Rat hippocampal neurons in culture: properties of GABA-activated Cl- ion conductance

1984 ◽  
Vol 51 (3) ◽  
pp. 500-515 ◽  
Author(s):  
M. Segal ◽  
J. L. Barker
Keyword(s):  
Hippocampal Neurons ◽  
Membrane Conductance ◽  
Membrane Current ◽  
Membrane Properties ◽  
Current Response ◽  
Gamma Aminobutyric Acid ◽  
Ion Conductance ◽  
Dissociated Cell Culture ◽  
The Mean ◽  
Response Spectral Analysis

The actions of gamma-aminobutyric acid (GABA) on the membrane properties of rat hippocampal neurons maintained in dissociated cell culture were studied using intracellular recording techniques. All the neurons tested were responsive to GABA applied by pressure from micropipettes containing 10-20 microM GABA. The response consisted of a marked increase in conductance associated with a potential change. The inversion potential was sensitive to the Cl- ion gradient across the cells. It was about -60 mV when measured during recordings utilizing K acetate-filled microelectrodes, about -15 mV when measured during recordings with KCl-filled microelectrodes, and about +15 mV when measured with KCl electrodes in a medium containing low [Cl-]o. These results indicate that the membrane conductance evoked by GABA primarily involves Cl- ions. There were no apparent differences between the inversion potential of responses elicited at the level of the cell body and those evoked on processes. The two-electrode voltage-clamp technique was used to study the membrane mechanisms underlying these responses. GABA generated current responses that were associated with an increase in both conductance and membrane current variance. At a given potential both the conductance change and increase in variance were directly proportional to the amplitude of the current response. Spectral analysis of the membrane current variance evoked by GABA revealed that many of the computed spectra could be fitted by a single Lorentzian equation, suggesting that GABA activates two-state (open-closed) Cl- ion channels whose durations are exponentially distributed. The mean duration of these channel openings was estimated to be 22.9 +/- 2.1 ms, while the average conductance was estimated to be 19.8 +/- 2.7 pS in 13 cells. Large-amplitude GABA responses evoked at -70 mV frequently faded in amplitude, often by as much as 50%, with little or no change in the associated conductance. Since the membrane current variance decreased in direct relation to the fading current response, while estimates of channel lifetime did not change, the results suggest that the fading is due to a shift in the Cl- gradient. Responses to constant amounts of GABA evoked at different membrane potentials showed that the macroscopic conductance activated by GABA varied with membrane potential. Often 4-5 times more conductance was generated at depolarized (0 to +10 mV) relative to hyperpolarized potentials (-60 to -70 mV).(ABSTRACT TRUNCATED AT 400 WORDS)

Rat hippocampal neurons in culture: voltage-clamp analysis of inhibitory synaptic connections

1984 ◽  
Vol 52 (3) ◽  
pp. 469-487 ◽  
Author(s):  
M. Segal ◽  
J. L. Barker
Keyword(s):  
Ion Channel ◽  
Time Constant ◽  
Hippocampal Neurons ◽  
Reversal Potential ◽  
Membrane Current ◽  
Synaptic Conductance ◽  
Resting Potential ◽  
Fluctuation Analysis ◽  
Current Response ◽  
Kinetics Of

Inhibitory postsynaptic potentials (IPSPs) recorded at room temperature in cultured rat hippocampal neurons had the same reversal potential as Cl--dependent voltage responses to gamma-aminobutyric acid (GABA). The IPSPs had a relatively short latency and long duration and could be evoked for hours without change in their properties. They were consistently depressed by picrotoxin applied near cell bodies of the neurons under study. Postsynaptic cells exhibiting IPSPs were voltage clamped with two electrodes for the purpose of studying the properties of evoked inhibitory postsynaptic currents (IPSCs). The IPSCs shared the same reversal potential and sensitivity to [Cl-]i as was observed with membrane current responses to GABA. They were both depressed by picrotoxin, with little if any change in the kinetics of ion channel activity either estimated from fluctuation analysis of drug-depressed current responses to GABA or calculated from semilogarithmic plots of IPSC decay. The decay of the IPSC was well fitted by a single exponential with a time constant of about 20 ms, which corresponded closely to the estimated average duration of an ion channel activated by GABA. IPSC decay was sensitive to the potential at which the cell was held, increasing by up to 50% in some cells clamped at positive potentials relative to values obtained at the level of the resting potential. IPSCs were enhanced in amplitude by diazepam, which also prolonged their time constant of decay. Diazepam potentiated membrane current responses to GABA and fluctuation analysis of potentiated responses indicated that the drug effects could be accounted for by an increase both in estimated channel duration and channel frequency. IPSCs were also altered by pentobarbital, which markedly prolonged their time constant of decay with little if any change in their amplitude. Pentobarbital enhanced current responses to GABA, an effect that could be accounted for primarily in terms of a pronounced increase in estimated channel lifetime. None of the drugs used in the present study affected the elementary conductance estimated from fluctuation analysis of GABA-evoked current response. The results suggest that IPSPs and IPSCs evoked in these cultured hippocampal cells are mediated by GABA, about 1,700 Cl- ion channels are activated at the peak of the synaptic conductance, and clinically important drugs act postsynaptically on the kinetics of the channels so as to change the amplitude and/or time course of the synaptic conductance.

scholarly journals The CXCL12/CXCR4 signaling is required for DPP4 regulation of spontaneous inhibitory postsynaptic currents (sIPSCs)

2020 ◽  
Author(s):  
Yusong Zhang ◽  
Zhiguo Chen ◽  
Zhishan Yang ◽  
Yadi Han
Keyword(s):  
Hippocampal Neurons ◽  
Mean Amplitude ◽  
Gamma Aminobutyric Acid ◽  
Cxcr4 Antagonist ◽  
Inhibitory Postsynaptic Currents ◽  
Cxcr4 Signaling ◽  
Postsynaptic Currents ◽  
The Mean

Abstract Background: Previous data suggested that dipeptidyl peptidase-IV (DPP4) involved in the occurrence of febrile seizure (FS), but its potential mechanism remains to be determined. Here, we investigated whether DPP4 regulated gamma-aminobutyric acid (GABA) mediated spontaneous inhibitory postsynaptic currents (sIPSCs) via the downstream C-X-C Motif Chemokine Ligand 12 (CXCL12)/ C-X-C chemokine receptor type 4 (CXCR4) signaling in cultured hippocampal neurons submitted to hyperthermia(39.5-40°C). Methods: Whole cell patch- clamp method was used to test sIPSC in vitro after DPP4 inhibition or CXCL12 administration. The level of CXCL12 and CXCR4 was tested using western blot analysis. The effect of CXCR4 antagonist AMD3100 (5 mg/ml, i.c.v) on seizures were tested using electroencephalogram (EEG) in a FS model. Results: We found that pharmacological DPP4 inhibitor sitagliptin (Sita,100μM) treatment or siRNA-mediated DPP4 knockdown enhanced the mean amplitude and frequency of sIPSCs in vitro. DPP4 knockdown with siRNA increased protein level of CXCL12 and CXCR4. Furthermore, CXCL12 (10 nM) treatment enhanced inhibitory transmission by increasing the mean frequency and amplitude of sIPSCs in vitro. AMD3100 administration decreased seizure severity by increasing hippocampal GABA content in vivo. Conclusions: Our data suggest that CXCL12/CXCR4 signaling is required for DPP4 regulation of sIPSCs, supporting that DPP4 played a key role in the pathogenesis of FS.

Developmental study of benzodiazepine effects on monosynaptic GABAA-mediated IPSPs of rat hippocampal neurons

1993 ◽  
Vol 70 (3) ◽  
pp. 1076-1085 ◽  
Author(s):  
C. Rovira ◽  
Y. Ben-Ari
Keyword(s):  
Hippocampal Neurons ◽  
Receptor Agonist ◽  
Hippocampal Slices ◽  
Pyramidal Cells ◽  
Developmental Study ◽  
Type I ◽  
Gamma Aminobutyric Acid ◽  
Adult Rats ◽  
Young Rats ◽  
In Cells

1. The effects of type I (BZ1) and type II (BZ2) benzodiazepine receptor ligands on monosynaptic gamma-aminobutyric acid (GABA)A-mediated inhibitory postsynaptic potentials (IPSPs) and on responses to exogenously applied GABA were studied using intracellular recordings from CA3 pyramidal cells of rat hippocampal slices taken at different postnatal stages [postnatal day 4 (P4)-P35)]. 2. The effects of midazolam, a BZ1 and BZ2 receptor agonist, were tested on the monosynaptic IPSPs at different stages. Monosynaptic, bicuculline-sensitive IPSPs were evoked by hilar stimulation in presence of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) antagonists [6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) and D(-)2-amino-5-phosphonopentanoic acid (50 microM)]. Midazolam at 300 nM maximally increased the duration and amplitude of monosynaptic GABAA-mediated IPSPs in neurons from pups (P4-P6, n = 6) and young (P7-P12, n = 8) and adult (P25-P35, n = 9) rats. All the effects of midazolam on IPSPs were reversed by the antagonist Ro 15-1788 (10 microM). 3. The effect of midazolam was also tested on the response to exogenously applied GABA (5 mM) in the presence of tetrodotoxine [TTX (1 microM)]. In neurons from young rats (n = 9), midazolam (1 nM-1 microM) did not change the responses to exogenously applied GABA, whereas in adult rats (n = 8) midazolam maximally increased GABA currents at 30 nM. 4. The effect of zolpidem, a BZ1 receptor agonist, was tested on monosynaptic IPSPs and GABA currents at different stages. Zolpidem (10 nM-1 microM) was inactive in cells from young rats (n = 12). In neurons from adult rats, zolpidem maximally increased the duration and amplitude of the monosynaptic IPSPs at 300 nM (n = 5) and the amplitude of GABA current at 30-100 nM (n = 5). 5. Methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) (300 nM), an inverse agonist of BZ1 and BZ2 receptors, decreased the amplitude and duration of monosynaptic IPSPs in neurons from pups (n = 3) and young (n = 4) and adult (n = 5) rats. In all cases, full recovery was obtained after exposure to R0 15-1788 (10 microM). DMCM (300 nM-10 microM) failed to reduce GABA responses in cells from young (n = 3) or adult (n = 7) rats. 6. Results indicate that the regulation by benzodiazepine of GABAA-mediated IPSPs varies with the developmental stage.(ABSTRACT TRUNCATED AT 400 WORDS)

BICUCULLINE/BACLOFEN-INSENSITIVE GABA RESPONSE IN CRUSTACEAN NEURONES IN CULTURE

1994 ◽  
Vol 191 (1) ◽  
pp. 167-193
Author(s):  
C Jackel ◽  
W Krenz ◽  
F Nagy
Keyword(s):  
Reversal Potential ◽  
Membrane Conductance ◽  
Gamma Aminobutyric Acid ◽  
Action Potential Firing ◽  
Patch Clamp Technique ◽  
Conformationally Restricted ◽  
Whole Cell Patch Clamp ◽  
Gabac Receptor ◽  
Potential Firing ◽  
Sr 95531

Neurones were dissociated from thoracic ganglia of embryonic and adult lobsters and kept in primary culture. When gamma-aminobutyric acid (GABA) was applied by pressure ejection, depolarizing or hyperpolarizing responses were produced, depending on the membrane potential. They were accompanied by an increase in membrane conductance. When they were present, action potential firing was inhibited. The pharmacological profile and ionic mechanism of GABA-evoked current were investigated under voltage-clamp with the whole-cell patch-clamp technique. The reversal potential of GABA-evoked current depended on the intracellular and extracellular Cl- concentration but not on extracellular Na+ and K+. Blockade of Ca2+ channels by Mn2+ was also without effect. The GABA-evoked current was mimicked by application of the GABAA agonists muscimol and isoguvacine with an order of potency muscimol>GABA>isoguvacine. cis-4-aminocrotonic acid (CACA), a folded and conformationally restricted GABA analogue, supposed to be diagnostic for the vertebrate GABAC receptor, also induced a bicuculline-resistant chloride current, although with a potency about 10 times lower than that of GABA. The GABA-evoked current was largely blocked by picrotoxin, but was insensitive to the GABAA antagonists bicuculline, bicuculline methiodide and SR 95531 at concentrations of up to 100 µmol l-1. Diazepam and phenobarbital did not exert modulatory effects. The GABAB antagonist phaclophen did not affect the GABA-induced current, while the GABAB agonists baclophen and 3-aminopropylphosphonic acid (3-APA) never evoked any response. Our results suggest that lobster thoracic neurones in culture express a chloride-conducting GABA-receptor channel which conforms to neither the GABAA nor the GABAB types of vertebrates but shows a pharmacology close to that of the novel GABAC receptor described in the vertebrate retina.

Neural repetitive firing: a comparative study of membrane properties of crustacean walking leg axons

1975 ◽  
Vol 38 (4) ◽  
pp. 922-932 ◽  
Author(s):  
J. A. Connor
Keyword(s):  
Membrane Conductance ◽  
Firing Frequency ◽  
Membrane Properties ◽  
Constant Current ◽  
Repetitive Firing ◽  
Type I ◽  
Frequency Range ◽  
Type Iii ◽  
Range Type ◽  
Sucrose Gap

1. Repetitive activity and membrane conductance parameters of crab walking leg axons have been studied in the double sucrose gap. 2. The responses to constant current stimulus could be classified into three catagories; highly repetitive with wide firing frequency range, type I; highly repetitive with narrow frequency range, type II; and nonrepetitive or repetitive to only a limited degree, type III. The minimum firing frequency for type I axons was much greater than for other recording techniques. 3. Voltage-clamp currents in type III axons were qualitatively similar to those of squid or lobster axon. 4. The outward membrane currents of type I and II axons showed a transient phase in addition to the usual delayed current. The magnitude of this transient was a function of both the holding and test voltages. 5. The direction of the transient current reversed in potassium-rich saline. 6. The type I repetitive response in the walking leg axons appears to be generated by the same types of conductance changes that have been demonstrated in molluscan central neurons.

scholarly journals Silencing-Induced Metaplasticity in Hippocampal Cultured Neurons

2008 ◽  
Vol 100 (2) ◽  
pp. 690-697 ◽  
Author(s):  
Irina V. Sokolova ◽  
Istvan Mody
Keyword(s):  
Hippocampal Neurons ◽  
Channel Blocker ◽  
Recovery Period ◽  
Long Term Potentiation ◽  
Homeostatic Plasticity ◽  
Membrane Properties ◽  
Postsynaptic Currents ◽  
Short Period ◽  
Spiking Activity

Silencing-induced homeostatic plasticity is usually expressed as a change in the amplitude or the frequency of miniature postsynaptic currents. Here we report that, prolonged (∼24 h) silencing of mature (20–22 days in vitro) cultured hippocampal neurons using the voltage-gated sodium channel blocker tetrodotoxin (TTX) produced no effects on the amplitude or frequency of the miniature excitatory postsynaptic currents (mEPSCs). However, the silencing changed the intrinsic membrane properties of the neurons, resulting in an increased excitability and rate of action potentials firing upon TTX washout. Allowing neurons to recover in TTX-free recording solution for a short period of time after the silencing resulted in potentiation of mEPSC amplitudes. This form of activity-dependent potentiation is different from classical long-term potentiation, as similar potentiation was not seen in nonsilenced neurons treated with bicuculline to raise their spiking activity to the same level displayed by the silenced neurons during TTX washout. Also, the potentiation of mEPSC amplitudes after the recovery period was not affected by the N-methyl-d-aspartate receptor blocker d-2-amino-5-phosponopentanoic acid or by the calcium/calmodulin-dependent kinase II (CaMKII) inhibitor KN-62 but was abolished by the L-type calcium channel blocker nifedipine. We thus conclude that the potentiation of mEPSC amplitudes following brief recovery of spiking activity in chronically silenced neurons represents a novel form of metaplasticity that differs from the conventional models of homeostatic synaptic plasticity.

Membrane Properties Related to the Firing Behavior of Zebrafish Motoneurons

2003 ◽  
Vol 89 (2) ◽  
pp. 657-664 ◽  
Author(s):  
Robert R. Buss ◽  
Charles W. Bourque ◽  
Pierre Drapeau
Keyword(s):  
Action Potential ◽  
Action Potentials ◽  
Membrane Conductance ◽  
Membrane Properties ◽  
High Threshold ◽  
Calcium Dependent ◽  
Larval Zebrafish ◽  
Plateau Potential ◽  
Potassium Conductances ◽  
Action Potential Burst

The physiological and pharmacological properties of the motoneuron membrane and action potential were investigated in larval zebrafish using whole cell patch current-clamp recording techniques. Action potentials were eliminated in tetrodotoxin, repolarized by tetraethylammonium (TEA) and 3,4-diaminopyridine (3,4-AP)-sensitive potassium conductances, and had a cobalt-sensitive, high-threshold calcium component. Depolarizing current injection evoked a brief (approximately 10–30 ms) burst of action potentials that was terminated by strong, outwardly rectifying voltage-activated potassium and calcium-dependent conductances. In the presence of intracellular cesium ions, a prolonged plateau potential often followed brief depolarizations. During larval development (hatching to free-swimming), the resting membrane conductance increased in a population of motoneurons, which tended to reduce the apparent outward rectification of the membrane. The conductances contributing to action potential burst termination are hypothesized to play a role in patterning the synaptically driven motoneuron output in these rapidly swimming fish.

Glycine regulation of synaptic NMDA receptors in hippocampal neurons

1996 ◽  
Vol 76 (5) ◽  
pp. 3415-3424 ◽  
Author(s):  
K. S. Wilcox ◽  
R. M. Fitzsimonds ◽  
B. Johnson ◽  
M. A. Dichter
Keyword(s):  
Nmda Receptor ◽  
Nmda Receptors ◽  
Hippocampal Neurons ◽  
Time Course ◽  
Hippocampal Slices ◽  
Maximal Effect ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Dissociated Cell Culture ◽  
Cultured Hippocampal Neurons

1. Although glycine has been identified as a required coagonist with glutamate at N-methyl-D-aspartate (NMDA) receptors, the understanding of glycine's role in excitatory synaptic neurotransmission is quite limited. In the present study, we used the whole cell patch-clamp technique to examine the ability of glycine to regulate current flow through synaptic NMDA receptors at excitatory synapses between cultured hippocampal neurons and in acutely isolated hippocampal slices. 2. These studies demonstrate that the glycine modulatory site on the synaptic NMDA receptor is not saturated under baseline conditions and that increased glycine concentrations can markedly increased NMDA-receptor-mediated excitatory postsynaptic currents (EPSCs) in hippocampal neurons in both dissociated cell culture and in slice. Saturation of the maximal effect of glycine takes place at different concentrations for different cells in culture, suggesting the presence of heterogenous NMDA receptor subunit compositions. 3. Bath-applied glycine had no effect on the time course of EPSCs in either brain slice or culture, indicating that desensitization of the NMDA receptor is not prevented by glycine over the time course of an EPSC. 4. When extracellular glycine concentration is high, all miniature EPSCs recorded in the cultured hippocampal neurons contained NMDA components, indicating that segregation of non-NMDA receptors at individual synaptic boutons does not occur.

Role of chloride-homeostasis in the inhibitory control of neuronal network oscillators

1996 ◽  
Vol 75 (6) ◽  
pp. 2654-2657 ◽  
Author(s):  
W. Jarolimek ◽  
H. Brunner ◽  
A. Lewen ◽  
U. Misgeld
Keyword(s):  
Glycine Receptor ◽  
Membrane Conductance ◽  
Burst Activity ◽  
Synaptic Activity ◽  
Gamma Aminobutyric Acid ◽  
Chloride Homeostasis ◽  
Outward Transport ◽  
Whole Cell Recordings ◽  
Rhythmic Burst

1. Spontaneous synaptic activity in networks formed by dissociated neurons from embryonic rat midbrain was analyzed in tight seal whole cell recordings. 2. Application of furosemide (0.5 mM) to the cell and its surrounding area increased the frequency of spontaneous synaptic currents. Incubation of the culture with furosemide resulted in “rhythmic” burst activity. 3. Furosemide (0.1-0.5 mM) changed equilibrium potentials of inhibitory postsynaptic currents, gamma-aminobutyric acid-A (GABAA) or glycine receptor-mediated Cl- currents by a blockade of Cl(-)-outward transport. Furosemide did not alter the slope conductance of GABAA receptor-mediated currents. Membrane conductance and cell excitability were also unaffected. 4. We conclude that furosemide locked the activity of the network in “burst activity” mode through impairment of inhibition resulting from the disturbance of Cl- homeostasis.

Development and properties of synaptic mechanisms in a network of rat hypothalamic neurons grown in culture

1990 ◽  
Vol 64 (3) ◽  
pp. 715-726 ◽  
Author(s):  
D. Swandulla ◽  
U. Misgeld
Keyword(s):  
Spontaneous Activity ◽  
Voltage Clamp ◽  
Lucifer Yellow ◽  
Drug Application ◽  
Growth Cones ◽  
Current Response ◽  
Gamma Aminobutyric Acid ◽  
Network Growth ◽  
Zero Current ◽  
Current Potential

1. Dissociated neurons from embryonic rat hypothalamus (E14-15) were cultured on a glial background monolayer for up to three months. Dendrites of cells 7-14 days in culture (DIC) intracellularly stained with the fluorescent dye Lucifer yellow were thin and smooth, and multiple growth cones could be observed. The length of the dendrites of older cells did not differ much, but dendrites were thicker and branched more profoundly, forming a complicated network. Growth cones were rare, but few spine-like protrusions could be observed. 2. Randomly occurring depolarizing potentials were recorded in 60% of the cells 7-14 DIC and in 90% of the cells 21 DIC. Activity became phasic when the gamma-aminobutyric acid (GABA) antagonists picrotoxin or bicuculline were applied. After 21 DIC the majority of the cells showed burst discharges, whereas only approximately 10% of the cells 7 DIC exhibited bursting. 3. With low [Cl] in the recording pipette, spontaneous activity consisted of hyperpolarizing and depolarizing potentials at -40-mV membrane potential. Some spontaneous activity persisted with Na channels blocked by tetrodotoxin (TTX, 0.3-1 microM), and when reducing the external [Ca]o from 5 to 0.3 mM. Picrotoxin blocked part of the activity, and the remaining activity was blocked by kynurenic acid. 4. Bursts of action potentials were superimposed on rhythmically occurring clusters of excitatory synaptic potentials (EPSPs), which had a steep rising phase and decayed within hundreds of milliseconds. Bursts of similar appearance could be triggered by brief (10 ms) depolarizing current injections, and a few cells had properties indicative for endogenous pacemakers. 5. From 7 DIC on, all cells responded to GABA and to the GABA agonist muscimol. Under voltage clamp, zero current potential depended on the Cl gradient across the membrane and corresponded to the zero current potential of picrotoxin-sensitive postsynaptic currents. 6. After 21 DIC all cells responded to glutamate and its agonist quisqualate. Under voltage clamp, nanomolar concentrations of quisqualate (100-500 nM) induced long-lasting inward currents, which did not decay substantially during prolonged drug application. Quisqualate concentrations greater than 1 microM induced a diphasic inward-current response consisting of an initial fast current transient followed by a maintained current component. With internal Cs replacing K and Na and external TTX (0.3 microM), both current components reversed sign at approximately 8 mV, as predicted by the Nernst equation for currents through channels that were permeable for monovalent cations. 7. Focal applications of GABA and muscimol elicited larger currents when applied near the soma than when applied to the dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)

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