Early ionic and membrane potential changes caused by the pesticide rotenone in striatal cholinergic interneurons

Mitochondrial metabolism impairment has been implicated in the pathogenesis of several neurodegenerative disorders. In the present work, we combined electrophysiological recordings and microfluorometric measurements from cholinergic interneurons obtained from a rat neostriatal slice preparation. Acute application of the mitochondrial complex I inhibitor rotenone produced an early membrane hyperpolarization coupled to a fall in input resistance, followed by a late depolarizing response. Current-voltage relationship showed a reversal potential of -80 +/- 3 mV, suggesting the involvement of a potassium (K+) current. Simultaneous measurement of intracellular sodium [Na+]i or calcium [Ca2+]i concentrations revealed a striking correlation between [Na+]i elevation and the early membrane hyperpolarization, whereas a significant [Ca2+]i rise matched the depolarizing phase. Interestingly, ion and membrane potential changes were mimicked by ouabain, inhibitor of the Na+-K+ATPase, and were insensitive to tetrodotoxin (TTX) or to a combination of glutamate receptor antagonists. The rotenone effects were partially reduced by blockers of ATP-sensitive K+ channels, glibenclamide and tolbutamide, and largely attenuated by a low Na+-containing solution. Morphological analysis of the rotenone effects on striatal slices showed a significant decrease in the number of choline acetyltransferase (ChAT) immunoreactive cells. These results suggest that rotenone rapidly disrupts the ATP content, leading to a decreased Na+-K+ATPase function and, therefore, to [Na+]i overload. In turn, the hyperpolarizing response might be generated both by the opening of ATP-sensitive K+ channels and by Na+-activated K+ conductances. The increase in [Ca2+]i occurs lately and does not seem to influence the early events.     

Ionic mechanisms of the effects of phenibut and GABA unrelated to changes in the function of chloride channels

It is established that beta-phenyl-GABA (phenibut) and partly GABA elicit direct depolarization of the isolated spinal cord motoneurons. The depolarizing effect of phenibut and a depolarizing component of GABA action do not alter in the presence of picrotoxin (10(-5) mol/l) and in the chloride-deficient medium. This depolarizing phenibut effect which is not bound with activation of GABAA-receptors and chloride channels coupled with them does not alter in Na+-deficient medium, enhances in the medium with excess of K+ ions (10 mol/l) and in presence of imidazol (5 . 10(-4) mol/l) and is completely abolished in the Ca2+-deficient medium with 2 mmol/l of Mn2+ or in the presence of 10(-4) mol/l theophylline. It is supposed that phenibut and partly GABA diminish intracellular concentration of cAMP via GABAB-receptor activation and decrease functional activity of voltage-dependent Ca2+-ionic channels and Ca2+-activated outward K+-currents.     

Changes in ionic conductances induced by cAMP in Helix neurons

Adenosine 3',5'-cyclic monophosphate (cAMP) was injected by a fast and quantitative pressure injection method into voltage-clamped identified Helix neurons. The intracellular elevation of cAMP caused an inward current which was not accompanied by a significant change in membrane conductance in a negative potential range with little activation of voltage-dependent membrane conductances. Near resting potential Na+ ions were the main carrier of the cAMP-induced inward current as measured with ion-selective microelectrodes. TTX did not affect the Na+ influx. K+ and less effective Ca2+ could substitute for Na+ in carrying the inward current. In the presence of Na+, divalent cations such as Ca2+ and Mg2+, and also La3+ exerted an inhibitory influence on the cAMP-induced inward current, and Ca2+ as measured with ion-selective microelectrodes did not contribute significantly to the current. Thus, the inward current was of a non-specific nature. Simultaneously to this cAMP action, the membrane permeability for K+ ions was decreased by cAMP. This effect became particularly obvious when K+ currents were activated by long-lasting, depolarizing voltage steps. In this situation a reduced K+ efflux following cAMP injection was observed by means of K+-selective microelectrodes located near the external membrane surface. Outward K+ currents were less reduced by cAMP if external Ca2+ was replaced by Ni2+. The nearly compensatory increase and decrease of two membrane conductances in the same neuron explained the lack of change in the cell input resistance despite the considerable depolarizing action of intracellularly elevated cAMP.     

Death of subcortical cholinergic neurons in certain neurodegenerative disorders may not be due to an overstimulation of N-methyl-D-aspartate receptors

Stimulation of N-methyl-D-aspartate (NMDA) receptors increases the activity of enzymes involved in the synthesis of nerve cell specific neurotransmitters. In the present study this phenomenon has been used to identify the neurons in the septal-diagonal band region having NMDA receptors. Exposure of cultures relatively enriched in subcortical cholinergic neurons to a depolarizing concentration of K+ (40 mM) significantly enhanced the expression of choline acetyltransferase (ChAT) activity. In contrast, when these septal cells were treated with as much as 100 microM NMDA no significant increase was observed in the activity of choline acetyltransferase, although there was a marked enhancement in glutamate decarboxylase activity. The results would indicate that subcortical cholinergic neurons do not possess excitatory amino acid receptors of the NMDA subtype, and that therefore neurotoxicity mediated through NMDA receptors may not be involved in the death of cholinergic neurons in degenerative disorders of the brain, such as Down's syndrome and Alzheimer's disease, which entail major losses of these neurons.     

The role of platelet membrane potential in the initiation of platelet aggregation

The membrane potential of human platelets, and the role of this potential in platelet aggregation, was assessed using the noncovalent, fluorescent probe DiS-C3-5. High K+ and Gramicidin depolarised the cells, whereas valinomycin in standard (4 mMK+) solution produced a hyperpolarisation. Very small changes in potential were observed when choline Cl replaced NaCl. These findings indicate that platelets possess a relatively K+-perm-selective membrane. The resting potential calculated from the "valinomycin null point" (the K+ concentration gradient at which valinomycin did not change the potential) was approximately -60 mV. Other factors that contribute to the platelet membrane potential include a significant Cl- permeability, demonstrated by replacing Cl- with methylsulphate, and an electrogenic Na+ pump, demonstrated using strophanthidin. Little or no change in potential was observed upon addition of ADP, collagen, U44069 or thrombin. Neither strong depolarisation with high K+ or gramicidin nor hyperpolarisation with valinomycin induced platelet aggregation or altered platelet responses to agonists. It is concluded that the information transduction mechanisms involved in platelet activation do not include changes in platelet membrane potential.     

Effect of prolonged hypoxia on Na+ channel mRNA subtypes in the developing rat cortex.

Voltage-gated Na+ channels are regulated in response to oxygen deprivation in the mammalian cortex. Past investigations have demonstrated that Na+ channel protein expression is up-regulated in the immature brain exposed to prolonged hypoxia. Since it is unknown as to which Na+ channel subtype(s) is involved in this regulation, we used RT-PCR to assess the effect of hypoxia on Na+ channel I, II and III alpha-subunit mRNA expression in the developing rat cortex. Na+ channel II mRNA tended to increase during early development, whereas Na+ channel I and III did not change or slightly decreased with age. Hypoxic exposure for 1-day had no effect on Na+ channel expression, while 5-day hypoxia significantly increased Na+ channel III density, with a slight increase in Na+ channel I and no appreciable change in Na+ channel II. These results suggest that Na+ channel subtype expression in the developing cortex is differentially regulated in response to prolonged hypoxic exposure.     

Active and Passive Membrane Properties of Spinal Cord Neurons that Are Rhythmically Active during Swimming in Xenopus Embryos

Cellular properties have been examined in ventrally located Xenopus spinal cord neurons that are rhythmically active during fictive swimming and presumed to be motoneurons. Resting potentials and input resistances of such neurons are - 75 +/- 2 mV (mean +/- standard error) and 118 +/- 17 M ohm respectively. Most cells fire a single impulse, 0.5 to 2.0 ms in duration and 48.5 +/- 1.8 mV in amplitude, in response to a depolarizing current step. A minority fire several spikes of diminishing amplitude to more strongly depolarizing current. Cells held above spike, threshold fire on rebound from brief hyperpolarizing pulses. Spikes are blocked by 0.1 to 1.0 microM tetrodotoxin (TTX) and are therefore Na+-dependent. Current/voltage (I/V) plots to injected current are approximately linear near the resting potential but become non-linear at more depolarized levels. Cells recorded in TTX with CsCI-filled microelectrodes show a linearized I/V plot at depolarized membrane potentials suggesting the normal presence of a voltage-dependent K+ conductance activated at relatively depolarized levels. Most cells recorded in this way but without TTX fire long trains of spikes of near constant amplitude, pointing to a role of the K+ conductance in limiting firing in normal cells. Spike blockage with TTX reveals, in some cells, a transient depolarizing Cd2+-sensitive and therefore presumably Ca2+-dependent potential that increases in amplitude with depolarization. Cells in TTX, Cd2+, and strychnine, and recorded with CsCI-filled microelectrodes to block active conductances respond to hyperpolarizing current steps with a two component exponential response. The cell time constant (tau0) obtained from the longer of these by exponential peeling is relatively long (mean 15.7 ms). These findings contribute to an increased understanding of the cellular properties involved in spinal rhythm generation in this simple vertebrate.     

The Effect of GHRP-6 on the Intracellular Na+ Concentration of Rat Pituitary Cells in Primary Culture

The objective of the present study was to further investigate the ionic mechanism of the action of GHRP-6 on male rat pituitary cells in culture. A synthetic hexapeptide, GHRP-6 stimulates the secretion of growth hormone both in vivo and in vitro. It is generally accepted that Ca2+ and protein kinase C but not cAMP are involved in the signal transduction pathway of the action of GHRP-6. Ca2+-influx through voltage-gated Ca2+ channels and mobilization of internal stored Ca2+ are thought to be responsible for an increase in cytosolic Ca2+ concentration. For activation of the voltage-gated Ca2+ channels, however, it is not determined whether the membrane Na+ permeability plays a role. To answer this question, we measured intracellular Na+ concentration of the pituitary cells with ion imaging technique. We found that GHRP-6 increased [Na+]i; the Na+ response depended on the presence of extracellular Na+ and was blocked by Gd3+, known as a blocker of nonselective cation channels but not by tetrodotoxin, a blocker of the voltage-gated Na+ channel; thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase, had no effect on the response; Ca2+ chelating agent, BAPTA had no inhibitory effect on the response; ouabain, an inhibitor of Na+-K+ ATPase, did not block the rise in [Na+]i induced by GHRP-6; somatostatin, which hyperpolarizes the cells by activating K+ channels, suppressed the response. These data clearly showed that GHRP-6 increased [Na+]i in the rat pituitary cells including somatotrophs. The rise in [Na+]i is likely to be due to an increase in the membrane Na+ permeability which should depolarize the cells, thereby activating the voltage-gated Ca2+ channels. This process leads to an influx of Ca2+ and subsequent increase in [Ca2+]i which results in an exocytotic release of GH.     

Receptors for gamma-aminobutyric acid (GABA) on aplysia neurons

Aplysia neurons show 5 different types of response (three excitatory and two inhibitory) to iontophoretic application of gamma-aminobutyric acid (GABA). Four of these are associated with a membrane conductance increase, but one is associated with a conductance decrease. The most common response is a fast hyperpolarization which reverses at about--58 mV and is sensitive to manipulation of external Cl- concentration, and thus is due to a specific increase in Cl- conductance. There is an infrequent, slower hyperpolarizing response which does not reverse above about--80 mV and is insensitive to external Cl-. This response appears to result from a conductance increase to K+. Two types of depolarizing responses are associated with conductance increases. These responses differ in their latency, duration and sensitivity to curare. The more frequent is relatively rapid (peak at 1-2 sec) and is depressed by curare at high concentrations. In other neurons, GABA causes a slower response, peaking at 6-10 sec, which is not curare-sensitive. Usually for both types of response, the voltage and conductance changes are completely abolished by perfusion with Na+-free seawater, and the responses cannot be reversed with depolarization. In other neurons such as L11, the response can be reversed with depolarization, and appears to result from a conductance increase to both Na+ and Cl-. In neuron R15, GABA causes a slow depolarizing response (peak at about 9 sec) which is associated with a decreased membrane conductance, probably to K+. The classical GABA antagonists, picrotoxin and bicuculline, block Cl- responses but no others, while the fast Na+ and Cl- responses are depressed by curare. Strychnine does not affect any GABA response. The multiplicity of GABA responses, the specificity of their organization and the fact that only some neurons have receptors for GABA, argue that GABA may have a role as a neurotransmitter in Aplysia. Furthermore, the existence of several types of excitatory GABA response suggests that GABA may function both as an inhibitory and excitatory neurotransmitter.     

Effects of ions and ionic channel activators or blockers on release of alpha-MSH from perifused rat hypothalamic slices.

The involvement of sodium and chloride ions in the process of alpha-melanocyte-stimulating hormone (a-MSH) release from hypothalamic neurons was investigated using perifused rat hypothalamic slices. Three different stimuli were found to increase a-MSH release from hypothalamic slices: high K+ concentration (50 mM), veratridine (50 microM), and the Na+/K(+)-ATPase inhibitor ouabain (1 mM). Spontaneous or K(+)-evoked a-MSH release was insensitive to the specific Na+ channel blocker tetrodotoxin (TTX; 1.5 microM) and to the blocker of K+ channels tetraethylammonium (TEA; 30 mM) or 4-aminopyridine (4-AP; 4 mM). In contrast, blockage of ouabain-sensitive Na+/K(+)-ATPase increased the resting level of a-MSH and caused a dramatic potentiation of K(+)-evoked a-MSH release. The Na+ channel activator veratridine (50 microM) triggered a-MSH release. This stimulatory effect was blocked by TTX and prolonged by TEA application, indicating the occurrence of voltage-sensitive Na+ and K+ channels on a-MSH neurons. Replacement of Na+ by impermeant choline ions from 95 to 60 mM did not alter K(+)-evoked a-MSH release. Conversely, dramatic reduction of the external Na+ concentration to 16 mM caused a robust increase of a-MSH secretion from hypothalamic neurons, likely through activation of the Na+/Ca2+ exchange system. These data indicate that the depolarizing effect of K+ results from direct activation of voltage-operated Ca2+ channels. The lack of effect of TEA on basal a-MSH release prompted us to investigate the possible involvement of chloride ions in the regulation of the spontaneous activity of a-MSH neurons. Substitution of Cl- for impermeant acetate ions did not affect basal or K(+)-evoked a-MSH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Levetiracetam does not modulate neuronal voltage-gated Na+ and T-type Ca2+ currents.

This study investigated whether the mechanism of action of levetiracetam (LEV) is related to effects on neuronal voltage-gated Na+ or T-type Ca2+currents. Rat neocortical neurones in culture were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study voltage-gated Na+ current. Additionally, visually identified pyramidal neurones in the CA1 area of rat hippocampal slices were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study low-voltage-gated (T-type) Ca2+ current. LEV (10 microM-1 mM) did not modify the Na+ current amplitude and did not change (200 microM) the steady-state activation and inactivation, the time to peak, the fast kinetics of the inactivation and the recovery from the steady-state inactivation of the Na+ current. Likewise, LEV (32-100 microM) did not modify the amplitude and did not change the steady-state activation and inactivation, the time to peak, the fast kinetics of the inactivation and the recovery from the steady-state inactivation of the T-type Ca2+current. In conclusion, neuronal voltage-gated Na+ channels do not appear directly involved in the antiepileptic mechanism of action of LEV, and LEV was devoid of effect on the low-voltage-gated (T-type) Ca2+ current in hippocampal neurones.     

Dendritic electrogenesis in rat hippocampal CA1 pyramidal neurons: functional aspects of Na+ and Ca2+ currents in apical dendrites

The regenerative properties of CA1 pyramidal neurons were studied through differential polarization with external electrical fields. Recordings were obtained from somata and apical dendrites in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), DL-2-amino-5-phosphonovaleric acid (APV), and bicuculline. S+ fields hyperpolarized the distal apical dendrites and depolarized the rest of the cell, whereas S÷ fields reversed the polarization. During intradendritic recordings, S+ fields evoked either fast spikes or compound spiking. The threshold response consisted of a low-amplitude fast spike and a slow depolarizing potential. At higher field intensities the slow depolarizing potential increased in amplitude, and additional spikes of high amplitude appeared. During intrasomatic recordings, S+ field evoked repetitive firing of fast spikes, whereas S÷ fields evoked a slow depolarizing, potential on top of which high- and low-amplitude spikes were evoked. Tetrodotoxin (TTX) blocked all types of responses in both dendrites and somata. Perfusion with Ca²⁺-free, Co²⁺-containing medium increased the frequency and amplitude of fast spikes evoked by S+ field and substantially reduced the slow depolarizing potential evoked by S÷ fields. Antidromic stimulation revealed that an all-or-none dendritic component was activated in the distal apical dendrites by back-propagating somatic spikes. The dendritic component had an absolute refractory period of about 4 ms and a relative refractory period of 10–12 ms. Ca²⁺-dependent spikes in the dendrites were followed by a long-lasting afterhyperpolarization (AHP) and a decrease in membrane input resistance, during which dendritic excitability was selectively reduced. The data suggest that generation of fast Na⁺ currents and slow Ca²⁺ currents in the distal part of apical dendrites is highly sensitive to the dynamic state of the dendritic membrane. Depending on the mode and frequency of activation these currents can exert a substantial influence on the input-output behavior of the pyramidal neurons. © 1996 Wiley-Liss, Inc.     

The effect of verapamil on GABA and dopamine release does not involve voltage-sensitive calcium channels

The effect of verapamil, which belongs to the group of drugs collectively referred to as 'organic Ca2+ channel blockers', was investigated on the basal and stimulated release of the neurotransmitters dopamine and GABA in rat striatum synaptosomes. Verapamil inhibits the Na(+)-dependent release of GABA in response to depolarization with an IC50 of 25 microM, whereas it is unable to modify the Na2(+)-independent, Ca2(+)-dependent fraction of GABA release induced by high K+ depolarization. Verapamil does not modify the basal release of GABA but stimulates the basal release of dopamine in a dose-dependent manner (ED50 5 microM). This verapamil-induced outflow of dopamine is independent of Ca2+ and occurs in the presence of tetrodotoxin, indicating that it is not mediated by voltage-sensitive Ca2+ or Na+ channels of the presynaptic membrane. Dopamine release induced by verapamil is cumulative with that induced by depolarizing agents (high K+ or veratridine). As verapamil, pimozide, a neuroleptic of the diphenylbutylpiperidine type, increases the basal and stimulated release of dopamine. We conclude that the opposite effects of verapamil of GABA and dopamine release are due to differences in the releasable fractions of these 2 types of neurotransmitters. Besides, none of these effects are directly linked with the blockade of voltage-operated Ca2+ channels of the presynaptic membrane.     

Subcellular mechanism and site of action of ionic lanthanum at the motor nerve terminal.

The mechanism by which ionic lanthanum (La3+) increases and subsequently decreases spontaneous transmitter release was investigated by recording miniature endplate potentials (MEPPs) at frog neuromuscular junctions. Addition of tetrodotoxin and Co2+ delayed the onset of MEPP frequency increase but did not otherwise prevent the response. Dinitrophenol substantially reduced but did not eliminate the increase, whereas 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8) completely abolished it. Thus, La3+ does not act by depolarizing the terminal or by substituting for Ca2+ at transmitter release sites. Instead, it appears to enter the terminal through Na+ channels and promote Ca2+ release from intracellular organelles. The profound depletion of transmitter with time may be due to the high turnover of transmitter coupled with the inhibition of metabolic processes by La3+.     

Acetylcholine and choline in rat adrenals and brain cortex prisms incubated at elevated concentrations of choline in the medium

Experiments were performed with rat adrenals and brain cortex prisms incubated in vitro in order to clarify whether it is possible to increase their acetylcholine (ACh) content by adding a high concentration of choline to the medium and whether the additional ACh formed can be released by subsequent depolarization. After 60 min incubation with 0.5 mmol/l choline, the concentration of ACh in the adrenals was increased by 116% (compared to the incubation without added choline), while in cortical prisms the observed increase (by 37%) was statistically non-significant. The content of ACh in both tissues was raised by paraoxon during incubations without added choline, but paraoxon did not augment the increased concentration of ACh in tissues incubated with added 0.5 mmol/l choline. The ACh that accumulated in the adrenals during 60 min preincubations with added choline could be released during subsequent depolarizing incubations; the release was Ca2+ independent. In contrast to brain cortex prisms and to the adrenals preincubated without choline, no resynthesis of ACh occurred during the period of depolarization in the adrenals preincubated with 0.5 mmol/l choline. Large amounts of choline accumulated in both tissues during incubations with 0.5 mmol/l choline and the accumulated choline could be released by depolarization; the release of choline from the adrenals was Ca2+ independent. Free choline was produced in the adrenals (presumably from choline esters) during the periods of depolarization. The reason for differences between the effects of increased concentrations of choline on ACh in the adrenals and in brain cortex is not known.(ABSTRACT TRUNCATED AT 250 WORDS)     

Depolarization of mouse forebrain minces with veratridine and high K+: failure to stimulate the Ca2+ independent, spontaneous release of acetylcholine from the cytoplasm due to hydrolysis of the acetylcholine stored there

Both high K+ and veratridine, depolarizing agents with different mechanisms of action, lowered the ACh content of the cytoplasmic (S3) fraction of mouse forebrain minces incubated in a Ca2+-free Krebs solution, without stimulating ACh release or altering the level of ACh in the vesicle-bound (P3) fraction. Veratridine increased the level of choline in the P3 fraction by the same amount as it reduced the level of ACh in the S3 fraction, and these changes did not occur in the presence of tetrodotoxin (TTX). Pretreatment of minces in normal Krebs increased the ACh but not the choline content of the S3 fraction. Following this expansion of the S3 ACh content, veratridine caused an even greater loss of S3 ACh, and increased the Ca2+-independent release of ACh slightly. Under these conditions, veratridine also stimulated the Ca2+ independent release of choline, and this increase exceeded that obtained for the Ca2+-independent release of ACh. Preincubation in normal Krebs with paraoxon did not alter the S3 ACh content after 5 min, but raised it by 78% after 30 min. Under the latter conditions of pretreatment, veratridine then stimulated the Ca2+-independent release of ACh even more, but did not stimulate the release of choline. These results suggest that depolarization of brain tissue does not facilitate the Ca2+-independent release of ACh from the cytoplasm because a portion of ACh stored there is hydrolyzed. When the cytoplasmic level of ACh is sufficiently elevated prior to depolarization, then some ACh escapes hydrolysis and is released independently of Ca2+. It is suggested that the depolarization-induced hydrolysis of cytoplasmic ACh may be mediated by an intraterminal form of AChE and may, in addition to the hydrolysis of extracellular ACh, provide substrate for the formation and release of ACh by the vesicle-bound fraction.     

Developmental increase in the sensitivity to magnesium of NMDA receptors on CA1 hippocampal pyramidal cells

The N-methyl-D-aspartate (NMDA) receptor is involved in processes, such as associative learning, that are particularly important during early postnatal development. It has been suggested that the activity and regulation of this receptor changes during development. Activation of the NMDA receptor is normally limited by Mg2+ present in the extracellular fluid of brain. We have found that Mg2+ less potently antagonizes the depolarizing action of NMDA in developing rats than in adults. A grease-gap method was used to record depolarizations evoked in CA1 hippocampal pyramidal cells by the excitants NMDA and AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate). In the adult CA1 area, Mg2+ shifted the NMDA concentration-response curve to the right in a manner consistent with voltage-dependent open channel block (uncompetitive antagonism) in a preparation with significant receptor reserve. The potency of Mg2+ increased during development; a greater than two-fold change in the EC50 for Mg2+ was observed between 10-15 days of age and adulthood. A concentration of 10 mM reduced the maximum response of CA1 pyramidal cells to NMDA in adult rats, but not in developing rats. In addition, Mg2+ often enhanced the maximum depolarizations evoked by NMDA in 10- to 15-day-old rats, but very seldom in adults. No significant developmental changes in AMPA-induced depolarizations were observed in the presence or absence of Mg2+. These results suggest that synaptically released glutamate will readily activate NMDA receptors during early development and that its ability to do this declines with the maturation of the brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Edrophonium-induced inward membrane current in single neurons physically isolated from Aplysia californica

The action of edrophonium on Aplysia neurons was studied using a concentration clamp technique which combines internal perfusion and a rapid drug application. Edrophonium elicited a dose-dependent inward current in the concentration range 10(-6) to 10(-4) M. At higher concentrations (10(-3) and 10(-2) M), the amplitude of the current often decreased and there was a rapid decay of the current. At these high concentrations, the current increased immediately after washing the neuron with normal solution. These results suggest that edrophonium blocks the ion channel which it opens. Removal of Na+ from the external solution greatly reduced the current amplitude by more than 90%. Removal of Ca2+ also reduced the amplitude of the response; however an increase of Ca2+ did not augment the response. These results suggest that Ca2+ does not carry the current, but is necessary for generation of an Na+-dependent inward current. Edrophonium, 10(-2) M, which completely blocked the current it induced within 20 s, did not significantly affect the voltage-dependent Na+ current. Tetrodotoxin, 1 x 10(-6) M, did not affect the edrophonium response. Hexamethonium, 1 x 10(-4) M, did not change the response elicited by edrophonium, while it significantly reduced the ACh response mediated by Na+. In some neurons edrophonium elicited an inward current, but ACh induced an outward current. Therefore the Na+ channels opened by edrophonium appear to be distinct from both the voltage-gated and ACh receptor-operated Na+ channels.     

Mediobasal hypothalamic neurons are excited by the iontophoretic application of sodium

In the course of studies on the responsiveness of mediobasal hypothalamic neurons to the iontophoretic application of cortisol, it was found that positive currents applied to a sodium chloride (1 M) barrel alone, but not to a choline chloride (1 M) barrel, frequently increased the firing of these neurons. Subsequently, systematic examination demonstrated that out of 102 MBH neurons 52 (51%) increased their firing by at least 30% with application of NaCl, using currents no greater than 10 nA. No such effect was obtained in response to Na application from a dilute solution (0.05 or 0.1 M). When glutamate was absent from the electrodes, the incidence of Na+ sensitivity fell to 17%, despite the routine use of backing currents to the glutamate barrel. K+ ions were more active than Na+ ions in producing excitation. When Na+ sensitivity was found, however, Na+ effects were produced by currents greater than K+ currents producing equivalent excitation. Like glutamate, K+ ions were capable of greatly enhancing responses to Na+. Comparison was made between cortisol and Na+ sensitivity in 70 MBH neurons; 28 cells responded to both, and 24 of them were inhibited by cortisol. Thus Na+ sensitivity is a frequent characteristic of MBH neurons inhibited by cortisol, and was present in 83% of cortisol-sensitive cells in this region. Iontophoresis of Na+ is commonly used as a control in pharmacological studies of the nervous system. Even more common is the case of concentrated NaCl solutions for recording. These procedures may not be as inert as previously thought, particularly in the hypothalamus.     

Neurotransmitters of the cerebellar glomeruli: uptake and release of labeled gamma-aminobutyric acid, glycine, serotonin and choline in a purified glomerulus fraction and in granular layer slices

We have studied some properties of the uptake and release of labeled gamma-aminobutyric acid (GABA), glycine, serotonin and choline in a purified fraction of glomeruli and in slices of the granular layer of the rat cerebellum. The uptake of both GABA and glycine into the glomerulus particles was dependent on the presence of Na+ in the medium. In contrast, the uptake of both serotonin and choline was Na+-independent. In slices of the granular layer also a slight Na+-dependence was observed for both serotonin and choline uptake; imipramine and hemicholinium partially inhibited the uptake of serotonin and choline, respectively. Choline uptake into the glomerulus particles showed two components, with apparent Km values of 16.8 and 102 microM. GABA release was stimulated by K+-depolarization about 100% (peak stimulation) and this value was reduced to 50% when Ca2+ was omitted. The release of glycine was stimulated more rapidly and notably than GABA (200%) and this stimulation was completely abolished in the absence of Ca2+. Serotonin release from the glomerulus particles was only slightly stimulated by depolarization, but this stimulation was strictly Ca2+-dependent. In slices of the granular layer, this stimulation was considerably larger (about 40%) and it was also almost totally dependent on Ca2+. In contrast, after loading with labeled choline the release of radioactivity from both the glomerulus particles and the cerebellar slices was not stimulated at all by K+-depolarization, either in the presence or in the absence of Ca2+. Most of the radioactivity released spontaneously corresponded to choline, and only a small proportion (8-14%) to acetylcholine. From the results of the release experiments and taking into account the pertinent data from the literature, it is concluded that GABA and glycine are probably the transmitters of different populations of Golgi axon terminals, whereas serotonin might be the transmitter of at least a certain population of the mossy fiber giant terminals, in the rat cerebellar glomeruli. In contrast, acetylcholine does not seem to have any transmitter role in the synaptic structures of the glomeruli.