Increase In Membrane Conductance Research Articles

Melatonin generates an outward potassium current in rat suprachiasmatic nucleus neurones in vitro independent of their circadian rhythm

The present study investigated the membrane mechanisms underlying the inhibitory influence of melatonin on suprachiasmatic nucleus (SCN) neurones in a hypothalamic slice preparation. Perforated-patch recordings were performed to prevent the rapid rundown of spontaneous firing rate as observed during whole cell recordings and to preserve circadian rhythmicity in SCN neurones. In current-clamp mode melatonin (1 μM or 1 nM) application, in the presence of agents that block action potential generation and fast synaptic transmission, resulted in a membrane hyperpolarisation accompanied with a decrease in input resistance in the majority of SCN neurones (71–86%). The amplitude of the hyperpolarisation was not found to be significantly different between circadian time 5–12 and 14–21. In voltage-clamp mode melatonin (1 μM or 1 nM) induced an outward current accompanied with an increase in membrane conductance. The current was found to be mainly potassium driven with voltage kinetics resembling those of an open rectifying potassium conductance. Investigations into the signal transduction mechanism revealed melatonin-induced inhibition of SCN neurones to be sensitive to pertussis toxin but independent of intracellular cAMP levels and phospholipase C activity. The present study shows that melatonin, at night-time physiological concentrations, reduces the neuronal excitability of the majority of SCN neurones independent of the time of application in the circadian cycle. Thus in vivo melatonin may be important for circadian time-keeping by amplifying the circadian rhythm in SCN neurones, by lowering their sensitivity to phase-shifting stimuli occurring at night.

Ion channels induced in planar lipid bilayers by the Bacillus thuringiensis toxin Cry1Aa in the presence of gypsy moth (Lymantria dispar) brush border membrane.

The apical brush border membrane, the main target site of Bacillus thuringiensis toxins, was isolated from gypsy moth (Lymantria dispar) larval midguts and fused to artificial planar lipid bilayer membranes. Under asymmetrical N-methyl-d-glucamine-HCl conditions (450 mm cis/150 mm trans, pH 9.0), which significantly reduce endogenous channel activity, trypsin-activated Cry1Aa, a B. thuringiensis insecticidal protein active against the gypsy moth in vivo, induced a large increase in bilayer membrane conductance at much lower concentrations (1.1-2.15 nm) than in receptor-free bilayer membranes. At least 5 main single-channel transitions with conductances ranging from 85 to 420 pS were resolved. These Cry1Aa channels share similar ionic selectivity with P(Cl)/P(NMDG) permeability ratios ranging from 4 to 8. They show no evidence of current rectification. Analysis of the macroscopic current flowing through the composite bilayer suggested voltage-dependence of several channels. In comparison, the conductance of the pores formed by 100-500 nm Cry1Aa in receptor-free bilayer membranes was significantly smaller (about 8-fold) and their P(Cl)/P(NMDG) permeability ratios were also reduced (2- to 4-fold). This study provides a detailed demonstration that the target insect midgut brush border membrane material promotes considerably pore formation by a B. thuringiensis Cry toxin and that this interaction results in altered channel properties.

Mechanism of Postinhibitory Rebound in Molluscan Neurons1

Postinhibitory rebound (PIR) is an intrinsic property of many neurons but the underlying mechanism is not well understood. We studied PIR and its relationship to spike adaptation in B-cells isolated from the buccal ganglia of Aplysia. These neurons exhibit PIR following inhibitory synaptic input and following direct membrane hyperpolarization. Hyperpolarizing and depolarizing voltage clamp pulses from the resting potential evoke slow changes in membrane current that persist in the form of tail currents following the pulses. A subtraction method was used to isolate slow tail currents for study. Current-voltage measurements indicate that slow outward tail currents following depolarizing pulses result from increases in membrane conductance, while inward tail currents following hyperpolarizations to −50 and −60 mV result from conductance decreases. The reversal potential of both outward and inward tail current is between −60 and −70 mV. Tail currents activated by pulses more positive than −60 mV are sensitive to the external K concentration and blocked by injection of Cs and TEA. When Ca2 influx is prevented by bathing cells in Ca2 free saline or by adding Co2 or Ni2 , the tail currents are reduced but a significant fraction of the current is insensitive to these treatments. More negative conditioning pulses activate a second component of inward tail current that is weakly sensitive to K but more strongly effected by substitution of N-methyl glucamine or Li for external Na . We conclude that both PIR and adaptation result from slow changes in a voltage dependent, non-inactivating K conductance that is active at voltages near the resting potential and is not tightly coupled to Ca2 influx. In addition, a second inward current is activated by large hyperpolarizing pulses that results from an increase in Na and K conductance. This second process is likely to contribute to PIR under particular circumstances.

PGE 2 Activation of Apical Membrane Cl − Channels in A6 Epithelia: Impedance Analysis

Measurements of transepithelial electrical impedance of continuously short-circuited A6 epithelia were made at audio frequencies (0.244 Hz to 10.45 kHz) to investigate the time course and extent to which prostaglandin E 2 (PGE 2) modulates Cl − transport and apical membrane capacitance in this cell-cultured model epithelium. Apical and basolateral membrane resistances were determined by nonlinear curve-fitting of the impedance vectors at relatively low frequencies (<50 Hz) to equations (Păunescu, T. G., and S. I. Helman. 2001. Biophys. J. 81:838–851) where depressed Nyquist impedance semicircles were characteristic of the membrane impedances under control Na +-transporting and amiloride-inhibited conditions. In all tissues (control, amiloride-blocked, and amiloride-blocked and furosemide-pretreated), PGE 2 caused relatively small (<∼3 μA/cm 2) and rapid (<60 s) maximal increase of chloride current due to activation of a rather large increase of apical membrane conductance that preceded significant activation of Na + transport through amiloride-sensitive epithelial Na + channels (ENaCs). Apical membrane capacitance was frequency-dependent with a Cole-Cole dielectric dispersion whose relaxation frequency was near 150 Hz. Analysis of the time-dependent changes of the complex frequency-dependent equivalent capacitance of the cells at frequencies >1.5 kHz revealed that the mean 9.8% increase of capacitance caused by PGE 2 was not correlated in time with activation of chloride conductance, but rather correlated with activation of apical membrane Na + transport.

Insulin Stimulates Membrane Conductance in a Liver Cell Line: EVIDENCE FOR INSERTION OF ION CHANNELS THROUGH A PHOSPHOINOSITIDE 3-KINASE-DEPENDENT MECHANISM

Activation of insulin receptors stimulates a rapid increase in the ion permeability of liver cells. To evaluate whether this response involves insertion of ion channels, plasma membrane turnover was measured in a model liver cell line using the fluorescent membrane marker FM1-43. Under basal conditions, the rate of constitutive membrane turnover was approximately 2%min(-1), and balanced exocytosis and endocytosis maintained the total cell membrane area constant. Exposure to insulin stimulated a transient increase in membrane turnover of up to 10-fold above constitutive rates. The response was concentration-dependent (0.001-10 microm). Insulin also caused a parallel increase in membrane conductance as measured by whole-cell patch clamp recording due to opening of Cl(-)- and K(+)-selective ion channels. The insulin-stimulated membrane turnover did not appear to involve the constitutive recycling compartments, suggesting that a distinct pool of vesicles may be involved. The effects of insulin on membrane turnover and membrane conductance were inhibited by blockers of phosphoinositide 3-kinase LY294002 and wortmannin or by disrupting microtubule assembly with nocodazole. Taken together, these findings indicate that insulin stimulates recruitment of new membranes through phosphoinositide 3-kinase-dependent mechanisms. Thus, regulated insertion of a separate population of ion channel-containing vesicles may represent one mechanism for mediating the changes in membrane conductance that are essential for the cellular response to insulin.

D2-like dopamine receptor activation excites rat dorsal raphe 5-HT neurons in vitro.

The aim of the present study was to investigate the effect of dopamine (DA) on the excitability of dorsal raphe nucleus (DRN) 5-hydroxytryptamine (5-HT) neurons using the patch-clamp technique in brain slices. Bath application of DA (1-300 microM) produced a concentration-dependent membrane depolarization in all 5-HT neurons examined. This effect persisted in the presence of tetrodotoxin (TTX; 1 microM) and low extracellular calcium. Moreover, blockade of ionotropic glutamate receptors with 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 2-amino-5-phosphonopentanoic acid (AP5) did not prevent DA-induced depolarization, indicating that it was mediated by a direct effect of DA on 5-HT neurons. The DA-induced depolarization was not antagonized by selective alpha1-adrenergic receptor antagonists, prazosin and WB 4101, but by a nonselective DA receptor antagonist, haloperidol. In addition, the selective D2-like receptor agonist quinpirole and antagonist sulpiride mimicked and blocked DA-induced depolarization, respectively. These results indicate that DA-induced membrane depolarization in DRN 5-HT neurons is mediated by the activation of D2-like DA receptors. The DA-induced membrane depolarization and inward current were associated with an increase in membrane conductance. Examination of the current-voltage (I-V) relationship for the DA-induced inward current revealed that the amplitude of the current increased with membrane hyperpolarization and reversed polarity at a potential near -15 mV. These data suggest that DA-induced depolarization in DRN 5-HT neurons is not mediated by a decrease in potassium conductance, but most likely by the activation of a nonselective cation current.

Dynamics of pavement cell-chloride cell interactions during abrupt salinity change in Fundulus heteroclitus.

Freshwater-adapted killifish (Fundulus heteroclitus) opercular epithelia were dissected and subjected to blood-side hypertonic bathing solution in Ussing-style chambers to simulate the increase in blood osmolality during migration to sea water. Conversely, seawater-acclimated killifish opercular epithelia were subjected to hypotonic bathing solutions to simulate the initial stages of migration to fresh water. Freshwater-acclimation (hypertonic stress) induced a rapid (approximately 30 min) increase in membrane conductance (G(t)) from 3.10+/-0.56 to 7.52+/-1.15 mS x cm(-2) (P<0.01, N=27), whereas seawater-acclimation (hypotonic stress) induced a rapid decrease in G(t) from 8.22+/-1.15 to 4.41+/-1.00 mS x cm(-2) (P<0.01, N=27; means +/- S.E.M.). Control seawater-acclimated membranes had a density of apical crypts (where chloride cells are exposed to the environment; detected by scanning electron microscopy) of 1133+/-96.4 crypts x mm(-2) (N=12), whereas the hypotonically shocked specimens had a lower crypt density of 870+/-36.7 crypts x mm(-2) (P<0.01 N=10; means +/- S.E.M.). Hypertonic shock of freshwater membranes increased crypt density from 383.3+/-73.9 (N=12) to 630+/-102. 9 crypts x mm(-2) (P<0.05; N=11; means +/- S.E.M.). There was no change in density of chloride cells, as detected by fluorescence microscopy; hence, osmotic stress changes the degree of exposure, not the number of chloride cells. Cytochalasin D (5.0 micromol x l(-1)) completely blocked the conductance response to hypotonic shock and the reduction in apical crypt density measured by scanning electron microscopy, while phalloidin (33 micromol x l(-1)), colchicine (3x10(-4)mol x l(-1)) and griseofulvin (1.0 micromol x l(-1)) were ineffective. Actin imaging by phalloidin staining and confocal microscopy revealed extensive actin cords in pavement cell microridges and a ring of actin at the apex of chloride cells. We conclude that the actin cytoskeleton of chloride cells is required to maintain crypt opening and that osmotic shock causes chloride cells to adjust their apical crypt size.

Angiotensin II induces inward currents in subfornical organ neurones of rats.

The action of angiotensin II on subfornical organ (SFO) neurones was studied using whole-cell current and voltage-clamp recordings in rat slice preparations. In the current-clamp mode, membrane depolarization in response to angiotensin II was accompanied by an increased frequency of action potentials and an increased membrane conductance. In the voltage-clamp mode, angiotensin II elicited inward currents in a dose-dependent manner. The net angiotensin II-induced inward currents were voltage-independent, with a mean reversal potential of -29.8 +/- 6.2 mV. Amplitudes of the angiotensin II-induced inward currents were decreased during perfusion with a low sodium medium. The angiotensin II-induced inward currents were blocked by the AT1 antagonist losartan, and were partially blocked by the AT2 antagonist PD-123319. Neurones which were sensitive to angiotensin II were found in the peripheral region of the SFO, whereas neurones in the central region were less sensitive to angiotensin II. These results suggest that angiotensin II induces inward currents, with opening of nonselective cation channels through mainly AT1 receptors in a subpopulation of SFO neurones of rats.

Electrophysiological and hemolytic activity elicited by the venom of the jellyfish Cassiopea xamachana

In this study, we determined hemolysis activity in human and sheep erythrocytes, and characterized the electrical responses in Xenopus oocyte membrane elicited by the venom of the jellyfish Cassiopea xamachana ( Cx). The Cx venom produced hemolysis in both species, being more potent on human red cells. The electrophysiological study showed that the Cx venom elicited three different responses in the oocytes. One current was generated in all the oocytes tested and corresponded with a slow inward current ( I Cx ) associated with an increase in membrane conductance. I Cx was concentration-dependent and had a reversal potential of −10.3±0.4 mV. Ionic substitution studies indicated that the conductive pathway was mainly permeable to cations and non-selective. The oocyte membrane resistance was completely recovered after washout of the venom, this suggested that the effect was due to generation of a specific membrane conductance as opposed to a possible non-specific membrane breakdown. A comparative study with three distinct native cationic channels present in the oocyte membrane [i.e. (1) hemi-gap-junction channels, (2) mechanosensitive channels, and (3) the ouabain-sensitive channel activated by palytoxin], showed that I Cx might correspond to opening of mechanosensitive channels or to activation of an unknown cationic channel located in the oocyte membrane. The bioactive fraction eliciting I Cx were peptides and was separated from two other peptidic hemolytic fractions by chromatography.

Long-term effects of urea on urothelial barrier function

Under physiologic conditions, the bladder epithelium (urothelium) is bathed on the urine side (mucosal solution) by a solution that has a variable composition but typically is high in potassium, low in sodium, and has a urea concentration that varies between 100 mmol/L to 500 mmol/L. In contrast, the interstitial side (serosal solution) is bathed in a solution whose composition is similar to that of blood. Previous studies have determined the effect of short-term exposure of the urothelium to serosal urea. It was demonstrated that short-term exposure (<10 minutes) of the serosal side of the urothelium to urea (200 mmol/L or 500 mmol/L) resulted in an increase in the apical (urine facing) membrane ion permeability. On removal of the urea the apical membrane, conductance decreased to control values; thus, the urea effect is reversible. Of interest is that the increase in apical membrane permeability by serosal urea can be completely blocked by the simultaneous presence of mucosal urea. The present study investigates the long-term effects of both mucosal and serosal urea on the mammalian urothelium using electrophysiologic techniques. Addition of urea to the serosal solution (after a short delay) resulted in an exponential increase in the transepithelial conductance followed by a plateau phase and ending in a rapid and irreversible increase in conductance. The exponential increase in transepithelial conductance was due to an increase in cell membrane conductance, whereas the irreversible increase in conductance was due to an increase in the paracellular conductance. The duration of the initial delay and plateau phase was inversely related to the concentration of serosal urea. Addition of urea to both the mucosa and serosal sides of the urothelium resulted in an increase in the duration of the delay, a decrease in the magnitude of the exponential increase, and an increase in the duration of the plateau phase. Thus symmetric urea delays the loss of urothelial barrrier function caused by serosal urea. The ability of mucosal urea to delay the onset of the irreversible increase of urothelial conductance allows the urothelium to perform its primary function, which is to act as a barrier to the movement of water, electrolytes, and nonelectrolytes between the urine and plasma.

Differential regulation of GABA release and neuronal excitability mediated by neuropeptide Y1 and Y2 receptors in rat thalamic neurons.

1. Neuropeptide Y (NPY) produced inhibitory effects on neurons of the thalamic reticular nucleus (RT; n = 18) and adjacent ventral basal complex (VB; n = 22), which included hyperpolarization (approximately 4 mV), a reduction in rebound and regular spikes and an increased membrane conductance. These effects were mediated predominantly via NPY1 receptor activation of G-protein-activated, inwardly rectifying K+ (GIRK) channels. 2. NPY reduced the frequency of spontaneous GABAA receptor-mediated inhibitory postsynaptic currents (sIPSCs) in RT (by 60 +/- 7 %, n = 14) and VB neurons (by 25 +/- 11 %, n = 16), but had no effect on the kinetic properties of sIPSCs. After removal of the RT nucleus, the inhibitory effects of NPY on sIPSCs in VB neurons remained (29 +/- 7 %, n = 5). The synaptic effects were mediated via NPY2 receptors. 3. NPY inhibited the frequency of miniature IPSCs (mIPSCs) in RT and VB neurons (by 63 +/- 7 %, n = 5, and 37 +/- 8 %, n = 10, respectively) in the presence of tetrodotoxin (TTX) (1 microM) but not TTX (1 microM) and Cd2+ (200 microM). 4. NPY inhibited evoked IPSCs in both RT (by 18 +/- 3 %, n = 6) and VB (by 5 +/- 4 %, n = 6) neurons without change in short-term synaptic plasticity. 5. We conclude that NPY1 and NPY2 receptors are functionally segregated in the thalamus: NPY1 receptors are predominantly expressed at the somata and dendrites and directly reduce the excitability of neurons in both the RT and VB nuclei by activating GIRK channels. NPY2 receptors are located at recurrent (RT) and feed-forward GABAergic terminals (VB) and downregulate GABA release via inhibition of Ca2+ influx from voltage-gated Ca2+ channels.

Excitotoxicity of Lathyrus Sativus Neurotoxin in Leech Retzius Neurons

The effects of Lathyrus sativus neurotoxin were studied on the cell membrane potential and cellular cation composition in Retzius nerve cells of the leech Haemopis sanguisuga, with ion-selective microelectrodes using liquid ion-exchangers. Bath application of 10-4 mol/l Lathyrus sativus neurotoxin for 3 min depolarized the cell membrane potential and decreased the input resistance of directly polarized membrane in Retzius neurons. At the same time the cellular Na+ activity increased and cellular K+ activity decreased with slow but complete recovery, while the intracellular Ca2+ concentration was not changed. Na+-free Ringer solutions inhibited the depolarizing effect of the neurotoxin on the cell membrane potential. Zero-Ca2+ Ringer solution or Ni2+-Ringer solution had no influence on the depolarizing effect of the neurotoxin on the cell membrane potential. It is obvious that the increase in membrane conductance and depolarization of the cell membrane potential are due to an influx of Na+ into the cell accompanied by an efflux of K+ from the cell.

Activation of Background Membrane Conductance by the Tyrosine Kinase Inhibitor Tyrphostin A23 and Its Inactive Analog Tyrphostin A1 in Guinea Pig Ventricular Myocytes

The effects of the tyrosine kinase (TK) inhibitor tyrphostin A23 and its inactive analog tyrphostin A1 on background membrane conductance were investigated in guinea pig ventricular myocytes. TK-inhibiting A23 reversibly increased membrane conductance under conditions designed to minimize Na+, Ca2+, K+, and Na+-K+ pump currents. Similar stimulatory action was obtained with TK-inactive A1. The tyrphostin-induced current was inhibited by omitting external Na+ or Ca2+, suppressed by chelating internal Ca2+, blocked by external Cd2+ and Ni2+, and insensitive to changes in internal Cl- concentration. We conclude that tyrphostins have a direct, TK-independent action that increases membrane conductance probably by stimulating Na+-Ca2+ exchange.

Characterisation of inhibitory and excitatory postsynaptic currents of the rat medial superior olive.

The medial superior olive (MSO) is part of the binaural auditory pathway, receiving excitatory projections from both cochlear nuclei and an inhibitory input from the ipsilateral medial nucleus of the trapezoid body (MNTB). We characterised the excitatory and inhibitory synaptic currents of MSO neurones in 3- to 14-day-old rats using whole-cell patch-clamp methods in a brain slice preparation.A dual component EPSC was mediated by AMPA and NMDA receptors. The AMPA receptor-mediated EPSC decayed with a time constant of 1.99+/-0.16 ms (n = 8). Following blockade of glutamate receptors, a monosynaptic strychnine-sensitive response was evoked on stimulation of the MNTB, indicative of a glycine receptor-mediated IPSC. GABAA receptors contributed to IPSCs in rats under 6 days old (bicuculline blocked 30% of the IPSC). In older rats little or no bicuculline-sensitive component was detectable, except in the presence of flunitrazepam. These glycinergic IPSCs showed a reversal potential that varied with changes in [Cl-]i, as predicted by the Nernst equation. The IPSC exhibited two developmentally relevant changes. (i) At around postnatal day 6, the GABAA receptor-mediated component declined, leaving a predominant glycine-mediated IPSC. The isolated glycinergic IPSC decayed with time constants of 7.8+/-0.3 and 38.3+/-1.7 ms, with the slower component contributing 7.8+/-0.6% of the peak amplitude (n = 121, 3-11 days old, -70 mV, 25 deg C). (ii) After day 11 the IPSC fast decay accelerated to 3.9+/-0.3 ms (n = 12) and the magnitude of the slow component declined to less than 1%. Spontaneous miniature glycinergic IPSCs (mIPSCs) were variable in amplitude and were of large conductance (1.83+/-0.19 nS, n = 8). The amplitude was unchanged on lowering [Ca2+]o. The time course of evoked and spontaneous miniature glycinergic IPSCs were compared. The 10-90% rise times were 0.7 and 0.6 ms, respectively. The evoked IPSC decayed with a fast time constant of 7.2+/-0.7 ms, while the mIPSC decayed with a fast time constant of 5.3+/-0.4 ms in the same seven cells.The glycinergic IPSC decay was voltage dependent with an e-fold change over 118 mV. The temperature dependence of the IPSC decay indicated a Q10 value of 2. Picrotoxin and cyanotriphenylborate had little or no effect on IPSCs from 6- to 14-day-old animals, implying homomeric channels are rare. We conclude that the MSO receives excitatory inputs mediated by AMPA and NMDA receptors and a strong glycinergic IPSC which has a significant contribution from GABAA receptors in neonatal rats. Functionally, the IPSC could increase membrane conductance during the decay of binaural glutamatergic EPSCs, thus refining coincidence detection and interaural timing differences.

The effect of hexadecaprenol on molecular organisation and transport properties of model membranes.

The Langmuir monolayer technique and voltammetric analysis were used to investigate the properties of model lipid membranes prepared from dioleoylphosphatidylcholine (DOPC), hexadecaprenol (C80), and their mixtures. Surface pressure-molecular area isotherms, current-voltage characteristics, and membrane conductance-temperature were measured. Molecular area isobars, specific molecular areas, excess free energy of mixing, collapse pressure and collapse area were determined for lipid monolayers. Membrane conductance, activation energy of ion migration across the membrane, and membrane permeability coefficient for chloride ions were determined for lipid bilayers. Hexadecaprenol decreases the activation energy and increases membrane conductance and membrane permeability coefficient. The results of monolayer and bilayer investigations show that some electrical, transport and packing properties of lipid membranes change under the influence of hexadecaprenol. The results indicate that hexadecaprenol modulates the molecular organisation of the membrane and that the specific molecular area of polyprenol molecules depends on the relative concentration of polyprenols in membranes. We suggest that hexadecaprenol modifies lipid membranes by the formation of fluid microdomains. The results also indicate that electrical transmembrane potential can accelerate the formation of pores in lipid bilayers modified by long chain polyprenols.

Quisqualate induces an inward current via mGluR activation in neocortical pyramidal neurons

Activation of metabotropic glutamate receptors (mGluRs) has multiple effects on the excitability of pyramidal neurons in rat frontal neocortex. Synaptic transmission and intrinsic excitability are both affected. During studies of the effects of quisqualate on synaptic activity, it was observed that quisqualate also induced a slow inward current. Whole-cell patch clamp recordings were obtained from layer II/III pyramidal neurons of neocortical slices in vitro. The bath solution contained APV, CNQX and bicuculline to block ionotropic glutamate and GABA A receptors. At a holding potential of −70 mV, quisqualate (2 μM) induced an inward current of about 60 pA. The response was reversible upon washing. This current was associated with an increase in membrane conductance and was still seen in the presence of TTX (0.5 μM). Bath application of the nonselective mGluR antagonist, ( R,S)-α-methyl-4-carboxyphenyglycine (MCPG, 200–500 μM) reduced the current by 70%. Other mGluR agonists (ACPD, DHPG, L-CCG-1 and L-AP4) did not induce a significant inward current at the concentrations tested. The current–voltage relation of the quisqualate-induced current was linear with a reversal potential near 0 mV suggesting involvement of nonselective cation channels. The quisqualate-induced inward current was markedly reduced (72%) with 200 μM GDP-β-S in the pipette solution, indicating that it is a postsynaptic phenomenon mediated by a G-protein dependent mechanism. These results suggest that mGluRs can directly increase the postsynaptic excitability of pyramidal cells.

Alpha 1-adrenoceptor-mediated excitation of substantia nigra pars reticulata neurons

The effect of noradrenaline was studied in principal neurons of the substantia nigra pars reticulata in rat brain slices using patch clamp recordings. Perfusion of noradrenaline or the α 1-adrenoceptor agonist phenylephrine increased the spontaneous firing activity of reticulata cells. The α 1-adrenoceptor antagonist prazosin counteracted the effects of noradrenaline. In contrast, the β-adrenoceptor agonist isoproterenol did not affect the activity of reticulata cells and the β-adrenoceptor antagonist pindolol did not prevent noradrenaline's effect. In whole-cell recordings, at −60 mV holding potential, noradrenaline caused a tetrodotoxin-resistant inward current with a time-course similar to the increase in firing activity. Analysis of the reversal potential of this current did not give homogeneous results. The net noradrenaline current could be associated with a conductance decrease or increase, or in some cases it did not reverse over a range from −120 to −30 mV. It is suggested that noradrenaline increases the excitability of substantia nigra reticulata cells through α 1-adrenoceptors. Both a reduction and an increase in membrane conductance may mediate this effect. The increase in the tonic firing of principal reticulata cells caused by noradrenaline may have significant consequences in regulating the final output of the basal ganglia and consequently in motor-related behaviours.

Cellular Mechanisms of Long-Lasting Adaptation in Visual Cortical NeuronsIn Vitro

The cellular mechanisms of spike-frequency adaptation during prolonged discharges and of the slow afterhyperpolarization (AHP) that follows, as occur in vivo with contrast adaptation, were investigated with intracellular recordings of cortical neurons in slices of ferret primary visual cortex. Intracellular injection of 2 Hz sinusoidal or constant currents for 20 sec resulted in a slow (tau = 1-10 sec) spike-frequency adaptation, the degree of which varied widely among neurons. Reducing either [Ca(2+)](o) or [Na(+)](o) reduced the rate of spike-frequency adaptation. After the prolonged discharge was a slow (12-75 sec) AHP that was associated with an increase in membrane conductance and a rightward shift in the discharge frequency versus injected current relationship. The reversal potential of the slow AHP was sensitive to changes in [K(+)](o), indicating that it was mediated by a K(+) current. Blockade of transmembrane Ca(2+) conductances did not reduce the slow AHP. In contrast, reductions of [Na(+)](o) reduced the slow AHP, even in the presence of pronounced Ca(2+) spikes. We suggest that the activation of Na(+)-activated and Ca(2+)-activated K(+) currents plays an important role in prolonged spike-frequency adaptation and therefore may contribute to contrast adaptation and other forms of adaptation in the visual system in vivo.

Membrane Mechanisms Underlying Contrast Adaptation in Cat Area 17In Vivo

Contrast adaptation is a psychophysical phenomenon, the neuronal bases of which reside largely in the primary visual cortex. The cellular mechanisms of contrast adaptation were investigated in the cat primary visual cortex in vivo through intracellular recording and current injections. Visual cortex cells, and to a much less extent, dorsal lateral geniculate nucleus (dLGN) neurons, exhibited a reduction in firing rate during prolonged presentations of a high-contrast visual stimulus, a process we termed high-contrast adaptation. In a majority of cortical and dLGN cells, the period of adaptation to high contrast was followed by a prolonged (5-80 sec) period of reduced responsiveness to a low-contrast stimulus (postadaptation suppression), an effect that was associated, and positively correlated, with a hyperpolarization of the membrane potential and an increase in apparent membrane conductance. In simple cells, the period of postadaptation suppression was not consistently associated with a decrease in the grating modulated component of the evoked synaptic barrages (the F1 component). The generation of the hyperpolarization appears to be at least partially intrinsic to the recorded cells, because the induction of neuronal activity with the intracellular injection of current resulted in both a hyperpolarization of the membrane potential and a decrease in the spike response to either current injections or visual stimuli. Conversely, high-contrast visual stimulation could suppress the response to low-intensity sinusoidal current injection. We conclude that control of the membrane potential by intrinsic neuronal mechanisms contributes importantly to the adaptation of neuronal responsiveness to varying levels of contrast. This feedback mechanism, internal to cortical neurons, provides them with the ability to continually adjust their responsiveness as a function of their history of synaptic and action potential activity.

A picoampere current generator for membrane electroporation

Electroporation is the electric-field induced formation of a voltage-dependent population of hydrophilic pores within a bilayer lipid membrane (BLM). This phenomenon can be observed as a transient increase in membrane conductance after the application of a voltage jump above a certain threshold. A very low current source is required to measure voltage fluctuations of a BLM due to voltage induced creation of dynamical pores during the electroporation. A current source able to drive a high-resistive load (on the order of 1012 Ω) by a constant current of 10 pA has been designed and realized. The current is maintained constant, with an uncertainty of 1.2% even when the resistive load fluctuates to 109 Ω over a very brief interval (10−3 s). These peculiar features have been obtained by designing a very simple circuit configuration, that gives an excellent match between the planned and the experimental behavior. The circuit bandwidth and the current precision are not the result of the circuit configuration, but are only limited by active components.