Modulation of the endogenous electrical activity of the bursting neuron in the snail Helix pomatia--I. The generator of the slow rhythms.

Electrical activity of the bursting neuron RPal of the snail Helix pomatia was found to be subjected to periodic modulation with an interval of about 100s. The modulation was produced by waves of membrane potential the amplitude of which could change and the bursting activity could be even transformed into the beating one. Clamping the membrane potential in this neuron revealed a periodic slow inward current which appears to cause the modulation of the neuron's electrical activity. The exogenous nature of the generator of the slow inward current in the neuron RPa1 and in other studied neurons was demonstrated. Decrease in the extracellular calcium ion concentration from 10 to 0.4 mM, with a simultaneous increase of magnesium ion concentration from 4 to 14 mM, caused no marked changes in the periodicity of the slow inward current. Decrease in the extracellular sodium ion concentration from 100 to 50 mM increased the interval between the phases of slow inward current and finally led to the complete disappearance of the current.It is suggested that a neurosecretory interneuronal communication exists in the snail brain which has a different mechanism from conventional synaptic transmission.     

Modulation of the endogenous electrical activity of the bursting neuron in the snail Helix pomatia--II. The membrane characteristics related to modulation of the endogenous activity of the neuron.

The relation of the amplitude of the slow inward current to the holding potential and the changes of membrane conductance during the development of the slow inward current under voltage clamp conditions were investigated on neurons RPa1 and RPa7. The amplitude of the slow inward current in neuron RPa1 linearly increased with membrane hyperpolarization from ?30 to ?90 mV. The equilibrium potential for the slow inward current found by extrapolation was about +45 mV. In neuron RPa1 rectangular hyperpolarizing pulses (7 mV, 2s), applied from a holding potential of ?60 mV, evoked inward currents before the development of the slow inward current and outward currents at the maximum of the slow inward current. These currents decreased with time. In the neuron RPa7 no relation of the amplitude of the slow inward current to holding potentials between ?58 and ?78 mV and no changes of membrane conductance during the development of the slow inward current were found.It is concluded that the slow inward current in neuron RPa1 is due to an increase of the membrane's sodium permeability. During the development of the slow inward current an additional potential- and time-dependent increase in permeability occurs upon hyperpolarization, obviously to potassium or chloride ions.     

The effects of capsaicin on action potential and outward potassium currents in a bursting neuron of the snail, Helix pomatia L

The effects of capsaicin (CAP) on membrane properties, action potentials (APs) and outward membrane currents were investigated using the single electrode current and voltage clamp. CAP (60-300 microM) depolarized the cell membrane and increased the input resistance (Rin). The burst frequency of the neuron simultaneously increased. CAP prolonged the APs and attenuated the outward currents with decreasing potency in the sequence of the early outward current (IA), an early and a late component of the delayed outward current.     

Inhibition of an identified snail postsynaptic neuron evoked by stimulation of a peptidergic interneuron initiating bursting pacemaker activity in another neuron

Beating neuron V8, whose activity is abolished upon interneuronal stimulation, has been found and identified in the CNS of the snail, Helix pomatia. It was found earlier that stimulation of the same interneuron initiates bursting pacemaker activity in another neuron. Under voltage clamp conditions interneuronal stimulation evoked a slow outward current in the V8 neuron with a latency of about 2 s. Intracellular injection of tetraethylammonium or Cs+ ions into the interneuron greatly increased the slow outward current amplitude. External application of CdCl2 (1 mM), LaCl3 (5 mM) or replacement of Ca2+ by Mg2+ ions reversibly abolished transmission between the interneuron and the V8 neuron. The neuronal membrane conductance increased by about three times on the maximum of the slow outward current development. Computer fitting showed that a decay of the slow outward current can be approximated by the sum of two exponentially decaying components with time constants of about 1 s and 16 s. Extrapolated reversal potential for a fast (1 s) component of the current was about -70 mV. A slow (16 s) component asymptotically decreased upon hyperpolarization and it was observed at even more negative potential than Ek. Theophylline (1 mM) and 3-isobutyl-1-methylxanthine (0.1 mM) reversibly increased the slow outward current amplitude, whereas imidazole (5 mM) and tolbutamide (5 mM) caused its reversible decrease. It is concluded that inhibitory monosynaptic transmission exists between the interneuron and the beating V8 neuron. Interneuronal simulation evokes an increase in the membrane potassium conductance and probably a decrease in stationary potential-dependent sodium conductance of the postsynaptic V8 neuron. The cellular cyclase system seems to be involved in the postsynaptic response.     

Control of the delayed outward potassium currents in bursting pace-maker neurones of the snail, Helix pomatia.

The net outward current in bursting pace-maker neurones of the snail (Helix pomatia) during sustained and repeated voltage clamp pulses was studied. The properties of currents remaining in cobalt-Ringer or after TEA injection were compared with those in untreated cells. 2. With sustained voltage clamp depolarizations the net outward current first increases to a maximum at 150 msec and then declines to 60% or less of its peak intensity. This depression, which is greater during repetition of short pulses (e.g. 100 msec pulses at 0-5 sec intervals), represents a true decrease in the outward flow of K (designated IK) and is not due to a decreased driving force resulting from extracellular K accumulation. The steady-state current-voltage (I-V) relationship for IK is N-shaped (Heyer & Lux, 1976). 3. A component of IK persists when Ca and Mg in the medium are replaced by Co (ICo-res). With voltage clamp depolarizations ICo-res increases rapidly to a maximum and then partially inactivates with voltage dependent time constants of hundredths or tenths of seconds. Repolarization removes the inactivation. Thus, repeated stimulation with short pulses does not increase the depression of ICo-res-ICo-res (e.g. measured during voltage steps from holding potentials of -50 to near 0 mV) is smaller in test pulses preceded by depolarization and larger in pulses preceded by hyperpolarization. The steady state I-V relationship is not N-shaped. ICo-res is blocked by intracellular injection of tetraethylammonium (TEA). 4. Repeated voltage clamp depolarization to near 0 mV with 100 msec pulses for neurones with large Ca currents in normal Ringer produces a long-term depression which is maximal with 300-400 msec repolarizations (to -50 mV) between pulses. This corresponds with stimulus parameters for the maximum Ca current (Heyer & Lux, 1976). Intracellular injection of Ca2+ (also Ba2+ and Co2+) likewise reduces the total net outward current and especially the delayed outward current under voltage clamp. 5. The component of IK which is removed by Co is identified as Ca dependent and designated IK(Ca). With single voltage clamp pulses IK(Ca) follows the approximate time course and voltage dependence of the slow inward Ca current (Iin slow; Heyer & Lux, 1976). Several lines of evidence suggest that Ca ions moving through the membrane activate IK(Ca). 6. Part of IK cannot be blocked by intracellular TEA injection. In different neurones the magnitude of the IK component resistant to TEA (ITEA-res) is approximately proportional to the relative magnitudes of Iin slow.ITEA-res does not inactivate with sustained depolarization and shows pronounced long-term depression with repetitive stimulation at intermediate intervals and an increased outward current at the onset of the second and subsequent pulses following short repolarizations. The steady-state I-V relationship is N-shaped. ITEA-res is abolished by extracellular Co. 7. A net inward current with low depolarizations can be measured after TEA injection...     

A calcium dependent inward current in frog skeletal muscle fibres

Summary Slow inward currents are here reported in frog skeletal muscle. The currents are turned on by depolarising the membrane beyound about –45 mV, and are blocked by replacement of Ca²⁺ by Mg²⁺ and in the presence of Ca²⁺ by Co²⁺ at 20mM.     

Blocking of the axon spikes by pentylenetetrazol in an identified neuron of the snail Helix lucorum, with consequent induction of epileptic activity in the cell body of the same neuron.

In an isolated preparation which consists of the buccal ganglia and the salivary nerve of Helix lucorum, neuron B2 can be identified by simultaneously recording the soma spike intracellularly and the axon spike extracellularly from the peripheral salivary nerve. The convulsant drug pentylenetetrazol (PTZ) eliminates the axon spike of the neuron within 1-5 min after its application while epileptic activity is induced in the soma of the same neuron for hours. It is interesting that almost all nerve activity in the peripheral nerve is blocked by PTZ.     

Contribution of restricted extracellular space to the inactivation of calcium current in the snail neuron

The mechanisms underlying the inactivation of calcium current (ICa) were investigated in isolated nerve cell bodies of Helix aspersa using a suction pipette technique that allowed voltage clamp and internal perfusion at the same time. ICa was recorded after eliminating the Na and K currents by removing Na+ and K+ both in external and internal solutions, and ICa inactivation due to intracellular Ca2+ accumulation was blocked by 5-25 mM EGTA. The inactivation rates of ICa, IBa and ISr corresponded to two exponential processes. The inactivation rates of the inward currents (IMn, ICd and IZn) less than 1/5 of ICa fitted a single exponential. However, when neurons were superfused with hypertonic external solution by adding 100 mM sucrose together with internal EGTA, the steady-state inactivation of ICa, IBa and ISr was reduced, and the inactivation processes changed to a single exponential similar to that of IMn, ICd and IZn. In contrast, internal perfusion with the hypertonic solution had no effect on the inactivation of ICa, IBa and ISr. Therefore, it was concluded that the inactivation process of ICa is dependent not only on the membrane voltage and the intracellular Ca2+ accumulation as described previously, but is also affected by the rapid fall in the concentration of Ca2+ in the restricted extracellular spaces (RES) which gets enlarged by the hypertonic external solution. The same is also true for IBa and ISr.     

The effects of intracellular protons on the electrical activity of single ventricular cells

Acidic or alkaline solutions were injected into single guinea-pig ventricular cells and the effects of cytoplasmic proton concentration ([H]_i) on their electrical activity were examined. Injections of low pH (3.7–4.7) solutions shortened the action potential and depressed the plateau by decreasingi _si and increasing the outward current at the plateau potentials. Injections of high pH (9.3–9.7) solutions had the opposite effects on the action potential and the membrane currents. A decremental oscillatory current, similar to the strophanthidin-induced transient inward current, was generated upon injecting large amount of low pH solutions. Injections of high pH solution abolished this current. Alterations in [Ca]_i also mimiced the changes of the action potential caused by H⁺-injections. Comparison of the effects of high pH and EGTA injections, however, disclosed marked different actions of these agents on the inactivation process ofi _si. Therefore, it may be concluded that a combination of a direct effect of the alterations in [H]_i and an effect due to changes in [Ca]_i secondary to proton injections, causes the above electrical changes of single ventricular cells.     

Modulation of the human mirror neuron system during cognitive activity

In this experiment we examined the relationship between the mirror neuron system and increased attention caused by task demands. Whole head MEG recordings were made from 13 participants who were asked to passively observe finger movement sequences, observe these sequences with the knowledge they would later have to perform the sequence presented, and finally, to perform a nonmotor mathematics task based on the finger-movement sequences. Beta-band (15-35 Hz) sensorimotor desynchronization was found in overlapping areas during passive observation and in a separate motor execution condition, indicating the activity of the human mirror neuron system. The beta desynchronization in these areas was enhanced relative to passive viewing when participants had to watch the stimuli to later imitate and when they performed the mathematics task, indicating that mirror neuron system activity can be modulated by attention.     

The effect of hyperpolarizing inputs on the dynamics of a bursting pacemaker neuron model in the pre-Bötzinger complex

The pre-Bötzinger complex (pre-BötC), a subregion of the ventrolateral medulla involved in respiratory rhythm generation, contains intrinsically bursting pacemaker neurons. A previous study proposed Hodgkin–Huxley type minimal models for pacemaker neurons and predicted the effect of a hyperpolarizing input on the dynamics of a model under certain conditions. In this model, bursting is explained by the dynamics of a persistent sodium current. In the present study, the effect of a hyperpolarizing input on the dynamics of a model was investigated under variable conditions. It was observed that immediately after an input of sufficient intensity and duration, an increase in the maximal value of the gating variable h of a persistent sodium current was brought about by a decrease in the timing of the hyperpolarizing input. This corresponds to an observation that immediately after the input, a monotonic increase in the number of spikes in the neuron model was brought about by a decrease in the timing of the hyperpolarizing input. In addition, the dependency of burst duration immediately after the input on the timing of the hyperpolarizing input varied depending on the condition of input. The present study is the first to elucidate that the influence of hyperpolarizing inputs on the number of spikes within a burst in a pacemaker neuron model in the pre-BötC is dependent on the timing of the hyperpolarizing input and to clarify the possible mechanism of this influence, thereby facilitating a detailed understanding of the dynamics of a pacemaker neuron model in the pre-BötC.     

A specific 70 kDa protein induced in the epileptogenic cortex of rats elicits bursting activity and inactivation of potassium current in snail neurons

The capability of a specific 70 kDa (P70) protein produced in cobalt-induced epileptogenic cortex of rat cerebrum in promoting epileptic bursting activity was examined in snail neurons, using the voltage clamp method in combination with the pressure injection technique. Injection of P70 into the neurons elicited bursting activity with paroxysmal depolarization shift together with a reduction in the delayed outward current which was suppressed by tetraethylammonium. These changes of membrane properties induced by P70 were dose-dependently inhibited by prior treatment with the anticonvulsant phenytoin. These findings indicate that P70 causes an inactivation of potassium conductance which may generate epileptic bursting activity.     

Two identified interneurons modulate the firing pattern of pacemaker bursting cells in Helix

Two interneurons modulating the firing pattern of two endogeneous bursting neurons have been identified in the visceral ganglion. They are characterized by large depolarizing afterpotentials and doublets due to the delayed firing of a dense layer of terminal processes converging onto the axon branches from the two bursters. Firing of the terminal processes upon soma or axon stimulation needs either subthreshold depolarization of the cell or conditioning nerve stimulation acting as a gating mechanism.     

A transient outward rectification in the cat sympathetic preganglionic neuron

Intracellular recordings were made from sympathetic pregangionic 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 concentation, 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.     

Properties of a facilitating calcium current in pace-maker neurones of the snail, Helix pomatia.

1. Simultaneous measurements of local voltage clamp currents from patches of soma membrane and K activity at the soma surface were used to analyse the time and voltage dependence of the slow inward current in bursting pace-maker neurones of the snail (Helix pomatia). 2. At low levels of depolarization (less than or equal to mV) a net inward current is recorded simultaneously with an efflux of K ions from the cell. 3. With larger depolarizations (20-170 mV from holding potential of -50 mV) the deficit in net outward charge transfer compared with K efflux and the appearance of inward-going tail currents following repolarization, reveal a persistent inward-going current also under these conditions. This inward current is carried primarily by Ca ions, as demonstrated by its voltage dependence (a minimum at about + 115 mV) and its disappearance in Co-Ringer. It is identified with the slow inward Ca current Iin slow (Eckert & Lux, 1976). 4. The inward current predicted from comparisons of current trajectories reaches a maximum at 15-20 msec (for depolarizations from -50 to 0 mV) and gradually declines with sustained depolarization. 5. Partial inactivation is removed by repolarization to -50 mV and the Ca dependent deficit is greater in the sum of repeated voltage clamp pulses than during sustained depolarization. It is largest for pulses of 25-100 msec duration, decreasing as pulse duration increases. 6. Responses to repeated activation with 100 msec pulses with different repolarization intervals reveal a minimum Iin slow at short intervals (e.g. 20 msec) due to failure to remove partial inactivation. At intermediate intervals (e.g. 200-400 msec) Iin slow shows facilitation. This is revealed in calculations of the net charge transfer and current deficits and is also shown in the tail currents following repolarization. The deficit increases progressively with repetitive stimulation. With longer intervals (e.g. 800-1000 msec) defacilitation during repeated stimulation after the first two pulses is revealed in calculations of deficits, current trajectories and in the tail currents. 7. Although facilitation depends on duration of repolarization between pulses, increasing intermediate hyperpolarizations from the holding potential of -50 mV are usually ineffective in increasing Iin slow. Strong preceding hyperpolarization can even decrease the magnitude of Iin slow and prevent its facilitation with repetitive stimulation,whereas preceding depolarizing pulses can increase Iin slow without preventing its facilitation with repetitive stimulation. 8. The properties of Iin slow are contrasted with previously described membrane conductances and compared with properties attributed to Ca fluxes in other systems.     

Adrenergic activity as a modulating factor in the genesis of myocardial hypertrophy in the rat

Sprague Dawley male rats (200-220 g) were subjected to a long-term treatment with low doses of catecholamines in an attempt to assess the effect of the adrenergic stimulation on ventricular weights, hemodynamics, and hydroxyproline content in the myocardium. Data are presented indicating that both exogenous catecholamines (i.e., isoproterenol) and endogenous catecholamines (released from tyramine) are able to bring about a degree of myocardial hypertrophy associated with a significant increase in the ventricular concentrations of hydroxyproline, without affecting hemodynamic parameters (blood pressures and heart rate). Thus the modulation of sympathetic tone in the genesis of cardiac hypertrophy is demonstrated, and a direct effect of the catecholamines on cardiac cells is postulated which is independent of hemodynamic changes.     

Interstitial cells of Cajal and electrical activity of smooth muscle in porcine ileum

Aim: To identify the interstitial cells of Cajal in the porcine ileum for the first time immunohistochemically and to examine the electrical properties of intestinal smooth muscle in the same region. Methods: In vitro intracellular microelectrode recordings were made from smooth muscle cells in cross‐sectional preparations from abattoir‐derived healthy porcine ileum. Immunohistochemical labelling of interstitial cells of Cajal was performed using an anti‐Kit antibody. Results: Slow waves were recorded in the circular muscle layer of all ileal preparations. The mean resting membrane potential of smooth muscle cells was ?61.0 ± 1.3 mV. Slow waves had a mean amplitude of 8.5 ± 0.5 mV, a frequency of 9.9 ± 0.1 cycles per minute and a duration of 5.6 ± 0.1 s. A waxing and waning pattern of slow wave activity was occasionally observed. In addition, higher frequency spiking activity associated with contractions was observed in some recordings. The L‐type calcium channel blocker nifedipine abolished both the spiking activity and the contractions, but had no significant effect on slow wave characteristics. Current‐injection manipulation of the resting membrane potential had no effect on slow wave amplitude, frequency or duration. Kit‐immunoreactive interstitial cells of Cajal were identified in the ileal samples and were present in the region of the myenteric plexus and in the circular and longitudinal muscle layers. Conclusion: This study recorded slow waves in vitro and demonstrated immunohistochemically the presence of interstitial cells of Cajal in the normal porcine ileum. This study forms a basis for future physiological and pathophysiological comparative studies of intestinal motility.