Intracellular Injection Of Chloride Ions Research Articles

Postsynaptic inhibition of hypoglossal motoneurons produces atonia of the genioglossal muscle during rapid eye movement sleep.

Hypoglossal motoneurons were recorded intracellularly to determine whether postsynaptic inhibition or disfacilitation was responsible for atonia of the lingual muscles during rapid eye movement (REM) sleep. Intracellular records were obtained of the action potentials and subthreshold membrane potential activity of antidromically identified hypoglossal motoneurons in cats during wakefulness, nonrapid eye movement (NREM) sleep, and REM sleep. A cuff electrode was placed around the hypoglossal nerve to antidromically activate hypoglossal motoneurons. The state-dependent changes in membrane potential, spontaneous discharge, postsynaptic potentials, and rheobase of hypoglossal motoneurons were determined. During quiet wakefulness and NREM sleep, hypoglossal motoneurons exhibited spontaneous repetitive discharge. In the transition from NREM sleep to REM sleep, repetitive discharge ceased and the membrane potential began to hyperpolarize; maximal hyperpolarization (10.5 mV) persisted throughout REM sleep. During REM sleep there was a significant increase in rheobase, which was accompanied by barrages of large-amplitude inhibitory postsynaptic potentials (IPSPs), which were reversed following the intracellular injection of chloride ions. The latter result indicates that they were mediated by glycine; IPSPs were not present during wakefulness or NREM sleep. We conclude that hypoglossal motoneurons are postsynaptically inhibited during naturally occurring REM sleep; no evidence of disfacilitation was observed. The data also indicate that glycine receptor-mediated postsynaptic inhibition of hypoglossal motoneurons is crucial in promoting atonia of the lingual muscles during REM sleep.

State-dependent phenomena in cat masseter motoneurons

In the present study we explored the mechanisms of carbachol-induced muscle atonia in the α-chloralose-anesthetized animal. We compared our findings to those that have been previously obtained in unanesthetized cats during muscle atonia occurring during natural active sleep. Accordingly, in cats anesthetized with a-chloralose, intracellular records were obtained from masseter motoneurons before and after carbachol-induced motor atonia. Following the induction of atonia, the membrane potential activity was dominated by high-frequency, discrete, hyperpolarizing potentials. These hyperpolarizing potentials were reversed in polarity by the intracellular injection of chloride ions and abolished by the application of strychnine. These findings indicate that they were inhibitory postsynaptic potentials (IPSPs) mediated by glycine. These IPSPs appeared exclusively during muscle atonia. In addition, masseter motoneurons were significantly hyperpolarized and their rheobase increased. There was a decrease in input resistance and membrane time constant. In the α-chloralose-anesthetized preparation, stimulation of the nucleus pontis oralis (NPO) induced IPSPs in masseter motoneurons following, but never prior to, the pontine injection of carbachol. Thus, this is the first demonstration that ‘reticular response-reversal’ may be elicited in an anesthetized preparation. Another state-dependent phenomenon of active sleep, the occurrence of IPSPs in motoneurons that are temporally correlated with ponto-geniculo-occipital (PGO) waves, was also observed in this preparation only after carbachol administration. Based on the data in this report, we conclude that the inhibitory system that mediates atonia during the state of active sleep can be activated in an animal that is anesthetized with a-chloralose. Specifically, the neuronal groups that generate spontaneous IPSPs, those that mediate the phenomenon of reticular response-reversal, and those involved in the generation of PGO waves are capable of being activated and remain functional during a-chloralose-anesthesia.

Activation of calcium-dependent chloride channels causes post-tetanic depolarization in rabbit parasympathetic neurons

Intracellular recordings were made from neurons in rabbit and feline vesical parasympathetic ganglia in vitro. In response to cathodal current injection (0.1–1 nA for 2–20 ms) the majority of rabbit neurons (229 out of 250) exhibited a single action potential that was followed by a fast and slow after-hyperpolarization (sAHP neuron). The remainder of the cells exhibited an action potential followed by only a fast after-hyperpolarization (fAHP neuron). fAHP neurons did not exhibit anomalous rectification and a spontaneous rhythmic hyperpolarization, which were common membrane properties in sAHP neurons. In response to a train of cathodal current pulses (5–20 Hz for 0.1–10 s), fAHP neurons exhibited action potentials followed by a post-tetanic depolarization (PTD). The PTD was associated with a decrease in membrane input resistance. The amplitude and duration of the PTD were a function of the number of action potentials in the train. The amplitude of the PTD was increased by membrane hyperpolarization and its estimated reversal potential was approximately −30 mV. Low-chloride solution and intracellular injection of chloride ions augmented the amplitude and duration of the PTD, whereas low-sodium, high-potassium and low-potassium solutions did not affect them. Tetraethylammonium (5–10 mM) and barium (0.5–1 mM) increased the amplitude and duration of the PTD. Nominal calcium-free solutions and ω-conotoxin (500 nM) abolished the PTD. The data suggest that activation of chloride channels by calcium influx through ω-conotoxin-sensitive calcium channels mediates the PTD. Repetitive stimulation of the pelvic nerve evoked a train of orthodromic action potentials followed by the PTD of fAHP neurons. (+)-Tubocurarine (10 μM) and hexamethonium (200 μM), but not atropine (1 μM), abolished orthodromic action potentials and the PTD, whereas these cholinergic antagonists did not depress the PTD evoked by direct action potentials. In summary, the data suggest that the PTD may function as a slow synaptic potential in fAHP neurons. This appears likely because neither slow excitatory nor inhibitory postsynaptic potentials are present in neurons of rabbit vesical parasympathetic ganglia. In contrast, slow inhibitory and excitatory postsynaptic potentials were recorded from neurons in feline vesical parasympathetic ganglia.

Intracellular study of generation mechanisms of induced wave in carp ( Cyprinus carpio) olfactory bulb

The membrane potential of carp mitral cells showed a rhythmic oscillation of 7–9 Hz which was composed of repeated cycles of depolarization and hyperpolarization during olfactory stimulation. This membrane potential oscillation was synchronous with, but in advance of, the induced wave by 16–47 msec on average. Intracellular injection of chloride ions from a KCl-filled micropipette reversed the polarity of the hyperpolarizing potentials to the depolarizing direction. These results suggest that the induced wave in the teleost olfactory bulb reflects rhythmic synaptic depolarization of the peripheral dendrites of the granule cells, which is generated by the reciprocal dendrodendritic interaction between the mitral cells and the granule cells.

Phase-dependent Modulation of a Cutaneous Sensory Pathway by Glycinergic Inhibition from the Locomotor Rhythm Generator in Xenopus Embryos.

In immobilized Xenopus laevis embryos two classes of sensory interneuron are excited by mechanosensory Rohon - Beard neurons and rhythmically inhibited during fictive swimming. Dorsolateral commissural (DLC) interneurons are inhibited in time with rhythmic motor discharge on the same side as their soma, while unidentified dorsolateral (DLX) interneurons are inhibited in the opposite phase of the swimming rhythm. The inhibition is abolished by bath application of strychnine sulphate at 1 - 10 microM, but not by the gamma-aminobutyric acid antagonists bicuculline (20 - 40 microM) or curare (70 - 100 microM). The inhibitory postsynaptic potentials (IPSPs) involve an increase in chloride conductance since they are reversed in sign to become depolarizing following intracellular injection of chloride ions. The conductance increase during inhibition was able to block impulses evoked by intracellular current in a phase-dependent manner, suggesting that postsynaptic inhibition is sufficient to account for the gating of afferent input to the spinal cord during swimming. An interneuron receives IPSPs that are predominantly in one phase of the rhythm, but most interneurons are also inhibited sporadically in the opposite phase. The amplitude and time course of the IPSPs closely follow the frequency of the swimming rhythm, with maximal inhibition occurring near the starts of episodes, when swimming frequency is at its highest. Towards the end of an episode, when swimming frequency declines, the level of inhibition is low, the membrane potential of the interneurons returns to rest between cycles, and IPSPs often fail to occur. Inhibition suppresses sensory excitation in a phase-dependent manner (cf. Sillar and Roberts, Nature, 331, 262 - 265, 1988). Sensory interneurons fire a single impulse in response to a brief sensory stimulus, but they will usually fire multiple impulses when depolarized with sufficient intracellular current. In some sensory interneurons a short-latency IPSP follows the impulse evoked by skin stimulation that could curtail impulse activity. However, when the inhibition is blocked by strychnine, sensory interneurons still fire a single short-latency impulse, favouring the conclusion that brief, synchronized afferent excitation elicits a single impulse in neurons that are capable of firing multiply. Since the inhibition of DLC interneurons occurs in phase with activity on the same side it probably originates from spiking in ipsilateral glycinergic commissural interneurons which have ipsilateral as well as contralateral projections. The inhibition of DLX interneurons in the opposite phase probably originates from the contralateral projections of commissural interneurons.

Diazepam potentiates postsynaptic inhibition in bulbar respiratory neurons of cats

The purpose of this study was to assess the effect of benzodiazepine on inhibitory postsynaptic potentials (IPSPs) of medullary respiratory neurons in decerebrate, paralyzed cats. Diazepam (0.05 and 0.1 mg/kg i.v.) reversibly increased the IPSP waves occurring during the inactive phase of the respiratory cycle in all inspiratory and postinspiratory neurons examined. Input resistances of these neurons were reduced at that phase. The reversal potential for the IPSP waves was unaltered. Intracellular injection of chloride ions reversed the IPSP to depolarization, and diazepam produced a purely depolarizing effect. The drug effects observed during the active phase of each neuron include a decrease in the firing rate and a shortening of the burst activity. The firing threshold and shape of these spikes, however, remained unaltered. These results suggest that diazepam depresses the bulbar respiratory neuronal activities specifically by potentiating the periodic postsynaptic inhibition.

Evidence of rhythmic inhibitory synaptic influences in hindlimb motoneurons during fictive locomotion in the thalamic cat.

Intracellular recordings of various motoneurons of proximal hindlimb muscles were performed on thalamic paralyzed cats, during fictive locomotion that was either spontaneous or evoked by stimulation of the subthalamic region. In motoneurons innervating sartorius (medialis and lateralis), vasti (intermedius, medialis and lateralis) and anterior biceps-semimembranous, one depolarization occurred in each locomotor cycle, alternating with a phase of repolarization that was synchronous with the activation of the antagonistic muscle nerve. This latter phase could be decreased or reversed by intracellular injection of chloride ions or current, revealing the presence of inhibitory inputs onto motoneurons. The pattern of membrane potential variations was more complex in motoneurons of rectus femoris and posterior biceps-semitendinosus muscles, but phases of chloride dependent inhibition were nevertheless identified, mainly during the sartorius nerve activation in the case of rectus femoris, and during the vasti and anterior biceps-semimembranosus nerve activations in the case of posterior biceps-semitendinosus. These inhibitory influences were shown to be controlled by the level of activity in exteroceptive afferents. The characteristics of the excitatory and inhibitory inputs to the hindlimb motoneurons identified here are discussed in relation with the organization of the central pattern generator for locomotion.

Effects of intracellular injection of chloride ions on inhibitory postsynaptic potentials and postburst hyperpolarization in cat sensorimotor cortex neurons

It was shown during acute experiments on cats immobilized with myorelaxants that intracellular injection of chloride ions into both pyramidal and non-pyramidal neurons of the sensorimotor cortex produces the early component only of IPSP, while the late phase and postburst hyperpolarization of pyramidal neurons are not very sensitive to this effect. It is deduced that membrane permeability to chloride ions increases during the early component of IPSP in pyramidal and nonpyramidal neurons of the cat sensorimotor cortex, while the late phase and postburst hyperpolarization is less dependent on chloride permeability.

Disfacilitation and long-lasting inhibition of neostriatal neurons in the rat.

Excitatory postsynaptic potentials evoked in rat neostriatal spiny projections neurons were followed by a long (100-300 ms) period of membrane hyperpolarization, followed in turn by a late depolarization. Concomitant with these changes in membrane potential were inhibition and subsequent excitation of spontaneous firing and excitatory activity evoked from substantia nigra and cerebral peduncle, but not from cortical stimulating sites. Thalamic-evoked excitatory activity was sometimes sensitive and sometimes insensitive to this inhibition, which has previously been believed to result from intrinsic inhibitory synaptic activity among neostriatal neurons. In intracellular recordings from neostriatal neurons in urethane anesthetized rats this long-lasting inhibitory response (1) exhibited alterations with intracellularly applied steady currents comparable to those of the EPSP, (2) failed to respond to intracellular injection of chloride ions, (3) was associated with either a decrease or no detectable change in the input conductance of the neurons, and (4) was abolished after lesions that interrupted polysynaptic pathways to neostriatum through intracortical and intrathalamic synaptic circuits. These findings indicate that the long lasting inhibitory portion of the responses of neostriatal neurons arises from a phasic inhibition of tonically active corticostriatal and thalamostriatal neurons and a concurrent decrease in the excitability of polysynaptic pathways converging on neostriatal neurons.

On the control of myotomal motoneurones during "fictive swimming" in the lamprey spinal cord in vitro.

Intracellular recordings have been made from myotomal motoneurones during "fictive swimming" in the in vitro preparation of the lamprey spinal cord, while monitoring the efferent burst activity in the ventral roots. The pattern of rhythmic activity in the motoneurones is described, as well as how synaptic inputs from the premotoneuronal level exert their control of motoneurone activity. (1) All motoneurones investigated displayed rhythmic, symmetric oscillations of their membrane potential during "fictive swimming". The period of depolarization occurred in phase with the burst discharge in the ventral root containing the motoneurone axon. (2) About one-third of the cells fired bursts of action potentials during the depolarized phase, while the remaining motoneurones exhibited subthreshold oscillations. (3) Intracellular injection of chloride ions reversed the sign of the hyperpolarized phase, demonstrating phasic active inhibition of the motoneurones during rhythmicity. (4) The depolarized phase was unaffected after chloride injection, showing that the motoneurones also received phasic active excitation. (5) "Pre-triggered" averaging of the motoneurone recording (using the ventral root spikes from other motoneurones for triggering), revealed that some degree of synchronous excitation of several motoneurones occurred, suggesting common excitation from the same premotor-interneurones. It is concluded that the rhythmic oscillations of membrane potential in lamprey myotomal motoneurones during "fictive locomotion" depend on phasic excitation alternating with phasic active inhibition. The premotoneuronal mechanism responsible for this control may consist of reciprocally organized groups of excitatory and inhibitory interneurones.

Repetitive synaptic potentials responsible for inhibition of spinal cord motoneurons during active sleep

High-gain intracellular recordings of spinal cord motoneurons were obtained in the chronically prepared cat during wakefulness, quiet sleep, and active sleep. Discrete hyperpolarizing postsynaptic potentials were found to occur repetitively throughout active sleep in conjunction with the presence of somatomotor atonia. These potentials were most prominent in the transition period from quiet to active sleep as well as during episodes of rapid eye movements during active sleep. Their polarity was reversed after the intracellular injection of chloride ions. We propose that these are inhibitory potentials which are responsible for or contribute significantly to the postsynaptic inhibition of spinal cord motoneurons during active sleep.

Substance P: ionic basis for depolarizing responses of mouse spinal cord neurons in cell culture

Intracellular recording methods were used to investigate the ionic basis for the postsynaptic actions of substance P (SP) on mouse spinal cord neurons grown in primary dissociated cell culture. SP and an analog, eledoisin-related peptide (ERP), were applied to single neurons by pressure ejection of peptide-containing solutions from blunt (2- to 10-micrometers) glass micropipettes. SP and ERP had similar excitatory actions, increasing spontaneous activity and depolarizing neurons by decreasing membrane conductance. Depolarizing responses were not inverted by intracellular injection of chloride ions, suggesting that SP responses did not result from decreased chloride conductance. SP and ERP responses were not abolished by extracellular tetraethylammonium ions (TEA+) but were reduced or eliminated by intracellular TEA+, suggesting that SP reduced a potassium conductance (gK). Finally, SP and ERP responses were larger when neurons were depolarized and smaller when the cells were hyperpolarized, and extrapolated reversal potentials for the peptide responses were 10 to 30 mV more negative than resting membrane potential. Thus, it was concluded that SP depolarized spinal cord neurons by decreasing a membrane potassium conductance. However, SP and ERP response polarity was not clearly reversed even at membrane potentials more negative than the expected potassium equilibrium potential. Moreover, extrapolated reversal potentials (RPeS) of SP responses varied linearly with the logarithm of extracellular potassium concentration ([K+]o) as predicted by the Nernst equation for potassium in [K+]o concentrations of 10, 15, 20, and 40 mM but were more depolarized than predicted for [K+]o concentrations of 1 and 5 mM. Since reduction of extracellular sodium by choline substitution did not alter the deviation from the Nernst equation for potassium, it was concluded that SP decreased a voltage-dependent potassium conductance which was absent at very negative potentials, present at resting membrane potential, and activated by membrane depolarization. Thus, SP decreases a conductance which appears similar to the muscarine-sensitive potassium conductance in sympathetic ganglion neurons (Brown, D. A., and P. R. Adams (1980) Nature 283: 673-676).

Effects of physostigmine on the inhibition of trigeminal motoneurones by cutaneous impulses in the cat.

Physostigmine (less than 0.3 mg/kg, i. v.) was administered systemically to cats, and the effect studied on the electrical activities of trigeminal motoneurones. Physostigmine depressed the monosynaptic reflex evoked by stimulation of the mesencephalic trigeminal tract and recorded from the masseter nerve, and potentiated the inhibition of this monosynaptic reflex by volleys in skin nerves. This effect began within 1–2 min after the injection of physostigmine, reached a maximum at 5–12 min and thereafter decayed gradually. Skin nerve stimulation induced inhibitory postsynaptic potentials (IPSPs) in trigeminal motoneurones. After physostigmine administration these IPSPs were increased in amplitude, and were accompanied by a prolonged, late hyperpolarization. The early phase of this IPSP and the late hyperpolarization showed different sensitivities to the intracellular injection of chloride ions and to polarizing currents, indicating different mechanisms for their production. The effect of physostigmine was blocked by atropine (1 mg/kg, i.v.) but not by dihydro-β-erythroidine (0.5 mg/kg, i.v.).