Increase In Cell Input Resistance Research Articles

Electrically Silent Divalent Cation Entries in Resting and Active Voltage-Controlled Muscle Fibers

Ca 2+ is known to enter skeletal muscle at rest and during activity. Except for the well-characterized Ca 2+ entry through L-type channels, pathways involved in these Ca 2+ entries remain elusive in adult muscle. This study investigates Ca 2+ influx at rest and during activity using the method of Mn 2+ quenching of fura-2 fluorescence on voltage-controlled adult skeletal muscle cells. Resting rate of Mn 2+ influx depended on external [Mn 2+] and membrane potential. At −80 mV, replacement of Mg 2+ by Mn 2+ gave rise to an outward current associated with an increase in cell input resistance. Calibration of fura-2 response indicated that Mn 2+ influx was too small to be resolved as a macroscopic current. Partial depletion of the sarcoplasmic reticulum induced by a train of action potentials in the presence of cyclopiazonic acid led to a slight increase in resting Mn 2+ influx but no change in cell input resistance and membrane potential. Trains of action potentials considerably increased Mn 2+ entry through an electrically silent pathway independent of L-type channels, which provided 24% of the global Mn 2+ influx at +30 mV under voltage-clamp conditions. Within this context, the nature and the physiological role of the Ca 2+ pathways involved during muscle excitation still remain open questions.

Involvement of TRP-like channels in the acute ischemic response of hippocampal CA1 neurons in brain slices

During a period of acute ischemia in vivo or oxygen–glucose deprivation (OGD) in vitro, CA1 neurons depolarize, swell and become overloaded with calcium. Our aim was to test the hypothesis that the initial responses to OGD are at least partly due to transient receptor potential (TRP) channel activation. As some TRP channels are temperature-sensitive, we also compared the effects of pharmacological blockade of the channels with the effects of reducing temperature. Acute hippocampal slices (350 μm) obtained from Wistar rats were submerged in ACSF at 36 °C. CA1 neurons were monitored electrophysiologically using extracellular, intracellular or whole-cell patch-clamp recordings. Cell swelling was assessed by recording changes in relative tissue resistance, and changes in intracellular calcium were measured after loading neurons with fura-2 dextran. Blockers of TRP channels (ruthenium red, La 3+, Gd 3+, 2-APB) or lowering temperature by 3 °C reduced responses to OGD. This included: (a) an increased delay to negative shifts of extracellular DC potential; (b) reduction in rate of the initial slow membrane depolarization, slower development of OGD-induced increase in cell input resistance and slower development of whole-cell inward current; (c) reduced tissue swelling; and (d) a smaller rise in intracellular calcium. Mild hypothermia (33 °C) and La 3+ or Gd 3+ (100 μM) showed an occlusion effect when delay to extracellular DC shifts was measured. Expression of TRPM2/TRPM7 (oxidative stress-sensitive) and TRPV3/TRPV4 (temperature-sensitive) channels was demonstrated in the CA1 subfield with RT-PCR. These results indicate that TRP or TRP-like channels are activated by cellular stress and contribute to ischemia-induced membrane depolarization, intracellular calcium accumulation and cell swelling. We also hypothesize that closing of some TRP channels (TRPV3 and/or TRPV4) by lowering temperature may be partly responsible for the neuroprotective effect of hypothermia.

Temperature Sensitivity of Dopaminergic Neurons of the Substantia Nigra Pars Compacta: Involvement of Transient Receptor Potential Channels

Changes in temperature of up to several degrees have been reported in different brain regions during various behaviors or in response to environmental stimuli. We investigated temperature sensitivity of dopaminergic neurons of the rat substantia nigra pars compacta (SNc), an area important for motor and emotional control, using a combination of electrophysiological techniques, microfluorometry, and RT-PCR in brain slices. Spontaneous neuron firing, cell membrane potential/currents, and intracellular Ca2+ level ([Ca2+]i) were measured during cooling by < or =10 degrees and warming by < or =5 degrees from 34 degrees C. Cooling evoked slowing of firing, cell membrane hyperpolarization, increase in cell input resistance, an outward current under voltage clamp, and a decrease of [Ca2+]i. Warming induced an increase in firing frequency, a decrease in input resistance, an inward current, and a rise in [Ca2+]i. The cooling-induced current, which reversed in polarity between -5 and -17 mV, was dependent on extracellular Na+. Cooling-induced whole cell currents and changes in [Ca2+]i were attenuated by 79% in the presence of 2-aminoethoxydiphenylborane (2-APB; 200 microM), and the outward current was reduced by 20% with ruthenium red (100 microM). RT-PCR conducted with tissue punches containing the SNc revealed mRNA expression for TRPV3 and TRPV4 channels, known to be activated in expression systems by temperature changes within the physiological range. 2-APB, a TRPV3 modulator, increased baseline [Ca2+]i, whereas 4alphaPDD, a TRPV4 agonist, increased spontaneous firing in 7 of 14 neurons tested. We conclude that temperature-gated TRPV3 and TRPV4 cationic channels are expressed in nigral dopaminergic neurons and are constitutively active in brain slices at near physiological temperatures, where they affect the excitability and calcium homeostasis of these neurons.

ATP inhibits the hypoxia response in type I cells of rat carotid bodies

Summary During hypoxia, ATP was released from type I (glomus) cells in the carotid bodies. We studied the action of ATP on the intracellular Ca(2+) concentration ([Ca(2+)](i)) of type I cells dissociated from rat carotid bodies using a Ca(2+) imaging technique. ATP did not affect the resting [Ca(2+)](i) but strongly suppressed the hypoxia-induced [Ca(2+)](i) elevations in type I cells. The order of purinoreceptor agonist potency in inhibiting the hypoxia response was 2-methylthioATP > ATP > ADP >> alpha, beta-methylene ATP > UTP, implicating the involvement of P2Y(1) receptors. Simultaneous measurements of membrane potential and [Ca(2+)](i) show that ATP inhibited the hypoxia-induced Ca(2+) signal by reversing the hypoxia-triggered depolarization. However, ATP did not oppose the hypoxia-mediated inhibition of the oxygen-sensitive TASK-like K(+) background current. Neither the inhibition of the large-conductance Ca(2+)-activated K(+) (maxi-K) channels nor the removal of extracellular Na(+) could affect the inhibitory action of ATP. Under normoxic condition, ATP caused hyperpolarization and increase in cell input resistance. These results suggest that the inhibitory action of ATP is mediated via the closure of background conductance(s) other than the TASK-like K(+), maxi-K or Na(+) channels. In summary, ATP exerts strong negative feedback regulation on hypoxia signaling in rat carotid type I cells.

The modulation by 5-HT of glutamatergic inputs from the raphe pallidus to rat hypoglossal motoneurones, in vitro.

Decreases in the activity of 5-HT-containing caudal raphe neurones during sleep are thought to be partially responsible for the resultant disfacilitation of hypoglossal motoneurones. Whilst 5-HT has a direct excitatory action on hypoglossal motoneurones as a result of activation of 5-HT2 receptors, microinjection of 5-HT2 antagonists into the hypoglossal nucleus reduces motor activity to a much lesser extent compared to the suppression observed during sleep suggesting other transmitters co-localised in caudal raphe neurones may also be involved. The aim of the present study was therefore to characterise raphe pallidus inputs to hypoglossal motoneurones. Whole cell recordings were made from hypoglossal motoneurones in vitro. 5-HT evoked a direct membrane depolarisation (8.45 +/- 3.8 mV, P < 0.001) and increase in cell input resistance (53 +/- 40 %, P < 0.001) which was blocked by the 5-HT2 antagonist, ritanserin (2.40 +/- 2.7 vs. 7.04 +/- 4.6 mV). Stimulation within the raphe pallidus evoked a monosynaptic EPSC that was significantly reduced by the AMPA/kainate antagonist, NBQX (22.8 +/- 16 % of control, P < 0.001). In contrast, the 5-HT2 antagonist, ritanserin, had no effect on the amplitude of these EPSCs (106 +/- 31 % of control, P = n.s.). 5-HT reduced these EPSCs to 50.0 +/- 13 % of control (P < 0.001), as did the 5-HT1A agonist, 8-OH-DPAT (52.5 +/- 17 %, P < 0.001) and the 5-HT1B agonist, CP 93129 (40.6 +/- 29 %, P < 0.01). 8-OH-DPAT and CP 93129 increased the paired pulse ratio (1.38 +/- 0.27 to 1.91 +/- 0.54, P < 0.05 & 1.27 +/- 0.08 to 1.44 +/- 0.13, P < 0.01 respectively) but had no effect on the postsynaptic glutamate response (99 +/- 4.4 % and 100 +/- 2.5 %, P = n.s.). They also increased the frequency (P < 0.001), but not the amplitude, of miniature glutamatergic EPSCs in hypoglossal motoneurones. These data demonstrate that raphe pallidus inputs to hypoglossal motoneurones are predominantly glutamatergic in nature, with 5-HT decreasing the release of glutamate from these projections as a result of activation of 5-HT1A and/or 5-HT1B receptors located on presynaptic terminals.

Opioid receptor regulation of muscarinic acetylcholine receptor-mediated synaptic responses in the hippocampus

A common feature of many synapses is their regulation by neurotransmitters other than those released from the presynaptic terminal. This aspect of synaptic transmission is often mediated by activation of G protein coupled receptors (GPCRs) and has been most extensively studied at amino acid-mediated synapses where ligand gated receptors mediate the postsynaptic signal. Here we have investigated how opioid receptors modulate synaptic transmission mediated by muscarinic acetylcholine receptors (mAChRs) in hippocampal CA1 pyramidal neurones. Using a cocktail of glutamate and γ-amino-butyric acid (GABA) receptor antagonists a slow pirenzepine-sensitive excitatory postsynaptic potential (EPSP M) that was associated with a small increase in cell input resistance could be evoked in isolation. This response was enhanced by the acetylcholine (ACh) esterase inhibitor physostigmine (1 μM) and depressed by the vesicular ACh transport inhibitor vesamicol (50 μM). The μ-opioid receptor agonists DAMGO (1–5 μM) and etonitazene (100 nM), but not the δ- and κ-opioid receptor selective agonists DTLET (1 μM) and U-50488 (1 μM), potentiated this EPSP M (up to 327%) without affecting cell membrane potential or input resistance; an effect that was totally reversed by naloxone (5 μM). In contrast, postsynaptic depolarizations and increases in cell input resistance evoked by carbachol (3 μM) were unaffected by DAMGO (1–5 μM) but were abolished by atropine (1 μM). Taken together these data provide good evidence for a μ-opioid receptor-mediated presynaptic enhancement of mAChR-mediated EPSPs in hippocampal CA1 pyramidal neurones.

Adrenergic responses in silent and putative inhibitory pacemaker-like neurons of the rat rostral ventrolateral medulla in vitro

Noradrenaline and adrenergic agonists were tested on pacemaker-like and silent neurons of the rat rostral ventrolateral medulla using intracellular recordings in coronal brainstem slices as well as in punches containing only the rostral ventrolateral medullary region. Noradrenaline (1–100 μM) depolarized or increased the frequency of discharge of all cells tested in a dose-dependent manner. The noradrenaline-induced depolarization was associated with an apparent increase in cell input resistance at low concentrations and a decrease or no significant change at higher concentrations. Moreover, it was voltage dependent and its amplitude decreased with membrane potential hyperpolarization. Noradrenaline caused a dose-related increase in the frequency and amplitude of spontaneous inhibitory postsynaptic potentials. The α1-adrenoceptor antagonist prazosin (0.5 μM) abolished the noradrenaline depolarizing response as well as the noradrenaline-evoked increase in synaptic activity and unmasked an underlying noradrenaline dose-dependent hyperpolarizing response associated with a decrease in cell input resistance and sensitive to the α2-adrenoceptor antagonist yohimbine (0.5 μM). The α1-adrenoceptor agonist phenylephrine (10 μM) mimicked the noradrenaline depolarizing response associated with an increase in membrane resistance as well as the noradrenaline-induced increase in synaptic activity. The α2-adrenoceptor agonists UK-14,304 (1–3 μM) and clonidine (10–30 μM) produced only a small hyperpolarizing response, whereas the β-adrenoceptor agonist isoproterenol (10–30 μM) had no effect. Baseline spontaneous postsynaptic potentials were abolished by strychnine (1 μM), bicuculline (30 μM) or both. However, only the strychnine-sensitive postsynaptic potentials had their frequency increased by noradrenaline or phenylephrine and they usually occurred with a regular pattern. Tetrodotoxin (1 μM) eliminated 80–95% of baseline spontaneous postsynaptic potentials and prevented the increase in synaptic activity evoked by noradrenaline and phenylephrine. Similar results were obtained in rostral ventrolateral medulla neurons impaled in both coronal slices and punches of the rostral ventrolateral medulla. It is concluded that noradrenaline could play an important inhibitory role in the rostral ventrolateral medulla via at least two mechanisms: an α2-adrenoceptor-mediated hyperpolarization and an enhancement of inhibitory synaptic transmission through activation of α1-adrenoceptors located on the somatic membrane of glycinergic interneurons. Some of these interneurons exhibit a regular discharge similar to the pacemaker-like neurons and might, at least in part, constitute a central inhibitory link in the baroreceptor–vasomotor reflex pathway.

A background sodium conductance is necessary for spontaneous depolarizations in rat pituitary cell line GH3.

The role of Na+ in the expression of membrane potential activity in the clonal rat pituitary cell line GH3 was investigated using the perforated patch variation of patch-clamp electrophysiological techniques. It was found that replacing bath Na+ with choline, tris(hydroxymethyl)aminomethane (Tris), or N-methyl-D-glucamine (NMG) caused the cells to hyperpolarize 20-30 mV. Tetrodotoxin had no effect. The effects of the Na+ substitutes could not be explained by effects on potassium or calcium currents. Although all three Na+ substitutes suppressed voltage-dependent calcium current by 10-20%, block of voltage-dependent calcium current by nifedipine or Co2+ did not result in hyperpolarization of the cells. There was no effect of the Na+ substitutes on voltage-dependent potassium currents. In contrast, all three Na+ substitutes influenced calcium-activated potassium currents [IK(Ca)], but only at depolarized potentials. Choline consistently suppressed IK(Ca), whereas Tris and NMG either had no effect or slightly increased IK(Ca). These effects on IK(Ca) also cannot explain the hyperpolarization induced by removing bath Na+. Choline always hyperpolarized cells yet suppressed IK(Ca). Furthermore, removing bath Na+ caused an increase in cell input resistance, an observation consistent with the loss of a membrane conductance as the basis of the hyperpolarization. Direct measurement of background currents revealed a 12-pA inward current at -84 mV that was lost upon removing bath Na+. These results suggest that this background sodium conductance provides the depolarizing drive for GH3 cells to reach the threshold for firing calcium-dependent action potentials.

CCK A receptors mediate slow depolarizations in cultured mammalian sympathetic neurons

The effect of cholecystokinin octapeptide (CCK-8) was examined in guinea-pig celiac ganglion (CG) neurons in primary culture using standard intracellular recording techniques. Sulfated CCK-8 (CCK-8S; 1 μM) evoked slow depolarizing responses in 94% of CG neurons tested. In contrast, membrane potential was not affected by nonsulfated CCK-8 (CCK-8NS; 1 μM), CCK tetrapeptide (CCK-4; 1 μM), or gastrin (1 μM). The selective CCK A receptor antagonist L 364,718 potently inhibited CCK-8S-induced slow depolarizations(IC 50 2.9 pM). In contrast, the selective CCK B receptor antagonist L 365,260 was a weak inhibitor of CCK-8S-induced slow depolarizations (IC 50 1.3 μM). The depolarizing responses to CCK-8S were associated with an average increase in cell input resistance of 61%. Single electrode voltage clamp experiments indicated that CCK-8S-induced depolarizations were associated with a slow inward shift in holding current. Thus, the present findings indicate that guinea-pig cultured CG neurons are endowed with excitatory CCK A receptors the activation of which elicits a decrease in membrane conductance, thereby resulting in slow depolarizations.

Pyramidal neurons in immature rat hippocampus are sensitive to β-adrenergic agents

The development of hippocampal neuronal sensitivities to the β-noradrenergic agent, isoproterenol, was examined in tissue from immature rats. The in vitro hippocampal slice preparation was used to assess intracellularly recorded responses from hippocampal neurons to pressure-pulse and bath application of noradrenergic drugs. Effects of the drug on individual hippocampal CA3 pyramidal neurons were compared across several stages of development, ranging from postnatal day 4–5 (P4–5) to maturity. Isoproterenol, pressure-pulse applied to CA3c pyramidal cells, produced a depolarization of membrane potential and an increase in cell input resistance in tissue as young as P7. Spike frequency adaptation (in trains of action potentials triggered by depolarizing pulses) was reduced, as were the slow after-hyperpolarizations following the spike trains. All agonist effects were blocked by timolol, a β-antagonist. Drug-induced changes in cell membrane and firing properties in immature tissue were qualitatively similar to β-receptor-mediated noradrenergic effects in adult tissue. These results indicate that the β-receptor-mediated component of the noradrenergic effect in rat hippocampus is physiologically functional by the seventh day of postnatal life; at earlier times (P4–5) these β-receptor-mediated noradrenergic actions are, at best, equivocal.

Effect of glutamate, aspartate and related derivatives on cerebellar purkinje cell dendrites in the rat: an in vitro study.

1. The responses of Purkinje cells to short duration (pulse) ionophoretic applications of L-aspartate (L-asp), L-glutamate (L-glu), N-methyl DL-aspartate (NMDLA) and quisqualic acid in their dendritic fields were studied in vitro on sagittal slices of lobules IX and X of the adult rat cerebellum.2. Pulse application of L-asp or L-glu evoked transient and dose-dependent increases in the firing rate of the simple spikes recorded extracellularly as single units. When the ionophoretic electrode was positioned in the dendritic field of the Purkinje cells, the lowest thresholds for L-glu and L-asp mediated excitations of the cells were as low as 25 and 35 pC respectively, with a latency for maximal responses as brief as 7 ms.3. In intracellular recordings these excitatory responses consisted of depolarizations of up to 18 mV in amplitude and with depolarizing slopes up to 0.52 mV/ms. They were generally unaccompanied by changes in cell input resistance in contrast to the marked decrease which occurred in response to steady applications of large doses of L-asp and L-glu.4. The spatial distribution of the excitatory sites confirmed that the dendritic sensitivity to L-glu was greater than that of the soma and showed that the same was true for L-asp. In 34% of cells the sensitivity for L-asp declined markedly in the upper region of the molecular layer, whereas it remained high for L-glu; no such differential sensitivity was detected in the remaining 66% of cells.5. Inhibitory responses, antagonized by 10(-5) M-bicuculline in the bath, were also induced in Purkinje cells by L-glu and L-asp when the ionophoretic electrode was withdrawn from the excitatory sites by as little as 8 mum and up to 40 mum upward or downward along the track of parallel fibres or positioned as far as 250 mum laterally.6. Whenever it was applied in the molecular layer, the pulse application of NMDLA elicited no excitatory response in Purkinje cells recorded extra or intracellularly. However, slow depolarizations accompanied by a slight increase in cell input resistance were obtained with steady applications of 20-50 nA of the drug for 20-30 s.7. In contrast, pulse application of quisqualic acid appeared to have the same type of fast excitatory effect on Purkinje cells as L-asp and L-glu, but its potency was greater and its action more prolonged. Furthermore, its steady application led to an abrupt and marked decrease in cell membrane resistance.8. The excitatory effects of L-asp, L-glu and quisqualic acid were antagonized by L-glutamic acid diethyl ester more consistently than by D-alpha-aminoadipate, suggesting together with previous observations that L-asp and L-glu act on Purkinje cells via quisqualic acid rather than via NMDLA receptors.

The effects of hydrogen ion on spinal neurons.

The effects of iontophoretically applied hydrogen ion (H+) on neuronal excitability were studied in the spinal cord of cats. The activity of extracellularly recorded neurons of the dorsal horn was either depressed or enhanced by H+ and in most cases of enhancement there was a preceding phase of depression. During intracellular recordings from motoneurons it was found that H+ application usually caused a hyperpolarization accompanied by an increase in cell input resistance. In a smaller number of cells the hyperpolarization was succeeded by a depolarization that was coupled with a decrease in input resistance. In some neurons it was shown that the depolarizing phase was more prominent and had a shorter latency when larger currents were used to eject H+. The varied reports from earlier studies of excitation or inhibition of neuronal excitability by H+ may be in part explainable by our observations that the effects may depend on the concentrations of H+ used.

The action of somatostatin on neurones of the myenteric plexus of the guinea-pig ileum.

1. Intracellular recordings were made from neurones in ganglia of the myenteric plexus of the guinea-pig ileum. 2. Somatostatin (10-300 nM) was applied to the neurones by adding it to the perfusing solution or by ejecting it (charges up to 500 nC) from an ionophoresis electrode onto the soma membrane. 3. By both methods of application, somatostatin either hyperpolarized or depolarized a proportion of neurones. Depolarizing responses were more often observed with ionophoretic application, and hyperpolarizing responses were more often observed with application by perfusion. Both responses were preserved in solutions containing zero Ca and elevated (6 mM) Mg. Some cells were both hyperpolarized and depolarized, depending on the method of administration. 4. The depolarizing responses to somatostatin were associated with an increase in cell input resistance; they became larger with membrane depolarization and smaller with membrane hyperpolarization, and reversed in polarity at a potential close to the potassium equilibrium potential. The hyperpolarizing responses to somatostatin were accompanied by a fall in cell input resistance.