Long-lasting Hyperpolarization Research Articles

Expression and biological significance of Ca2+-activated ion channels in human keratinocytes.

In whole-cell recordings from HaCaT keratinocytes, ATP, bradykinin, and histamine caused a biphasic change of the membrane potential consisting of an initial transient depolarization, followed by a pronounced and long-lasting hyperpolarization. Flash photolysis of caged IP3 mimicked the agonist-induced voltage response, suggesting that intracellular Ca2+ release and subsequent opening of Ca2+-activated ion channels serve as the common transduction mechanism. In contrast, cAMP- and PKC-dependent pathways were not involved in the electrophysiological effects of the extracellular signaling molecules. The depolarization was predominantly mediated by a DIDS- and niflumic acid-sensitive Cl- current, whereas a charybdotoxin- and clotrimazole-sensitive K+ current underlay the prominent hyperpolarization. Consistent with the electrophysiological data, RT-PCR showed that HaCaT keratinocytes express two types of Ca2+-activated Cl- channels, CaCC2 and CaCC3 (CLCA2), as well as the Ca2+-activated K+ channel hSK4. That the pronounced hSK4-mediated hyperpolarization bears significance on the growth and differentiation properties of keratinocytes is suggested by RNase protection assays showing that hSK4 mRNA expression is strongly down-regulated under conditions that allow keratinocyte differentiation. hSK4 might thus play a role in linking changes in membrane potential to the biological fate of keratinocytes.

Small-conductance calcium-activated potassium channels.

SK channels play a fundamental role in all excitable cells. SK channels are potassium selective and are activated by an increase in the level of intracellular calcium, such as occurs during an action potential. Their activation causes membrane hyperpolarization, which inhibits cell firing and limits the firing frequency of repetitive action potentials. The intracellular calcium increase evoked by action potential firing decays slowly, allowing SK channel activation to generate a long-lasting hyperpolarization termed the slow afterhyperpolarization (sAHP). This spike-frequency adaptation protects the cell from the deleterious effects of continuous tetanic activity and is essential for normal neurotransmission. Slow AHPs can be classified into two groups, based on sensitivity to the bee venom toxin apamin. In general, apamin-sensitive sAHPs activate rapidly following a single action potential and decay with a time constant of approximately 150 ms. In contrast, apamin-insensitive sAHPs rise slowly and decay with a time constant of approximately 1.5 s. The basis for this kinetic difference is not yet understood. Apamin-sensitive and apamin-insensitive SK channels have recently been cloned. This chapter will compare with different classes of sAHPs, discuss the cloned SK channels and how they are gated by calcium ions, describe the molecular basis for their different pharmacologies, and review the possible role of SK channels in several pathological conditions.

Effects of interleukin-1 beta on neurons in mammalian pelvic ganglia.

The effects of interleukin-1 beta (IL-1 beta) on the membrane potential and synaptic transmission were examined in neurons of mammalian pelvic ganglia. Bath-application of recombinant human IL-1 beta (6-300 pM) for 10 s-5 min produced a long-lasting hyperpolarization associated with increased input resistance in 11 neurons of rat major pelvic ganglia (MPG). In other 8 neurons, IL-1 beta (300 pM) produced a biphasic response that consists of an initial depolarization followed by a long-lasting hyperpolarization. IL-1 beta 163-171 (10-100 pM), a synthetic nonapeptide analog that contains the active domain of human IL-1 beta, mimicked the effect of IL-1 beta in MPG neurons. gamma-Aminobutyric acid (GABA, 300 microM) produced a depolarization followed by a hyperpolarization that was blocked by picrotoxin (100 microM). Db-cyclic guanosine monophosphate (db-cyclic GMP, 100 microM) also produced an initial depolarization followed by a long-lasting hyperpolarization. These results suggest that the IL-1 beta-induced biphasic response is mediated by a GABA receptor-cyclic GMP pathway. IL-1 beta and IL-1 beta 163-171 caused an initial facilitation followed by a long-lasting depression of the excitatory postsynaptic potential (EPSP) in rabbit VPG. The data suggest that IL-1 beta presynaptically depressed the EPSP by reducing the release of acetylcholine (ACh) from the pelvic nerve terminals.

PGE2 hyperpolarizes gallbladder neurons and inhibits synaptic potentials in gallbladder ganglia.

Gallbladder prostaglandin E2 (PGE2) levels are significantly elevated in pathophysiological conditions, resulting in changes in gallbladder motility or secretion that may involve actions of the prostanoid in intramural ganglia. This study was undertaken to examine the effects of PGE2 on neurons of the intramural ganglia of the guinea pig gallbladder. Application of PGE2 by microejection or superfusion elicited a complex triphasic change in the resting membrane potential (RMP). For example, application of PGE2 by microejection (100 microM) resulted in a brief hyperpolarization (mean duration 11.1 +/- 1.3 s), followed by a mid-phase repolarization toward or above RMP (mean duration 50.7 +/- 8.1 s), and finally a long-lasting hyperpolarization (mean duration 157.3 +/- 36.7 s). Associated with these PGE2-evoked alterations in RMP were changes in input resistance measured via injection of hyperpolarizing current pulses. An examination of the action potential afterhyperpolarization (AHP) during the PGE2-evoked response revealed an attenuation of both the amplitude and duration of the AHP. However, only a slight increase in excitability of gallbladder neurons in the presence of PGE2 was evident in response to depolarizing current pulses, and PGE2 did not cause the cells to fire spontaneous action potentials. Application of PGE2 reduced the amplitudes of both fast and slow excitatory synaptic potentials. These results suggest that increased prostaglandin production may decrease ganglionic output and therefore contribute to gallbladder stasis.

Superoxide Release is Involved in Membrane Potential Changes in Mouse Peritoneal Macrophages

Participation of reactive oxygen species (ROS) in the changes in macrophage membrane potential resulted from effects of different agonists has been studied. Treatment of macrophages with chemotactic peptide fMLP or platelet-activating factor (PAF) caused a brief depolarization followed by a long-lasting hyperpolarization. Lipopolysaccharide and interferon-γ only depolarized the plasma membrane. Chemiluminescence measurements indicated that only fMLP and PAF activated macrophages to release ROS. The hyperpolarization response of the cell was significantly decreased in the presence of superoxide dismutase (but not catalase). Moreover, the O 2 ̇ − -generating system, xanthine plus xanthine oxidase, caused a marked hyperpolarization. In all the cases, the hyperpolarization induced by fMLP, PAF and O 2 ̇ − -generating system was found to depend on the concentration of intracellular Ca 2+ and extracellular K +. Furthermore, in the presence of quinidine, a blocker of Ca 2+-dependent K + conductance fMLP and PAF caused only prolonged depolarization while the effect of O 2 ̇ − was reduced to a minimum. These data suggest that the macrophage hyperpolarization response to fMLP and PAF involves superoxide-mediated Ca 2+-dependent alteration of the relative membrane permeability to K +.

Evidence for cholinergic inhibitory and serotonergic excitatory neuromuscular transmission in the heart of the bivalve Mercenaria mercenaria

The heart of Mercenaria mercenaria is innervated bilaterally at the atria. A pair of cardiac nerves arise as a branch of the cerebro-visceral connective and run to the posterior end of the junction between each atrium and its efferent branchial vessel. Innervation evidently spreads over the heart, since both inhibitory and excitatory junctional potentials (IJPs and EJPs) can be recorded from the atria, the atrio-ventricular (AV) valve or the ventricle. The cardiac nerves contain inhibitory and excitatory axons. Neural stimulation can cause increases in the frequency or amplitude of beating, depending on the strength and frequency of stimulation. Electrical stimulation of the nerves to the incurrent and excurrent siphons causes bradycardia or tachycardia even after cutting the cerebro-visceral connective at a site anterior to the origin of the cardiac nerves. This may indicate a reflex pathway involving neurons whose cell bodies are located in the visceral ganglion. Neural depression of myocardial action potentials is mediated by discrete IJPs, which follow nerve stimuli one-to-one. IJPs can be recorded from the atria, the AV valve or the ventricle. A long-lasting hyperpolarization follows cessation of excitatory stimulation of the cardiac nerve. IJPs may be produced by cholinergic nerves and are mediated primarily by Cl-. They are blocked by Mytolon and by d-tubocurarine (dTC), but not by methylxylocholine. In low-[Cl-] solution, IJPs are inverted into depolarizing junctional potentials, which are blocked by Mytolon and dTC. Neurally induced tachycardia is mediated by discrete EJPs, which also follow stimuli applied to the cardiac nerve in a one-to-one manner. EJPs can be recorded from the atria, the AV valve and the ventricle. The myocardium and the AV valve were excited by application of serotonin. EJPs recorded from these sites were reduced in amplitude by methysergide (1-methyl-d-lysergic acid butanolamide), suggesting that the EJPs may be serotonergic. Just after entering the heart, at the posterior end of the junction with the efferent branchial vessel, the cardiac nerves contain thick processes which show serotonin-like immunoreactivity. These processes spread along the ramifications of the nerves, which extend to the atrium, the AV valve, the ventricle and even into the wall of the aorta. This study provides direct electrophysiological evidence for serotonergic EJPs and cholinergic IJPs, plus immunocytochemical evidence for neural processes containing serotonin, in the myocardium.

Functional characterization of the H-current in SCN neurons in subjective day and night: a whole-cell patch-clamp study in acutely prepared brain slices.

Neurons of the rat suprachiasmatic nucleus (SCN) exhibit a circadian rhythm in spontaneous firing rate. In this whole-cell patch-clamp study in slices, we examined the possibility that H-current (IH) contributes to the spontaneous firing rate of SCN neurons. Most of our experiments were performed during the subjective day, because this is the time epoch during which one would expect the largest excitatory effect of IH if it were to fluctuate in a circadian rhythm. Current-clamp experiments showed that blockade of IH by Cs+ (1 mM) did not influence the spontaneous firing rate and resting membrane potential. Voltage-clamp experiments revealed that IH, when activated at the resting membrane potential, is probably too small in magnitude and too slow in activation to make a significant contribution to the spontaneous firing rate. Both results suggest that IH does not significantly contribute to the spontaneous firing of SCN neurons. In addition, we investigated whether the kinetics and voltage dependence of IH were modulated in a circadian manner. However, no substantial day-night differences in IH were found. We conclude that IH, as recorded in whole-cell mode, does not contribute significantly to spontaneous firing in most SCN neurons and that this current, is more likely to be involved in 'rescuing' SCN neurons from large and long-lasting hyperpolarizations by depolarizing the membrane.

Prolonged inhibitory potentials in layer III projection cells of the rat medial entorhinal cortex induced by synaptic stimulation in vitro

The entorhinal cortex projects via layer III neurons directly to the hippocampal area CA1 and the subiculum. We studied the functional properties of the medial entorhinal cortex projection cells in horizontal hippocampal-entorhinal cortex combined slices. These cells displayed, upon single-shock synaptic stimulation, an excitatory postsynaptic potential followed by a fast and/or slow inhibitory postsynaptic potential. Short train repetitive stimulation subthreshold for generation of action potentials induced a slow hyperpolarization of up to 20 s. Pharmacological analysis shows that the slow hyperpolarization could be divided into three components: i) the first component, which lasted 1 s, was sensitive to GABA B receptor antagonists; ii) the second component lasting for about 6 s was sensitive to atropine, suggesting muscarinic acetylcholinergic nature of this responses; iii) a late component lasting for up to 20 s was sensitive to naloxone, suggesting a role for opioids in its generation. The finding that layer III projection neurons to the hippocampus proper develop long-lasting hyperpolarizations suggests possible control mechanisms for the output functions of the entorhinal cortex.

Function of the hyperpolarization-activated inward rectification in nonmyelinated peripheral rat and human axons.

The function of time-dependent, hyperpolarization-activated inward rectification was analyzed on compound potentials of nonmyelinated axons in the mammalian peripheral nervous system. Isolated rat vagus nerves and fascicles of biopsied human sural nerve were tested in a three-chambered, Vaseline-gap organ bath at 37 degrees C. Inward rectification was assessed by recording the effects of long-lasting hyperpolarizing currents on electrical excitability with the use of the method of threshold electrotonus (program QTRAC, copyright Institute of Neurology, London, UK) and by measuring activity-dependent changes in conduction velocity and membrane potential. Prominent time-dependent, cesium-sensitive inward rectification was revealed in rat vagus and human sural nerve by recording threshold electrotonus to 200-ms hyperpolarizing current pulses. A slowing of compound action potential conduction was observed during a gradual increase in the stimulation frequency from 0.1 to 3 Hz. Above a stimulation frequency of 0.3 Hz, this slowing of conduction was enhanced during bath application of 1 mM cesium. Cesium did not alter action potential waveforms during stimulation at frequencies < 1 Hz. Cesium-induced slowing in action potential conduction was correlated with membrane hyperpolarization. The hyperpolarization by cesium was stronger during higher stimulation frequencies and small in unstimulated nerves. These data show that a cesium-sensitive, time-dependent inward rectification in peripheral rat and human nonmyelinated nerve fibers limits the slowing in conduction seen in such axons at action potential frequencies higher than approximately 0.3 Hz.

Intracellular Characterization of Song-Specific Neurons in the Zebra Finch Auditory Forebrain

Auditory neurons in the forebrain nucleus HVc (hyperstriatum ventrale pars caudale) are highly sensitive to the temporal structure of the bird's own song. These "song-specific" neurons respond strongly to forward song, weakly to the song with the order of the syllables reversed, and little or not at all to reversed song. To investigate the cellular mechanisms underlying these responses, in vivo intracellular recordings were made from adult HVc neurons. Song-specific cells could be divided into those that responded strongly throughout autogenous song (tonic cells) and those that responded with bursts of action potentials at specific points during the song (phasic cells). Phasic cells were hyperpolarized during autogenous song, even though this stimulus also elicited the strongest response. Less hyperpolarization was seen to the same song with the syllables in reverse order, and none was seen to reversed song. The responses of both types of song-specific cells contained high-frequency bursts of action potentials. The bursts of the phasic cells showed attenuation of the action potential height and lack of full repolarization after each spike. This type of bursting was significantly correlated with the amount of hyperpolarization before each burst in phasic cells and nonauditory cells that generated such bursts spontaneously. These data suggest that song-specific neurons use longlasting hyperpolarization as a mechanism to integrate auditory context, an important component of temporal order selectivity. Hyperpolarization also may increase the precision of spike timing, which could be important for the neural code subserving song learning and production.

Afferent and Efferent Synaptic Transmissions in Hair Cells

Hair cells transduce mechanical information into electrical signal and, via afferent synapse, transmit it to the central nervous system (CNS). Hair cells receive cholinergic efferent innervation from the CNS, and a long-lasting membrane hyperpolarization is produced by activation of Ca2+-activated K+ channels. Acetylcholine may facilitate afferent synaptic transmission by suppressing K+ channels on the afferent nerve terminal.

The role of GABAB mechanisms in animal models of absence seizures.

Generalized absence seizures in humans are a unique type of epilepsy characterized by a synchronous, bilateral 3-Hz spike and wave discharge emanating from a cortical and thalamic network within the brain. The availability of a number of pharmacological and genetic animal models has provided us with the means with which to investigate the cellular and molecular mechanisms underlying these seizures. Over the last few years a significant amount of research in these models has focused on the role of the inhibitory GABAB receptors, which have been previously described in a number of brain areas as being responsible for a long-lasting hyperpolarization and depression in neurotransmitter release. Initial studies provided evidence that the GABAB receptor was capable of generating the low threshold calcium spike required for initiation of the burst firing, leading researchers to hypothesize that the GABAB receptors played a significant role in these seizures. Subsequent research took advantage of the new generation of GABAB antagonists that became available in the early 1990s and demonstrated that in a number of models the seizures could be abolished by the administration of one of these compounds. Further biochemical, molecular, and electrophysiological experiments have been carried out to determine the exact involvement of GABAB receptors and their mechanism of action. The current evidence and interpretations of this work are presented here.

Synaptic responsiveness of cortical and thalamic neurones during various phases of slow sleep oscillation in cat.

1. The fluctuations during various phases of the slow sleep oscillation (< 1 Hz) in synaptic responsiveness of motor cortical (Cx), thalamic reticular (RE) and thalamocortical (TC) neurones were investigated intracellularly in cats under ketamine-xylazine anaesthesia. Orthodromic responses to stimuli applied to brachium conjunctivum (BC) axons and corticothalamic pathways were studied. The phases of slow oscillation consist of a long-hyperpolarized, followed by a sharp depth-negative EEG deflection and a series of faster waves that are associated with the depolarization of Cx and RE neurones, while TC cells display a sequence of IPSPs within the spindle frequency. 2. BC-evoked bisynaptic excitatory postsynaptic potentials (EPSPs) in Cx and RE neurones were drastically reduced in amplitude during the long-lasting hyperpolarization and the early part of the depolarizing phase. By contrast, the BC-evoked monosynaptic EPSPs of TC cells were not diminished during the depth-positive EEG wave, but the hyperpolarization during this phase of the slow oscillation prevented TC neurones transferring prethalamic signals to the cortex. 3. At variance with the diminished bisynaptic EPSPs evoked in response to BC stimuli during the long-lasting hyperpolarization, Cx-evoked monosynaptic EPSPs in Cx cells increased linearly with hyperpolarization during this phase of the slow oscillation. Similarly, the amplitudes of Cx-evoked EPSPs in RE and TC cells were not diminished during the long-lasting hyperpolarization. 4. The diminished responsiveness of Cx and RE neurones to prethalamic volleys during the long-lasting hyperpolarization is attributed to gating processes at the level of TC cells that, because of their hyperpolarization, do not transfer prethalamic information to further relays.

Mechanisms of long-lasting hyperpolarizations underlying slow sleep oscillations in cat corticothalamic networks.

1. To explore the nature of the long-lasting hyperpolarizations that characterize slow oscillations in corticothalamic circuits in vivo, intracellular recordings were obtained under ketamine-xylazine anaesthesia from cortical (Cx) cells of the cat precruciate motor cortex, thalamic reticular (RE) cells from the rostrolateral sector, and thalamocortical (TC) cells from the ventrolateral (VL) nucleus. 2. Measurements in the three cell types showed input resistance (Rin) to be highest during the long-lasting hyperpolarizations that correspond to depth-positive waves of the cortical EEG. Rin was lowest during the early phase of high-amplitude depth-negative EEG waves and increased thereafter until the next cycle of the slow oscillation. 3. Spontaneous long-lasting hyperpolarizations were compared with those evoked by dorsal thalamic stimulation. Voltage versus current (V-I) plots showed similar membrane potential (Vm) ranges and slopes for spontaneous and evoked hyperpolarizations in both Cx and RE cells. V-I plots from TC cells had similar slopes, but Vm during evoked hyperpolarizations was displaced towards more negative values. 4. Intracellular injection of constant hyperpolarizing current in Cx cells increased the amplitude of the initial part of the depolarizing plateau of the slow oscillation, but decreased the amplitude of the last part. 5. These results suggest disfacilitation to be the dominant mechanism in the membrane of cortical and thalamic cells during the spontaneous long-lasting hyperpolarizations, which shape and synchronize slow oscillations in corticothalamic networks. In Cx and RE cells, the same mechanism underlies thalamically evoked long-lasting hyperpolarizations. By contrast, evoked responses in TC cells show a strong additional hyperpolarizing factor. We propose that GABAB processes are stronger in TC than in Cx neurones, thus rendering the thalamus an easier target for absence-type epileptic phenomena through potentiation of thalamic rebound capabilities.

GABA-induced responses in electrophysiologically characterized neurons within the rat rostro-ventrolateral medulla in vitro

Rostro-ventrolateral medulla (RVL) neurons were recorded using conventional intracellular recording techniques in brain slices maintained in vitro at 32°C and classified into 3 major groups. The first group included neurons having endogenous pacemaker-like (PL) activity with regular firing frequency (mean = 8 Hz) and a linear current-voltage relationship ( I–V). The second group of neurons were slowly and irregularly firing (IF) or quiescent, presenting membrane potential oscillations and their I–V usually displayed an inward rectification. These neurons had a relatively longer action potential duration. The third group included silent neurons (S) with no apparent membrane oscillations and they differed from the first two groups by having relatively shorter action potential duration and amplitude and lower cell input resistance. When recorded with KCl-filled electrodes, the majority of silent neurons displayed a time-dependent inward rectification. With KAc-filled electrodes, irregular slow hyperpolarizing and depolarizing spontaneous potentials could be recorded primarily on PL and IF neurons, respectively. Moreover, fast spontaneous inhibitory postsynaptic potentials (PSPs) were detected in about 15% of PL and S neurons. They generally exhibited a regular pattern and were depolarizing when KCl-filled electrodes were used for recording. The amplitude of these inhibitory PSPs was reversibly reduced by the GABAA antagonists bicuculline, SR 95531 and picrotoxin. With KAc-filled electrodes, pressure-applied GABA (20 mM) evoked complex responses. In PL neurons, it consisted of a fast hyperpolarization followed by a slower depolarization that were both sensitive to SR 95531 and picrotoxin. The response was terminated by a long-lasting hyperpolarization that was reduced, but not abolished, by the GABA B antagonist CGP 35348. In IF and S neurons, GABA application usually produced a fast followed by a slow monophasic hyperpolarization and depolarization, respectively. The fast component of these responses was sensitive to the GABA A antagonists. Pressure application of isoguvacine (10 mM) always induced monophasic responses in all types of neurons recorded. Baclofen (1–30 μM) reduced the firing frequency and hyperpolarized PL and IF neurons, an effect that was antagonized by CGP 35348 (50–100 μM); however, it had little effect on silent neurons. It is concluded that RVL neurons have heterogeneous electrophysiological characteristics. Their predominant synaptic input and GABA responsiveness might be additional criteria to identify the excitatory and inhibitory elements in the RVL circuitry. All neuronal types seem to have functional GABA A and GABA B receptors; however, only a subpopulation is under tonic inhibitory control in vitro, probably from local GABAergic pacemaker intemeurons. Our results further emphasize the role of GABA as an important neurotransmitter in the RVL network.

Inhibition of pacemaker current by the bradycardic agent ZD 7288 is lost use-dependently in sheep cardiac Purkinje fibres.

The inhibition of the pacemaker current (if) in sheep cardiac Purkinje fibers by ZD 7288 [4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium++ + chloride] is lost use-dependently. This disinhibition of if was investigated by using the two-microelectrode voltage-clamp technique. The pulse protocol consisted of a rest period (holding potential of about -50 mV, 1-10 micromol/l ZD 7288) followed by a train of test pulses (potential negative to -100 mV, stimulation frequency 0.05 Hz). At the beginning of the first test pulse there was an immediate reduction of if but inhibition was lost during continued stimulation. Activation of if is sigmoidal and the early delay in current activation was prolonged from 33 ms (no ZD 7288) to 424 ms (10 micromol/l ZD 7288). Therefore hardly any disinhibition occurred during short test pulses (0.5s). During longer test pulses (5 s, -120 mV, 10 micromol/l) disinhibition developed with a time constant of about 2 s. The inhibition of if by ZD 7288 was lost voltage-dependently. With 10 micro mol/l ZD 7288 the half-maximal disinhibition occurred at -92 mV and the slope factor of the disinhibition/voltage curve (Boltzmann relation) was 4.8 mV. The voltage-dependent disinhibition could be abolished largely by extracellular application of protease (0.5 mg/ml, 7 min). After prior disinhibition, reinhibition at the holding potential (about -50 mV) followed a bi-exponential time course indicating that inhibition may be produced by a fast (tau=0.7 min) and a slow component (tau=20-30 min). Increasing ZD 7288 concentration from 1 to 10 mu mol/l accelerated reinhibition, mainly by an increase of the amplitude (A) of the fast component. The ratio Afast/Aslow was 0.399 at 1 micromol/l and 2.65 at 10 micromol/l ZD 7288. The reinhibition of if was unchanged by shifting the holding potential from -50 mV to -20 mV. Trials to wash out the effects of 10 micromol/l ZD 7288 gave two results. The inhibition of if was slightly reversed after a wash-out of 1.5 h with drug-free solution. A second effect of the drug, the fast reinhibition, could be completely removed by wash-out. In summary if is inhibited by ZD 7288 at membrane potentials at which the virtual if gate is closed. Disinhibition occurs during long-lasting hyperpolarization but will hardly be operative in unclamped fibres under physiological conditions.

Motoneuron depolarizations, paroxysmal activity, and reflex changes induced by rapid cooling of toad spinal cord

Sucrose gap recordings from the ventral roots of isolated, hemisected South American toad spinal cords were used to investigate the effects on motoneurons of rapid cooling from 21–22°C to 6–8°C. Such cooling produced large ventral root depolarizations that slowly decayed to baseline. During the repolarization, a series of large, slow, paroxysmal, motoneuron depolarizations were seen. Upon rewarming, large, long-lasting hyperpolarizations (unaffected by dihydro-ouabain) were observed. Cold markedly increased the duration of reflexes evoked by dorsal root stimulation. Mg 2+ or tetrodotoxin reduced cold-induced motoneuron depolarizations by about 50%. Kynurenate or 1–2-amino-7-phosphono-heptanoic acid reduced cold-induced depolarizations by approximately the same amount and eliminated paroxysmal activity suggesting that synaptic effects caused by the release of excitatory amino acid transmitters and the subsequent activation of NMDA receptors are necessary for the generation of about one half of the cold-induced motoneuron depolarization, and for the production of paroxysmal motoneuron activity. Nonsynaptic mechanisms and cold-induced alteration of the resting membrane potential appear partly responsible for the remainder of the cold-induced depolarizations.

Cerebellar-responsive neurons in the thalamic ventroanterior-ventrolateral complex of rats: In vivo electrophysiology

In vivo intracellular recordings were obtained from identified thalamocortical neurons in the ventroanterior-ventrolateral complex in urethane-anesthetized rats. This thalamic nucleus has few interneurons. Neurons that responded to cerebellar stimulation were injected intracellularly with horseradish peroxidase or biocytin and examined with light and electron microscopy (see companion paper). Intrinsic membrane properties and voltage-dependent rhythmic activity of cerebellar-responsive ventroanterior-ventrolateral neurons were similar to those described previously for thalamic neurons. Thus, in addition to conventional “fast” Na +-dependent spikes, rat ventroanterior-ventrolateral neurons had “slow” Ca 2+-mediated low-threshold spikes and membrane conductances that supported rhythmic oscillations. Two modes of spontaneous activity were observed: (i) a tonic firing pattern that consisted of irregularly occurring fast spikes that predominated when the membrane potential was more positive than about − 60 mV, and (ii) a rhythmic firing pattern, observed when the membrane potential was more negative than about − 65 mV, composed of periodic (4–8 Hz) membrane hyperpolarizations and ramp depolarizations that often produced a low-threshold spike and a burst of fast spikes. In some neurons, spontaneous fast prepotentials were also observed, often with a relatively constant rate (up to 70 Hz). Cerebellar stimulation elicited excitatory postsynaptic potentials that in some cases appeared to be all-or-none and were similar in form to fast prepotentials. Stimulation of ipsilateral motor cortex elicited a short-latency antidromic response followed by a monosynaptic excitatory postsynaptic potential, which had a slower rise time than excitatory postsynaptic potentials evoked from cerebellum, suggesting that cortical inputs were electrotonically distal to cerebellar inputs. In the presence of moderate membrane hyperpolarization, the cortically evoked excitatory postsynaptic potential was followed by a long-lasting hyperpolarization (100–400 ms duration), a rebound depolarization and one or two cycles resembling spontaneous rhythmic activity. Membrane conductance was increased during the initial component of the long hyperpolarization, much of which was probably due to an inhibitory postsynaptic potential. In contrast, membrane conductance was unchanged or slightly decreased during the latter three-quarters of the long hyperpolarization. The amplitude of this component of the long hyperpolarization usually decreased when the membrane was hyperpolarized with intracellular current injection. Thus, both disfacilitation and an inhibitory postsynaptic potential may have contributed to the latter portion of the cortically-evoked long hyperpolarization. The cortically-evoked inhibitory postsynaptic potentials likely originated predominantly from feedforward activation of GABAergic neurons in the thalamic reticular nuclei. The disfacilitation probably resulted from activation of inhibitory circuits intrinsic to the cortex and/or corticothalamic circuits that transiently reduced tonic cortical excitatory drive onto thalamic neurons. The possibility that disfacilitation occurred following experimentally-induced synchronous activation of cortical circuitry suggests that the cerebral cortex provides a significant degree of tonic excitatory drive onto neurons in the ventroanterior-ventrolateral complex of rats. We conclude that the synaptic and intrinsic membrane properties of thalamic neurons in the ventroanterior-ventrolateral complex of rats are fundamentally similar to previously described properties of thalamocortical neurons of other species (e.g. felines) in related nuclei that possess GABAergic interneurons, in spite of the scarcity of GABAergic interneurons in rat ventroanterior-ventrolateral complex. Furthermore, rhythmic disfacilitation of cortical inputs to thalamus may contribute to the maintenance of the rhythmic activity in thalamocortical circuits that is prominent during different behavioral states.

Pharmacology and electrophysiology of a synchronous gaba-mediated potential in the human neocortex

Spontaneous synchronous field potentials of negative polarity (duration = 200–700 ms, interevent interval= 9.1 ± 2.9 s; n = 27slices) were recorded, during application of 4-aminopyridine (50 μM), from the superficial/middle layers of slices of human neocortex obtained in the course of neurosurgery for the relief of intractable seizures. The negative-going field potential corresponded to an intracellular long-lasting (duration = 200–1600 ms) depolarization that could be preceded by an excitatory postsynaptic potential-hyperpolarizing inhibitory postsynaptic potential sequence and followed by a long-lasting hyperpolarization. This synchronous activity continued to occur following blockade of excitatory synaptic transmission by excitatory amino acid receptor antagonists, but was greatly reduced and eventually disappeared during application of the GABA A receptor antagonist bicuculline methiodide. Simultaneous extracellular recordings from three sites in the slice located along an axis parallel to the pia showed that successive synchronous field potentials could originate from any of the three areas. They invaded the other two sites in c. 35.5% of the cases, while propagation to another site only or no propagation at all was observed, respectively, in 44.4% and 20% of instances. The velocity of lateral propagation of the synchronous field potential was 7.9 ± 2.5mm/s(range= 4.5−11.8mm/s, n = 6). The modalities of origin and propagation remained the same after blockade of excitatory amino acid receptors. Under these conditions, however, there was a higher incidence of non-propagation and the velocity was significantly lower than in control ( 5.6 ± 1.9mm/s;range= 2.8–7.7mm/s, n = 6). These data indicate that, in the human neocortex, 4-aminopyridine can reveal a synchronous field potential that correlates with an intracellular long-lasting depolarization and is mainly due to the activation of postsynaptic GABA A receptors. The action of excitatory amino acid receptors is not necessary for the generation and propagation of these GABA-mediated potentials. We propose that this potential represents a novel mechanism for synchronization and spread of neuronal activity, including seizure-like discharges in the human neocortex.

Feedback inhibition in the cat's lateral geniculate nucleus.

Feedback inhibition is generally believed to be a ubiquitous feature of brain circuitry, but few specific instances have been documented. An example in cats is the supposed feedback circuit involving relay cells of the lateral geniculate nucleus and cells of the perigeniculate nucleus (a part of the thalamic reticular nucleus): geniculate relay cells innervate the perigeniculate nucleus, which, in turn, provides an inhibitory, GABAergic projection back to the lateral geniculate nucleus. However, feedback inhibition at the single-cell level requires that a given perigeniculate cell project back onto the same geniculate relay cell that innervates it. We probed for this in an in vitro slice preparation of the cat's lateral geniculate nucleus. We evoked a single action potential in a geniculate cell via a brief, depolarizing pulse delivered through an intracellular recording electrode and looked for any evoked hyperpolarizations. For 6 of the 36 geniculate cells tested, we observed a long-lasting hyperpolarization after the action potential, and much of this was eliminated by application of bicuculline, suggesting synaptically activated inhibitory postsynaptic potentials. We interpreted this to be clear evidence that a given neuron may inhibit itself via circuitry mediating feedback inhibition in the cat's lateral geniculate nucleus.