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L M Nowak

Publications and source records attributed to L M Nowak.

18 recordsLinked to original sources
PubMed1998

Extracellular 3',5' cyclic guanosine monophosphate inhibits kainate-activated responses in cultured mouse cerebellar neurons.

The effects of extracellularly applied 3'-5' cyclic guanosine monophosphate (cGMP) on kainate responses from cultured cerebellar granule and Purkinje neurons were investigated using whole-cell and outside-out patch recording modes. Cerebellar granule cell responses to kainate were not homogeneous, nor were the effects of cGMP. Therefore, effects of cGMP are described for two groups of granule cells categorized on the basis of the underlying channel conductance estimated by variance analysis. Cells with high-noise kainate responses had average channel conductances of 5 to 7 picoseimens, whereas the average conductances of low-variance noise responses were 0.3 to 2.0 picoseimens. High-noise kainate responses were inhibited by externally applied cGMP (5-1000 microM) in a rapidly reversible and dose-dependent manner. IC50 values were estimated at approximately 150 microM cGMP for 25 microM kainate and approximately 500 microM cGMP for 100 microM kainate. Evidence that cGMP-mediated inhibition of high-noise kainate responses occurred by a competitive mechanism included the following: 1) cGMP-mediated inhibition was overcome by increasing agonist concentration. 2) The shape of kainate current-voltage (I-V) curves and their reversal potentials were unchanged in cGMP. 3) Neither the estimated conductance nor the kinetics of the kainate-activated channels was affected by cGMP. In contrast to the uniform effects of cGMP on the high-noise kainate responses, the effects on low-noise kainate responses were variable. Half of the low-noise kainate responses were inhibited by cGMP to a similar extent as the high-noise responses; however, the other 50% of cells exhibiting low-noise kainate responses appeared to be less sensitive to the cyclic nucleotide. Moreover, cGMP coapplication decreased the estimated conductances for some low-noise kainate responses and altered their noise kinetics, which suggests either that cGMP-sensitive and -insensitive kainate receptor channels are coexpressed in these cells or that cGMP-mediated inhibition is not competitive for this subgroup of glutamate receptor channels. Overall, these data indicate that there are direct inhibitory effects of extracellular cGMP on a large group of excitatory synapses in the CNS--effects that need to be taken into account when investigators utilize membrane-permeable cGMP analogs. Whether this cGMP-mediated inhibition has a functional role in brain is unknown.

Animals↗
PubMed1992

Slow voltage-dependent changes in channel open-state probability underlie hysteresis of NMDA responses in Mg(2+)-free solutions.

Many single-channel studies rely on the assumption that the channels are functioning under steady-state conditions. In examining the basis for nonlinear whole-cell current-voltage curves in Mg(2+)-free solutions we discovered that N-methyl-D-aspartate (NMDA) channels in excised patches reversibly shifted their open-state probability (Po) in a voltage-dependent way, exhibiting approximately 3- to 4-fold greater Po at positive potentials than at rest. Changes in Po were mainly attributable to shifts in frequency of channel opening. Po changed remarkably slowly (2-15 min), explaining the hysteresis of whole-cell current-voltage curves obtained in nonequilibrium conditions. The slow increase in Po provides a mechanism by which NMDA channels can substantially increase Ca2+ influx in cells depolarized for prolonged periods of time and may play a role in excitotoxicity.

Animals↗
PubMed1992

Effects of low doses of bicuculline on N-methyl-D-aspartate single-channel kinetics are not evident in whole-cell currents.

Bicuculline methiodide (BIC-Mel) (10-100 microM) altered the kinetics of N-methyl-D-aspartate (NMDA) responses in single-channel and whole-cell recordings. The principal effect of BIC-Mel (10-100 microM) on NMDA channels was a dose-dependent decrease in mean channel open time (tau o), accompanied by the introduction of a new closed time (tau B) of 14.0 +/- 3.5 msec (mean +/- standard deviation; n = 14) in closed time distributions, which was independent of BIC-Mel concentration. BIC-Mel (10-100 microM) increased the frequency of NMDA channel opening in a dose-dependent manner, offsetting the decrease in tau o, such that the total time spent in the open state per minute was unchanged, and thus the total charge/min through NMDA channels was unchanged. Similarly, the amplitudes of NMDA whole-cell current responses were not noticeably affected by 10-80 microM BIC-Mel, even though power spectra density analysis of the whole-cell NMDA-stimulated noise revealed changes in the underlying channel kinetics in the presence of BIC-Mel. Taken together, the effects of 10-80 microM BIC-Mel on NMDA responses were consistent with the predictions of the sequential block model; however, the effects of BIC-Mel exhibited no obvious voltage dependence. In addition to the low-dose effects of BIC-Mel, 100 and 200 microM BIC-Mel inhibited whole-cell NMDA responses. The inhibition by 100 microM BIC-Mel was not large, but it was augmented from 15% to 30% by increasing the NMDA concentration from 10 microM NMDA to 20 microM NMDA, indicating that channel activation was necessary for BIC-Mel-mediated inhibition. Preliminary single-channel experiments performed under conditions conducive to trapping of an open channel blocker at its binding site indicated that the effect of BIC-Mel on tau o persisted after the removal of the blocker, consistent with use dependence of the dissociation of BIC-Mel from the NMDA receptor-channel complex.

Animals↗
PubMed1991

Galanin inhibits a dihydropyridine-sensitive Ca2+ current in the RINm5f cell line.

Mechanisms of action of the neuropeptide galanin, a putative neuromodulator in the central and peripheral nervous systems, have been evaluated extensively in insulin-secreting cells isolated from pancreas and cell lines derived from pancreatic tumors. Galanin inhibits insulin secretion from these cells through several mechanisms, including activation of ATP-dependent K+ channels and inhibition of adenylyl cyclase leading to a decrease in cAMP. Here we report that galanin also inhibits a dihydropyridine-sensitive Ca2+ current. Both electrophysiological actions by galanin would result in less Ca2+ entry, as the action to increase K+ current would hyperpolarize the cells and the decrease in voltage-gated Ca2+ current would decrease Ca2+ influx at depolarized potentials where these channels are activated. These galanin actions would directly counter the two opposing electrophysiological responses to carbohydrate stimulation in RINm5f cells, which are to inhibit K+ current and to stimulate Ca2+ current. Given that stimulation of presynaptic nerve terminals in pancreas releases galanin, these results suggest that Ca(2+)-dependent insulin release from native pancreatic beta cells may also be regulated by similar neuropeptide effects.

Animals↗
PubMed1991

Multiple effects of tetraethylammonium on N-methyl-D-aspartate receptor-channels in mouse brain neurons in cell culture.

1. The mechanisms of tetraethylammonium (TEA) antagonism of N-methyl-D-aspartate (NMDA) responses were investigated in cultured mouse cortical neurons by analysing single-channel and whole-cell currents from patch clamp recordings. TEA (1-5 mM) decreased whole-cell NMDA responses. Kainate and quisqualate receptor-mediated responses were unaffected at these TEA concentrations. 2. In whole-cell recordings, increasing the NMDA concentration while keeping the TEA concentration constant resulted in greater inhibition by TEA. Thus, TEA-mediated inhibition of NMDA responses was not due to competitive antagonism, and the greater inhibition by a single dose of TEA as NMDA concentration was elevated indicated some form of non-competitive inhibition. In single-channel recordings, two inhibitory effects were seen in 1-5 mM-extracellular TEA: single-channel conductance (gamma) was decreased, and the frequency of channel events was decreased. These effects were not accompanied by any change in average channel open time. 3. Single-channel current-voltage (I-V) curves obtained in 2, 5, 10 and 30 mM-TEA indicated the decrease in NMDA channel conductance was voltage dependent with larger reduction occurring as patches were hyperpolarized. The data were well fitted by the Woodhull model with the dissociation constant (KD) showing an e-fold increase in inhibition for a 43-45 mV change in membrane potential. The 0 mV KD was 45 mM-TEA decreasing to about 11 mM at -60 mV. The TEA block site appeared to sense approximately 60% of the transmembrane potential field (delta = 0.6) for extracellular application of TEA. 4. The decrease in channel opening frequency seen in TEA was concentration dependent and generally more sensitive to extracellular TEA than the channel block effect. There was a 50% reduction in the number of NMDA channel openings observed in 5 mM-TEA. Increasing either NMDA or glycine concentrations in constant TEA concentration caused an additional decrease in the frequency of NMDA channel opening. In contrast to extracellular TEA, intracellular TEA had no noticeable effect on open-state probability. 5. NMDA single-channel currents were observed at positive potentials after completely replacing pipette Cs+ by 140 mM-TEA-Cl indicating TEA could serve as a current carrier through NMDA channels. Single channel I-V curves obtained with pipettes containing 70 or 140 nM-TEA in place of equivalent amounts of Cs+ were fitted by the Goldman-Hodgkin-Katz (GHK) equation over the range of -80 to +70 mV assuming a permeability of 0.45 compared with a Cs+ permeability of 1.0.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Mechanisms of blockade of excitatory amino acid receptor channels.

Ion channels coupled to NMDA, kainate and AMPA receptors are the target of pharmacological regulation by a variety of drugs and ions. While these channels are all nonselectively permeated by Na+ and K+ ions, the NMDA receptor-channel complex contains a number of pharmacological sites distinct from those found on the others. For example, Mg2+ ions rapidly and reversibly block open NMDA channels in a highly voltage-dependent manner. Its extreme voltage dependence suggests that the Mg2+ binding site lies deep within the ion channel pore. By contrast the voltage-dependent block of activated channels by the dissociative anesthetic 'slow channel blockers' has unusual characteristics. In the fourth article in our series on excitatory amino acids, John MacDonald and Linda Nowak analyse the characteristics of these two types of block and describe the hypotheses that have been put forward to explain the mechanisms involved.

Animals↗
PubMed1983

Muscarine-sensitive voltage-dependent potassium current in cultured murine spinal cord neurons.

Muscarine produced membrane depolarization and decreased membrane conductance of mouse spinal cord neurons in dissociated cell culture. When the neurons were voltage clamped, muscarine evoked inward currents which increased with membrane depolarization and decreased with membrane hyperpolarization. However, the muscarine-induced inward currents did not invert at large negative potentials, suggesting that muscarine decreased a voltage-dependent potassium current (m-current) [2]. Using the voltage-jump current-relaxation technique, m-current was demonstrated in spinal cord neurons and shown to be a muscarine-sensitive potassium current.

Action Potentials↗
PubMed1983

Flicker fusion characteristics of rod photoreceptors in the toad.

Critical fusion frequency (CFF) of toad rods was examined at various intensities using intracellular recordings. Data were compared to CFF measurements (as a function of intensity) obtained with the electroretinogram (ERG) and with a horizontal cell. In all instances the rod (and rod mediated) responses produced a curve which increased monotonically to a high frequency asymptote at about 6 Hz. The curves obtained with the ERG and the horizontal cell were double branched. The upper branch exhibited a Purkinje shift and thus must have been produced by cones. Even when using a red background to suppress cone sensitivity, there was no sign of a second rod mediated branch in the CFF curves from this retina.

Animals↗
PubMed1983

Ionic mechanism of muscarinic cholinergic depolarization of mouse spinal cord neurons in cell culture.

1. Muscarinic cholinergic actions were investigated in a population of large multipolar spinal cord neurons in primary dissociated cell culture using conventional intracellular recording and single-microelectrode voltage-clamp techniques. 2. Cholinergic agonists were applied to the surface of neuronal somata by pressure ejecting drug-containing bathing medium from small blunt (2-10 microns) glass micropipettes. Atropine was applied by diffusion from large (20-30 microns) blunt micropipettes positioned near the soma. 3. Muscarine increased action-potential firing and evoked slow sustained membrane depolarization. Action potentials but not slow membrane depolarizations were eliminated by the sodium channel blocker, tetrodotoxin. Muscarine-induced depolarizing responses were unaffected by the calcium channel blocker, cadmium. 4. Depolarizing responses evoked by selective and nonselective muscarinic cholinergic agonists were dose dependent, reversibly antagonized by atropine, and did not desensitize. 5. Muscarine depolarized neurons and decreased membrane conductance during recording with both 3 M KCl- and 4 M KAc-filled intracellular recording micropipettes. When membrane potential was held constant using the single-electrode voltage-clamp technique (KCl-filled micropipettes), muscarine and gamma-aminobutyric acid (GABA) evoked inward currents at resting membrane potential. GABA-induced inward current responses were decreased by depolarization and had reversal potentials near -30 mV, consistent with GABA increasing chloride conductance. Muscarine-induced inward current responses were increased by depolarization and had extrapolated reversal potentials near -80 mV, consistent with muscarine decreasing a potassium conductance. 6. Unlike GABA-induced currents, muscarine-induced currents evoked in normal Tris-buffered saline (5 mM potassium) did not vary as a linear function of membrane potential and did not reverse polarity in six of seven neurons near potassium equilibrium potential. However, in high-potassium medium (15 mM) muscarinic responses did reverse polarity and current was linearly related to membrane potential. Thus, the apparent voltage dependence of muscarine responses was probably due to voltage dependency of the potassium conductance and not due to potassium channel rectification. 7. Preliminary evidence (37) indicates that muscarine decreases a time- and voltage-dependent potassium current in some cultured spinal cord neurons. Whether reduction of m current can completely account for muscarine postsynaptic actions in these cells remains unclear. Muscarine may also block a small population of non-voltage-dependent potassium channels in addition to reducing m current.

Animals↗
PubMed1982

GABA and bicuculline actions on mouse spinal cord and cortical neurons in cell culture.

The neutral amino acid gamma-aminobutyric acid (GABA) produced membrane hyperpolarization and increased membrane chloride ion conductance of spinal cord (SC) and cortical (CTX) neurons in cell culture. GABA dose-response curves were obtained for SC neurons by pressure applying known concentrations of GABA from micropipettes with large tips (miniperfusion pipettes). GABA response threshold was about 2 micrometers and large responses were elicited at GABA concentrations greater than 10 micrometers. Bicuculline (BICUC) (0.1-10 micrometers) reversibly antagonized GABA responses on both SC and CTX neurons with a half maximal inhibitory concentration of about 1 micrometer. BICUC antagonism of GABA responses was competitive (Lineweaver-Burke analysis). These results are compared with data on GABA and BICUC displacement of [3H]GABA binding to membranes of SC and CTX neurons in cell culture. It is suggested that high affinity GABA receptors are likely to be relevant for postsynaptic GABA responses while low affinity GABA receptors may be presynaptic.

Animals↗
PubMed1982

Membrane depolarization and prolongation of calcium-dependent action potentials of mouse neurons in cell culture by two convulsants: bicuculline and penicillin.

The convulsant compounds bicuculline (BICUC) and penicillin (PCN) are antagonists of GABA-mediated synaptic inhibition. In addition, we have shown that BICUC and PCN produced membrane depolarization of mouse spinal cord neurons in primary dissociated cell culture by blocking a potassium conductance, a non-synaptic direct effect. Both compounds also prolonged calcium-dependent action potentials of mouse dorsal root ganglion and spinal cord neurons in cell culture. Thus, BICUC and PCN had both synaptic and non-synaptic actions. The possibility that both synaptic and non-synaptic actions of BICUC and PCN are involved in their convulsant mechanism of action is discussed.

Action Potentials↗
PubMed1982

Substance P: ionic basis for depolarizing responses of mouse spinal cord neurons in cell culture.

Intracellular recording methods were used to investigate the ionic basis for the postsynaptic actions of substance P (SP) on mouse spinal cord neurons grown in primary dissociated cell culture. SP and an analog, eledoisin-related peptide (ERP), were applied to single neurons by pressure ejection of peptide-containing solutions from blunt (2- to 10-micrometers) glass micropipettes. SP and ERP had similar excitatory actions, increasing spontaneous activity and depolarizing neurons by decreasing membrane conductance. Depolarizing responses were not inverted by intracellular injection of chloride ions, suggesting that SP responses did not result from decreased chloride conductance. SP and ERP responses were not abolished by extracellular tetraethylammonium ions (TEA+) but were reduced or eliminated by intracellular TEA+, suggesting that SP reduced a potassium conductance (gK). Finally, SP and ERP responses were larger when neurons were depolarized and smaller when the cells were hyperpolarized, and extrapolated reversal potentials for the peptide responses were 10 to 30 mV more negative than resting membrane potential. Thus, it was concluded that SP depolarized spinal cord neurons by decreasing a membrane potassium conductance. However, SP and ERP response polarity was not clearly reversed even at membrane potentials more negative than the expected potassium equilibrium potential. Moreover, extrapolated reversal potentials (RPeS) of SP responses varied linearly with the logarithm of extracellular potassium concentration ([K+]o) as predicted by the Nernst equation for potassium in [K+]o concentrations of 10, 15, 20, and 40 mM but were more depolarized than predicted for [K+]o concentrations of 1 and 5 mM. Since reduction of extracellular sodium by choline substitution did not alter the deviation from the Nernst equation for potassium, it was concluded that SP decreased a voltage-dependent potassium conductance which was absent at very negative potentials, present at resting membrane potential, and activated by membrane depolarization. Thus, SP decreases a conductance which appears similar to the muscarine-sensitive potassium conductance in sympathetic ganglion neurons (Brown, D. A., and P. R. Adams (1980) Nature 283: 673-676).

Action Potentials↗
PubMed1981

Substance P decreases a potassium conductance of spinal cord neurons in cell culture.

Substance P (SP) produced membrane depolarization and decreased membrane conductance of mouse spinal cord neurons in primary dissociated cell culture. SP-responses were abolished by intracellular tetraethylammonium suggesting that SP decreased potassium conductance. Reversal of SP-responses was not observed with membrane hyperpolarization suggesting that SP reduced a voltage-dependent potassium conductance that was activated by membrane depolarization.

Animals↗
PubMed1981

Bicuculline: a convulsant with synaptic and nonsynaptic actions.

The convulsant bicuculline (BICUC) had both synaptic and nonsynaptic actions on mouse spinal cord neurons in primary dissociated cell culture. BICUC antagonized postsynaptic responses to the inhibitory neurotransmitter GABA (a synaptic action) and produced direct membrane depolarization by blocking a membrane potassium conductance and directly prolonging calcium-dependent action potentials (nonsynaptic actions). In cultured spinal cord neurons, BICUC also produced paroxysmal depolarizing events (PDE), which might be equivalent to in vivo convulsant-induced bursting in spinal cord neurons or paroxysmal depolarizing shifts (PDS) in cortical neurons. Thus, the concentrations of BICUC producing both synaptic and nonsynaptic actions overlapped those producing paroxysmal activity. The results suggest that in addition to antagonism of GABA-mediated synaptic inhibition, direct nonsynaptic actions of BICUC may be required for some neurons to develop PDS.

Action Potentials↗
PubMed1981

Substance P and somatostatin actions on spinal cord neurons in primary dissociated cell culture.

In this paper, we have demonstrated that two peptides, SP and SRIF, which have been localized to and are released from small DRG neurons, have specific but different actions on mouse spinal cord neurons in PDC culture. SP has been shown to be excitatory by decreasing potassium conductance and to a lesser extent sodium conductance; SP also appeared to evoke release of neurotransmitter. SRIF has been shown to modify neurotransmitter release by a presynaptic action. The extensive experimental control that is possible over neurons in vitro permits precise measurements of specific membrane actions to be made and thus allows hypotheses of cellular mechanisms of peptide actions to be advanced. Predictions made based on these hypotheses should then be tested using intact preparations. A reasonable goal for such studies is a detailed understanding of the cellular and ionic mechanisms underlying actions in the CNS, PNS and gut.

Action Potentials↗