Transient Outward Rectification Research Articles

Pharmacological and Immunohistochemical Evidence for Serotonergic Modulation of Cholinergic Nucleus Basalis Neurons

Identified electrophysiologically by low threshold bursts and transient outward rectification, cholinergic nucleus basalis neurons were recorded and labelled intracellularly in guinea-pig basal forebrain slices. By means of a triple labelling immunofluorescent technique, serotonin-immunoreactive fibres were visualized in close proximity to the soma and dendrites of the biocytin-labelled, choline acetyl transferase (ChAT)-immunoreactive cells. By bath application, 5-hydroxytryptamine (5-HT) produced a direct hyperpolarization of the identified cells which was mimicked by 5-HT1A receptor agonists, suggesting that it may inhibit the tonic firing but also modulate the low threshold bursting of the cholinergic nucleus basalis neurons.

Intracellular recordings from neurons of the arcuate nucleus in superfused explants of rat hypothalamus.

The membrane properties of arcuate nucleus (ARN) neurons and their connections with extranuclear regions were examined in vitro, using intracellular recordings obtained in superfused explants of rat hypothalamus. Current-clamp analysis revealed that the electroresponsive properties of a large proportion of ARN neurons are influenced by the expression of various forms of low threshold rectification. When the initial membrane potential of a neuron was maintained negative to -70 mV, depolarizing current injection could precipitate an immediate bursting response, due to the activation of a low-threshold spike, or delay firing as a result of transient outward rectification. These properties represent a means by which changes in membrane potential may modulate the responsiveness of ARN neurons to synaptic input. Indeed, electrical stimulation revealed that in addition to efferent projections to the supraoptic nucleus, medial preoptic area, or neurointermediate pituitary, subgroups of ARN neurons may receive a reciprocal, or collateral innervation from each of these areas. This functional diversity of intrinsic and synaptic mechanisms provides ARN neurons with a high capacity for the complex local processing of afferent information.

Sensitivity of transient outward rectification to ion channel blocking agents in guinea-pig substantia nigra pars compacta neurones in vitro

The sensitivity of a transient outward rectifier in guinea-pig substantia nigra pars compacta (SNC) neurones in vitro was tested to a variety of ion channel blocking agents. This rectification was not blocked by TEA (10 mM), 4-aminopyridine (4-AP) (5 mM), apamin (0.2–1 μM) or Cd 2+ ions (200 μM). Cs + ions (5 mM) blocked the inward ‘anomalous’ rectifier but had no effect on the transient outward rectification. However, replacing Ca 2+ ions in the bath perfusate by Ba 2+ blocked the transient outward but not the inward rectifier. It is suggested that an atypical barium-sensitive conductance is responsible for the transient outward rectification of these neurones.

Properties of supraoptic magnocellular neurones isolated from the adult rat.

1. Magnocellular neurosecretory cells (MNCs) were isolated from the supraoptic nucleus of adult Long-Evans rats using an enzymatic procedure. Immunocytochemical staining with antibodies against vasopressin and oxytocin revealed that MNCs can be identified by size. The membrane properties of these cells were examined at 32-34 degrees C using intracellular recording methods. 2. Isolated MNCs displayed a mean (+/- S.E.M.; n = 109) resting membrane potential of -64.1 +/- 1.0 mV, an input resistance of 571 +/- 34 M omega, and a time constant of 8.7 +/- 0.4 ms. Measurements of specific resistivity and input capacitance revealed that the soma of these cells accounts for a mere 20% of their total somato-dendritic membrane in situ. 3. Voltage-current relations measured near -60 mV were linear negative to spike threshold. From more hyperpolarized membrane potentials, voltage responses to depolarizing current steps displayed transient outward rectification and delayed impulse discharge. 4. Action potentials (76.6 +/- 0.9 mV) triggered from an apparent threshold of -59.3 +/- 0.1 mV broadened progressively at the onset of spontaneous or current-evoked spike trains. Steady-state spike duration increased as a logarithmic function of firing frequency with a maximum near 25 Hz. These effects were abolished in Ca(2+)-free solutions. 5. In all cells, evoked spike trains were followed by a prolonged Ca(2+)-sensitive after-hyperpolarization. In contrast, only a small proportion (16%) of MNCs displayed spontaneous bursting activity or depolarizing after-potentials following brief current-evoked bursts. 6. Isolated MNCs responded to amino acids (glutamate and GABA) and to the neuropeptide cholecystokinin, indicating that receptors for these neurotransmitters are expressed postsynaptically by MNCs and are retained following dissociation. 7. Increasing the osmolality of the superfusing solution by 5-30 mosmol kg-1 caused a membrane depolarization associated with a decrease of input resistance and accelerated spontaneous spike discharge in each of thirty-six MNCs tested. Current-clamp analysis suggested that these responses resulted from the activation of a cationic conductance. Excitatory effects of hyperosmolality were not observed in non-magnocellular neurones (n = 6).

Evidence for voltage-activated outward currents in the neuropilar membrane of locust nonspiking local interneurons

Outward currents activated by depolarization were studied in the neuropilar membrane of locust nonspiking local interneurons, using the single-electrode voltage-clamp technique in situ. Preliminary observation of these currents in 272 neurons revealed two families. The first and most commonly observed (85% of recordings) showed a large transient current followed by a slowly decaying/late current. The second (15% of recordings) showed an additional outward current with a slow rate of activation, a peak within 100-150 msec, and a slow rate of inactivation. Only neurons of the first type were studied further. The transient current was activated by depolarization around -60 mV, with a time to peak of approximately 11 msec at -50 mV and less than 3 msec at -20 mV. This current decayed exponentially, with a time constant of 8.1 +/- 1.6 msec (n = 8 interneurons) at -30 mV. This time constant of inactivation did not appear to depend strongly on membrane voltage, in the range in which it was studied. A second and longer time constant of inactivation of 50-400 msec could not be assigned to either of the transient and late components of the outward current. The ratio of transient-to-late current varied between 1.6 and 5.4, with a mean of about 2.5. The reversal potential for the transient current could, on average, be shifted by 14 mV by a threefold increase in the bath K+ concentration, indicating that K+ is a charge carrier for the current. The transient current became inactivated with maintained depolarization and appeared half-inactivated at about -60 mV (slope factor k1/2 = 8 mV). This current was thus not fully inactivated at "resting" potential (average, -58 mV). Recovery from inactivation followed a single exponential time course, with a time constant of approximately 100 msec at -80 mV. The time course of recovery from inactivation of the transient current was well correlated with that of the recovery of transient outward rectification, as measured in current-clamp recording. Tetraethylammonium, at a bath concentration of 10 mM reduced the transient current by 70% and the delayed current by 60%. 4-Aminopyridine, at a bath concentration of 5 mM, had a significant effect in only two of five interneurons, reducing the transient current by approximately 85% and the late current by approximately 15%. Quinidine at a bath concentration of 100 microM was ineffective. Although these blockers did not allow a clear pharmacological separation of the currents, they were effective in reducing the outward rectification observed in current clamp during step depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiology of dopaminergic and non‐dopaminergic neurones of the guinea‐pig substantia nigra pars compacta in vitro.

1. The membrane properties of substantia nigra pars compacta neurones were studied using an in vitro slice preparation of guinea-pig midbrain. 2. Neurones were divided into two classes based on their electrophysiological properties: bursting neurones displayed a burst of several rapidly accommodating action potentials in response to relaxation of hyperpolarizing current injected through the microelectrode, while non-bursting neurones produced regularly spaced action potentials. These neuronal types were found to be electrophysiologically distinct from those recorded in the substantia nigra pars reticulata and the subthalamic nucleus. 3. Non-bursting neurones, which comprised ca 85% of the sampled cells, were characterized by a slow, pacemaker pattern of firing at rest, broad action potentials, a pronounced spike after-hyperpolarization, long membrane time constants, and strong transient outward and time-dependent inward rectification. 4. Bursting neurones, comprising ca 15% of the sample, displayed rapid firing rates at rest, fast action potentials, a shallow spike after-hyperpolarization and briefer membrane time constants. All of these parameters were significantly different from those of the non-bursting type. Bursting neurones lacked transient outward or time-dependent inward rectification. 5. Both types of cells were capable of generating pronounced calcium-dependent, low-threshold spikes in the presence of tetrodotoxin (TTX). However, only the non-bursting type displayed calcium-dependent rhythmic oscillations in membrane potential near resting potential in the presence of TTX. The firing rate, action potential shape and after-hyperpolarization of non-bursting neurones were strongly influenced by calcium-dependent currents. 6. The majority of cells were injected with biocytin, which allowed morphological reconstruction of the neurones and confirmation of their location within the pars compacta. Non-bursting neurones had variable soma shapes and their dendrites were mostly directed in a medio-lateral direction. Many cells extended some of their dendrites into the pars reticulata. Bursting neurones were mainly fusiform in shape with their dendrites oriented in a medio-lateral direction; a few had dendrites extending into the pars reticulata. 7. Thirty-six neurones were also double labelled using a combination of biocytin or Lucifer Yellow injection with tyrosine hydroxylase (TH) immunohistochemistry. Non-bursting neurones all displayed TH immunofluorescence, while none of the bursting neurones were TH positive.(ABSTRACT TRUNCATED AT 400 WORDS)

Properties of tuberomammillary histamine neurones and their response to galanin

Histaminergic neurones in the tuberomammillary nucleus possess electrophysiological properties which distinguish them from other neurones in their neighborhood. Their resting potential is -50 mV and they are spontaneously active at about 2 Hz in a slice preparation. They display a transient outward rectification and an anomalous inward rectification. Bath application of galanin (0.1 microM) reduced their firing rate significantly and hyperpolarized them slightly.

Membrane properties of rat magnocellular neuroendocrine cells in vivo

Membrane properties of rat magnocellular neuroendocrine cells (MNCs) were examined during intracellular recordings in vivo. Current-clamp experiments revealed sustained outward rectification positive to −55 mV and transient outward rectification of depolarizing responses elicited from negative holding potentials. Trains of impulses were associated with a progressive increase of spike duration. Such features, which were not observed in neighboring non-neuroendocrine cells, are similar to those of MNCs in slices or explants of rat hypothalalus. In contrast, however, cells recorded in vivo were characterized by intense synaptic inhibition and a lower specific impedance than commonly observed from MNCs impaled in vitro.

Electrophysiological characteristics of cholinergic and non-cholinergic neurons in the rat medial septum-diagonal band complex

We examined the electrophysiological properties of cholinergic and non-cholinergic neurons in the medial septum-diagonal band complex (MSDB) of the rat in the in vitro slice preparation. Cells were identified electrophysiologically, filled with Lucifer yellow, fixed and processed immunohistochemistry with fluorescent labeled anti-choline acetyltransferase (ChAT) antibody. Cholinergic and non-cholinergic neurons differed in action potential parameters, spike afterpotentials and in current-voltage relationships. In addition, cholinergic neurons expressed a potent transient outward rectification in response to a depolarizing current pulse.

Membrane properties of rat substantia gelatinosa neurons in vitro.

1. The membrane properties of substantia gelatinosa (SG) neurons in an in vitro adult rat transverse spinal cord slice preparation with attached dorsal root have been examined. Intracellular recordings were obtained from identified SG neurons. 2. Seventy-six percent of SG neurons exhibited a time-dependent anomalous rectification (AR) when the membrane was hyperpolarized from the resting potential. The time-dependent AR was blocked by cesium (Cs+, 2 mM) but not by barium (Ba2+, 2 mM). Application of Cs+ itself caused membrane hyperpolarization in those SG neurons that expressed the time-dependent AR. The activation of the time-dependent AR was maximal at potentials 5-10 mV below the resting membrane potential. 3. In a few SG neurons, the current-voltage relationship revealed a marked inward rectification, even though there was no detectable time-dependent anomalous rectification during hyperpolarization. Analysis of the Ba2+- and Cs+-sensitivity of these neurons confirmed that SG neurons expressed two distinct ARs, one of which is fast and Ba2+-sensitive and the other of which is time-dependent and Ba2+-insensitive. 4. Fifty-one percent of SG neurons exhibited a transient outward rectification when hyperpolarizing current pulses were applied from potentials more positive than -60 mV or when depolarizing pulses were applied from potentials more negative than -65 mV. The transient outward rectification persisted for 0.3-2 s when hyperpolarizing pulses were applied at -55 mV. 5. The transient outward rectification was associated with a decrease in membrane resistance and was enhanced in low K+ solutions. 4-aminopyridine (4-AP, 2 mM) reversibly blocked the transient outward rectification. 6. The time-dependent anomalous and transient outward rectifying currents exerted opposite effects on the firing properties of SG neurons. Activation of the time-dependent AR increased neuronal excitability. In neurons that exhibited the time-dependent AR, membrane depolarization caused the appearance of a rebound depolarization that resulted in the generation of spikes with only a short delay after application of the depolarizing pulse. In contrast, the transient outward rectifying current markedly delayed spike firing in response to depolarizing pulses. This delay was blocked by application of 4-AP. 7. The diversity in response properties of subpopulations of SG neurons may result in part from this heterogeneity in membrane properties.

Repetitive firing properties of medial pontine reticular formation neurones of the rat recorded in vitro.

1. Intracellularly recorded neurones in nucleus reticularis pontis caudalis of the medial pontine reticular formation (mPRF) in the in vitro slice preparation were analysed for repetitive firing properties in response to intracellularly applied constant-current pulses. 2. Three neuronal classes were defined by this procedure: (1) non-burst neurones, which had only a non-burst firing pattern; (2) low-threshold burst neurones, which had either a low-threshold burst pattern or a non-burst pattern; (3) high-threshold burst neurones, which had either a high-threshold burst pattern or a non-burst pattern. 3. Histological characterization of electrophysiologically identified mPRF neurones with carboxyfluorescein showed no definite morphological difference between the first two classes. There was a trend for low-threshold burst neurones to have larger somata. 4. The low-threshold burst was generated by a slow calcium-dependent low-threshold spike, revealed in the presence of tetrodotoxin. The size of the low-threshold spike and thus the number of fast action potentials in the low-threshold burst was controlled by at least five factors including: activation; inactivation; amplitude of low-threshold conductance available to be activated; delayed outward conductance; and early transient outward conductance. 5. The non-burst pattern examined in both non-burst and low-threshold burst neurones appeared to be controlled primarily by one or more calcium-dependent potassium conductances sensitive to the removal of calcium and tetraethyl-ammonium. In the presence of tetrodotoxin (TTX), the addition of antagonists to calcium-dependent potassium current revealed a slow high-threshold calcium spike which was distinguished from the low-threshold spike by its threshold, lack of inactivation (at potentials negative to -40 mV) and insensitivity to Mg2+. A long-duration after-hyperpolarization (greater than 0.5 s) was not observed in any of these cells. 6. An early transient outward rectification sensitive to 4-aminopyridine and probably mediated by A-current was apparent in low-threshold burst and non-burst neurones and affected both the low-threshold burst and non-burst firing patterns. 7. Alteration of resting membrane potential, such as occurs in vivo during the depolarization associated with desynchronized sleep, may inactivate the low-threshold spike and the transient outward conductance responsible for the distinctive responses observed from more hyperpolarized membrane potentials and produce a more homogeneous non-burst response pattern. Membrane potential effects on intrinsic conductances thus may furnish an important mechanism for changes in mPRF neuronal responsivene

Membrane properties of histaminergic tuberomammillary neurones of the rat hypothalamus in vitro.

1. Intracellular recordings were obtained from neurones of the tuberomammillary nucleus in an in vitro explant of the rat hypothalamus. 2. Tuberomammillary neurones were spontaneously active (2.1 +/- 0.6 Hz) at the resting potential which was around -50 mV. Action potential amplitude was 75 +/- 8 mV (n = 9); mean mid-amplitude duration was 1.8 +/- 0.4 ms (n = 9). 3. The mean input resistance of tuberomammillary neurones was 176 +/- 42 M omega (n = 30), and the mean membrane time constant was 19.8 +/- 5.3 ms (n = 30). These neurones exhibited inward rectification with hyperpolarization from the resting potential, and transient outward rectification at the offset of hyperpolarizing electrotonic pulses. 4. Action potentials were followed by an after-hyperpolarization of 300-600 ms duration and 12-18 mV amplitude. This after-hyperpolarization had a reversal potential around -80 mV, was abolished by intracellular loading with caesium, and was reduced but not abolished by bath application of either cadmium, cobalt or nickel. 5. Tetrodotoxin abolished spontaneous action potentials. Further addition of tetraethylammonium ions revealed a regenerative spike which was reversibly blocked by the addition of cobalt. 6. That tuberomammillary neurones exhibiting these properties were indeed histaminergic was confirmed in five cases by intracellular ionophoresis of Lucifer Yellow and subsequent double labelling by immunofluorescent localization of the histamine synthetic enzyme L-histidine decarboxylase.

Interactions between grafted serotonin neurons and adult host rat hippocampus.

We have followed the development of physiological and functional properties of serotonin-containing raphe neurons grafted into an adult host hippocampus as a model system for graft-host interactions. These raphe cells have no clear identifying properties on the day of grafting: they develop them while growing in the host. Raphe neurons, recorded 1 month after grafting, possess adult normal physiological properties. These include high input resistance, slow membrane time constant, lack of inward rectification, a transient outward rectification, broad spikes having a Ca2+ component, lack of accommodation, and a large afterhyperpolarization. The graft is first innervated by host fibers and later projects to the host tissue. When stimulated, postsynaptic hyperpolarized responses are recorded in hippocampal neurons. In the freely moving rat, raphe grafts can restore sleep-wakefulness variations in an evoked population response of the hippocampus to afferent stimulation, which is eliminated by depletion of serotonin. These studies illustrate that grafted serotonin neurons develop functional relations with a host brain.

A transient outward rectification in the cat sympathetic preganglionic neuron.

Intracellular recordings were made from sympathetic preganglionic neurons of the intermediolateral nucleus, in the slice of the T3 segment of the cat spinal cord. Depolarization to -60 mV from membrane potentials more negative than -75 to -80 mV caused a transient outward rectification which decayed in 0.5-2.0s. The rectification was sensitive to changes in K concentration, was abolished by 4AP (2 mM), and was unaffected by low Ca (0.25 mM), Co (2 mM), Ba (1 mM), TEA (20 mM) or intracellular Cs. These properties suggest that the rectification is due to a transient K-current similar to the A-current described in other neurons.

Fetal raphe neurons grafted into the hippocampus develop normal adult physiological properties

Embryonic midbrain raphe was grafted into serotonin-deficient adult rat hippocampus. Serotonin-containing neurons in the graft survive for at least 6 months after grafting. Grafted neurons develop physiological properties, not present on the day of grafting, identical to those of normal adult serotonin-containing neurons. These include (a) high input resistance and slow membrane time constant, (b) lack of inward rectification in response to hyperpolarizing current pulses and (c) a potent, 4-aminopyridine-sensitive transient outward rectification. The grafted neurons innervate the host tissue with axons that have a slow conduction velocity and refractoriness. It is suggested that grafted CNS neurons may possess normal physiological properties.