide: evidence for a G-protein-mediated inward current

Vasoactive intestinal polypeptide (VIP) caused a reversible increase in the firing rate of locus coeruleus (LC) neurons. Voltage-clamp at −60 mV revealed that VIP induced an inward current, associated with a small increase in conductance. The inward current persisted in the presence of Co²⁺ (to block Ca²⁺ channels) or tetrodotoxin (to block fast voltage-dependent Na⁺ channels). Substitution (80%) of Na⁺ with choline or Tris reduced the VIP-elicited inward current by approximately 75%. Changing external K⁺ concentrations did not alter the effect of VIP. The inward current induced by VIP became irreversible after the intracellular administration of GTPγS, a hydrolysis-resistant analog of GTP which can cause a prolonged activation of G-proteins. The intracellular application of GDPβS, which can interfere with G-protein activation, attenuated the effect of VIP. Pertussis toxin, an inactivator of certain G-proteins, did not block the effect of VIP. We conclude that VIP directly excites LC neurons by inducing a largely Na-dependent inward current. As this effect became irreversible in the presence of intracellular GTPγS, was attenuated by GDPβS, and was not eliminated by pertussis toxin, mediation through a pertussis toxin-insensitive G-protein is suggested.     

Voltage clamp analysis of the effect of excitatory amino acids and derivatives on Purkinje cell dendrites in rat cerebellar slices maintained in vitro

A voltage clamp analysis of the effects ofl-aspartate,l-glutamate and related derivatives on Purkinje cell dendrites was performed in rat cerebellar slices maintained in vitro. Short iontophoretic pulse applications ofl-aspartate andl-glutamate in the dendritic field of Purkinje cells induced dose-dependent inward currents with fast onset and recovery. Quisqualate application also gave rise to well developed inward currents with fast onset and slow recovery, whereas N-methyl-d,l-aspartate had no or little effect on Purkinje cell membranes unless prolonged (several seconds) applications were used. Steady applications of low doses of N-methyl-d,l-aspartate much more severely depressedl-aspartate thanl-glutamate mediated responses, whereas inward currents due to quisqualate were unaffected. Inward currents due to quisqualate were often more reduced than those due tol-aspartate by steady applications of 2-amino-5-phosphonovalerate, and the antagonistic action of this drug on responses due tol-glutamate was very weak. These results suggest that receptors of Purkinje cells for glutamate and aspartate are different, and are also different from N-methyl-d-aspartate and quisqualate receptors.     

Activation by N-acetyl-L-aspartate of acutely dissociated hippocampal neurons in rats via metabotropic glutamate receptors

PURPOSE: We previously reported that an increase in the N-acetyl-L-aspartate (NAA) level due to the lack of aspartoacylase gene was found in the brain of the tremor rat (tm/tm), which is a mutant with a causative gene named tm that shows epileptic seizures. Therefore, NAA is suggested to be one of the factors involved in the induction of epileptic seizures. Patch-clamp studies were performed to determine whether NAA produces an excitatory effect on acutely dissociated rat hippocampal neurons. METHODS: Acutely dissociated hippocampal neurons were prepared from normal Wistar rats aged 3-4 weeks. NAA-induced currents were investigated by using the whole-cell voltage-clamp recording technique. RESULTS: Application of NAA at concentrations of 100 nM to 1 mM through a U-tube for 2 s produced an inward current in a concentration-dependent manner at a holding potential of -60 mV. When the current-voltage relation was examined, the reversal potential of the NAA-induced current was found to be approximately 0 mV. The NAA-induced current was inhibited by bath application of the metabotropic glutamate receptor (mGluR) antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG) and by intracellular application of guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS), a nonhydrolyzable GDP analogue. However, the NAA-induced current remained unaffected by glutamic acid diethyl ester, a non-N-methyl-D-aspartate (NMDA)-subtype ionotropic glutamate receptor antagonist, or the voltage-dependent ion channel blockers tetrodotoxin, CdCl2, and tetraethylammonium-chloride. Conversely, the mGluR agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) also induced an inward current, with a reversal potential of 0 mV. The ACPD-induced current also was inhibited by MCPG. CONCLUSIONS: These results suggest that NAA acts on the G protein-coupled mGluRs to induce an inward current that results in excitation of the neurons, thereby contributing to the occurrence of epileptic seizures.