Baclofen inhibition of dorsal root-evoked inhibitory postsynaptic currents in substantia gelatinosa neurons of rat spinal cord slice

We investigated the action of baclofen, an agonist for GABA_B receptor, on the dorsal root-evoked inhibitory postsynaptic currents (IPSCs) in substantia gelatinosa (SG) neurons in the adult rat spinal cord slice. Most of the dorsal root-evoked IPSCs in SG were mediated by Aδ fibers. Baclofen (10 μM) reduced the amplitude of Aδ fiber-evoked glycine receptor-mediated IPSCs to 20.9±3.0% (n=13), and GABA_A receptor-mediated IPSCs to 18.6±3.4% (n=12) of the controls, respectively. The results further suggest modulatory role of GABA_B receptor in spinal dorsal horn.     

GABAB受体对大鼠海马CA1区锥体细胞突触传递的作用

目的研究激活GABA_B受体对大鼠海马CA1区锥体细胞突触传递的影响。方法对成年大鼠海马脑片CA1区锥体细胞采用“盲法”全细胞电压钳记录,分别检测和分析巴氯芬(10μmol/L)对自发性的兴奋性突触后电流(EPSCs)和抑制性突触后电流(IPSCs)的影响。结果巴氯芬可显著降低符氨酸能EPSCs和γ-氨基丁酸能IPSCs的频率(P＜0．01),各自达58%±7%(n=17)和42%±10%(n=15),而对它们的幅度无显著性影响。结论巴氯芬对海马CA1区锥体细胞EPSCs和IPSCs的抑制作用属于突触前抑制,推测GABA_B受体所介导的这种抑制作用对CA1区神经元兴奋性的传出具有抑制作用,从而对癫痫的产生有控制作用。     

Electrophysiological evidence for a rapid membrane action of the gonadal steroid, 17β-estradiol, on CA1 pyramidal neurons of the rat hippocampus

The rapid electrophysiological effects of 17β-estradiol on CA1 pyramidal neurons(n = 86) were investigated utilizing intracellular recording from the rat hippocampal slice preparation. Bath application of 17β-estradiol, but not 17α-estradiol, caused a reversible depolarization and increased input resistance with a latency of less than 1 min in 19.8% of CA1 neurons tested. There was no significant difference in the percentage of estradiol-responsive cells between male and female rats. Estradiol-responsive cells were identified from prepubertal female rats, as well as females in all stages of the estrous cycle. 17β-estradiol had no effect on the slow afterhyperpolarization or accommodative properties of CA1 neurons. In 2 out of 4 cells tested, the specific antiestrogen, tamoxifen, blocked the excitatory response to 17β-estradiol.     

Protein tyrosine kinase inhibitors reduce high-voltage activating calcium currents in CA1 pyramidal neurones from rat hippocampal slices

We have investigated the effects of protein tyrosine kinases (PTKs) inhibitors on high-threshold voltage activating (HVA) calcium currents in CA1 pyramidal neurones, whole-cell patch-clamp recorded from rat hippocampal slices. Genistein (100 μM) and tyrphostin B42 (100 μM), two PTKs inhibitors, reduced the steady-state barium current (I_Ba). On the other hand, daidzein and genistin (100 μM), two inactive analogues of genistein, had no effect on I_Ba amplitude. The inhibition induced by genistein was more pronounced at negative potentials. In order to characterize the calcium channels subtypes inhibited by PTKs inhibitors, we examined the effect of genistein in the presence of different calcium channel blockers. When L-type calcium channels were blocked by nifedipine, genistein induced a strong inhibition of the nifedipine-resistant I_Ba, suggesting an effect on non-L-type channels. Genistein did not antagonize the depressant effect of ω-Conotoxin-GVIA, a selective N-type calcium channel blocker, suggesting that N-type channels were not blocked by genistein. ω-Conotoxin-MVIIC (3–10 μM), a selective P/Q-type calcium channel blocker, greatly antagonized the depressant effect of genistein. Our results suggest that PTKs inhibitors reduce P-/Q-type, but not L- or N-types calcium currents in neurones of the CNS. The possible modulation of calcium channels by endogenous PTKs is discussed.     

Chronic corticosterone treatment maintains synaptic activity of CA1 hippocampal pyramidal cells: Acute high corticosterone administration increases action potential number

The hypothalamic-pituitary-adrenocortical (HPA) axis controls the levels of plasma corticosterone (CT) in the rat and the levels of cortisol in man. CT is important in maintaining homeostasis and regulating energy production. Homeostasis is maintained by basal activation of the hippocampal-HPA axis. In response to stress CT secretion is increased. CT activation of receptors in the hippocampus provides feedback inhibition of the HPA axis to return the system to basal activity. There are two types of CT receptors: the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). CT has a 10-fold higher affinity for MR than GR. Normal basal levels of CT occupy the majority of the MR. During the diurnal surge of CT and following the presentation of a stressful stimulus, the MR and GR are both maximally occupied. To begin to understand how CT influences the hippocampal-HPA axis, intracellular recording techniques in the hippocampal brain slice preparation were used to determine how high concentrations of CT may alter cell characteristics and/or evoked synaptic activity. Two treatment groups were used, i.e., adrenalectomized (ADX) and ADX with CT pellet replacement (ADX + CT) that produced plasma blood levels equal to that seen in a normal rat in the morning. Acute administration of 100 nM CT decreased action potential threshold and the number of action potentials elicited by a depolarizing current pulse in cells from both the ADX and ADX + CT treated rats. The amplitude of the evoked excitatory postsynaptic potentials (EPSP) or inhibitory postsynaptic potentials (IPSP) declined in cells recorded from ADX animals and ADX rats acutely treated with high concentrations of CT (ADWCT). The EPSPs and IPSPs were stable in cells from ADX + CT rats exposed to high CT concentrations. We conclude from these data that chronic CT treatment is necessary to maintain EPSP and IPSP amplitude. Also, acute administration of a high concentration of CT alters the output of the CA1 hippocampal pyramidal cells by increasing the number of action potentials elicited by a depolarizing current pulse but does not alter the amplitude of subthreshold EPSPs and IPSPs. © 1995 Wiley-Liss, Inc.     

Opioids suppress spontaneous and NMDA-induced inhibitory postsynaptic currents in the dorsal raphe nucleus of the rat in vitro

Recently, local injection of morphine in the dorsal raphe nucleus (DRN) has been shown to increase serotonin release in the forebrain of unanesthetized rats. This study investigated the site of action of opioids in rat brain slices containing the DRN. Postsynaptic currents (PSCs), measured intracellularly under voltage clamp, were induced in serotonergic neurons with bath and microiontophoretic applications of NMDA to activate local neurons. Met-enkephalin (ENK) suppressed spontaneous and NMDA-induced GABAergic inhibitory PSCs. This effect, which was mimicked by the μ agonist DAMGO but not the κ-agonist U50488 or the δ-agonist DPDPE, was reversed by the μ antagonist CTOP. ENK also suppressed spontaneous and NMDA-induced glutamatergic excitatory PSCs. By searching with focal microiontophoretic NMDA applications, GABAergic and glutamatergic cells projecting on serotonergic neurons were found in the DRN and the adjacent periaqueductal gray. Consistent with the reduction in PSCs, ENK inhibited/hyperpolarized the great majority (81%) of non-serotonergic neurons recorded extra- and intracellularly in the DRN; the ENK effect reversed polarity at −99±9 mV, close to the potassium reversal potential. In contrast, ENK inhibited/hyperpolarized only 28% of serotonergic neurons; in the affected cells, the ENK effect, blocked by CTOP, had its reversal potential shifted with change of extracellular potassium in agreement with the value predicted by the Nernst equation for a potassium conductance; serotonin occluded the ENK inhibition. Taken together, these results indicate that opioids inhibit both local GABAergic and glutamatergic cells projecting onto DRN serotonergic neurons.     

Characterization of spontaneous excitatory synaptic currents in pyramidal cells of rat prelimbic cortex

Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded with the whole-cell patch-clamp technique from 41 pyramidal cells in the layers V-VI of the prelimbic (PL) cortex. The sEPSCs occurred randomly and the averaged frequency in 41 cells was 1.81+/-0.27 Hz. The amplitude distribution was skewed toward larger events and could be adequately fitted by a sum of two or three Gaussian distributions, but they could not be fitted by a sum of Gaussian distributions with equidistant separation in all cells studied (n=24). In eight of 24 cells, after the transformation of the amplitudes into logarithms, the skewed histogram became bell-shaped and could be adequately fitted by a single Gaussian distribution, whereas in the other 16 cells, after the transformation the histograms were still skewed. However, for those latter cells, when the logarithms were transformed into difference, the distribution of the differences in 15 of 16 cells became bell-shaped and could be adequately fitted by a single Gaussian distribution. The pie distribution of different rise times within one cell in 1 ms bin showed that there were four different patterns of the rise time distribution. The amplitude distribution of the sEPSCs was unchanged in 10 of 22 cells after TTX, but in the other 12 cells, it was changed significantly. However, for these cells although TTX had a marked effect, it could not change the skewed distribution into a single Gaussian distribution in case of both original and transformed data.     

Heterogeneous distribution of tetrodotoxin-sensitive calcium-conducting channels in rat hippocampal CA1 neurons

Voltage-dependent Ca2+ currents (ICas) in neurons can be classified into T-, N- and L-types. In the CA1 pyramidal neurons freshly isolated from rat hippocampus we found an additional tetrodotoxin (TTX)-sensitive Ca2+ current (termed 'TTX-ICa') which passed through the Na+ channel. The TTX-ICa showed a heterogeneous distribution in the dorsal site of Ca1 region.     

Effects of berberine on potassium currents in acutely isolated CA1 pyramidal neurons of rat hippocampus

The effects of berberine, an isoquinoline alkaloid with antiarrhythmic action, on voltage-dependent potassium currents were studied in acutely isolated CA1 pyramidal neurons of rat hippocampus by using the whole-cell patch-clamp techniques. Berberine blocked transient outward potassium current (IA) and delayed rectifier potassium current (IK) in a concentration-dependent manner with EC50 of 22.94+/-4.96 microM and 10.86+/-1.06 microM, Emax of 67.47+/-4.00% and 67.14+/-1.79%, n of 0.77+/-0.08 and 0.96+/-0.07, respectively. Berberine 30 microM shifted the steady-state activation curve and inactivation curve of IA to more negative potentials, but mainly affected the inactivation kinetics. Berberine 30 microM positively shifted the steady-state activation curve of IK. These results suggested that blockades on K+ currents by berberine are preferential for IK, and contribute to its protective action against ischemic brain damage.     

IPSPs elicited in CA1 pyramidal cells by putative basket cells in slices of adult rat hippocampus

CA1 basket cells are identifiable by an axonal arbour largely confined to, and spanning, the entire depth of stratum pyramidale where they innervate pyramidal somata and proximal dendrites. Basket cells display a range of electrophysiological properties and the inhibitory postsynaptic potentials (IPSPs) they elicit in pyramidal cells vary widely in duration. To determine whether these parameters are correlated, we used paired intracellular recordings, with biocytin filling, in pyramidal cells of adult hippocampal slices, and studied gamma-aminobutyric acid (GABAA) IPSPs (n = 43) elicited by putative basket cells (n = 35) with axons largely confined to stratum pyramidale in simultaneously recorded pyramidal cells. Fast-spiking interneurons elicited relatively brief IPSPs, while IPSPs elicited by burst-firing cells were amongst the slowest. Regular spiking interneurons elicited fast and slow GABAA IPSPs, but any one interneuron elicited IPSPs with remarkably similar durations in two to four pyramidal targets. However, with different types of target for a single putative basket cell, IPSPs elicited in postsynaptic interneurons were briefer than in pyramidal cells. Vertical oriens cells with somata in stratum oriens and a narrow, sparse axonal arbour in stratum pyramidale in transverse hippocampal slices, elicited IPSPs whose rise times and half widths clustered around intermediate values. Durations of IPSPs in pyramidal cells thus correlate, to a degree, with the physiological properties of presynaptic basket cells. The seven-fold range of durations observed (10-70 ms half widths) may underlie contributions made by different basket cells to hippocampal rhythms of different frequencies.     

Sevoflurane-induced ionic current in acutely dissociated CA1 pyramidal neurons of the rat hippocampus

The electrophysiological properties of sevoflurane (Sev)-induced current (I_Sev) were investigated in CA1 pyramidal neurons freshly dissociated from the rat hippocampus by using the nystatin perforated patch recording configuration under voltag-clamp condition. Within the range of Sev concentrations from 3 · 10⁻⁴ to 2 · 10⁻³ M, I_sev was an inward current which consisted of an initial transient peak component and a successive steady-state plateau component. The peak current component increased in a concentration-dependent manner with a conductance increase. The application of Sev over 2 · 10⁻³ M, however, suppressed the peak and steady-state current components with a concomitant decrease in conductance and elicited a transient inward current (‘hump’ current) immediately after wash out. The current-voltage relationship for I_Sev showed some outward rectification suggesting a slight voltage-dependency of the I_sev. The reversal potential of I_Sev (E_Sev) was close to the E_Cl and shifted by 52 mV for a 10-fold change in extracellular Cl⁻ concentrations, indicating that I_Sev is passing through Cl⁻ channels. The single channel conductance obtained from the analysis of the variance of I_Sev fluctuations was 15.3 ± 1.3pS.     

Hyperexcitability of CA3 pyramidal cells in mice lacking the potassium channel subunit Kv1.1

PURPOSE: To investigate further the membrane properties and postsynaptic potentials of the CA3 pyramidal cells in mice that display spontaneous seizures because of a targeted deletion of the Kcna1 potassium channel gene (encoding the Kv1.1 protein subunit). METHODS: Intracellular recordings were obtained from CA3 pyramidal cells in hippocampal slices prepared from Kcna1-null and control littermates. CA3 pyramidal cells were activated: orthodromically, by stimulating mossy fibers; antidromically, by activating Schaffer collaterals; and by injecting intracellular pulses of current. Responses evoked under these conditions were compared in both genotypes in normal extracellular medium (containing 3 mM potassium) and in medium containing 6 mM potassium. RESULTS: Recordings from CA3 pyramidal cells in Kcna1-null and littermate control slices showed similar membrane and action-potential properties. However, in 33% of cells studied in Kcna1-null slices bathed in normal extracellular medium, orthodromic stimulation evoked synaptically driven bursts of action potentials that followed a short-latency excitatory postsynaptic potential (EPSP)-inhibitory PSP (IPSP) sequence. Such bursts were not seen in cells from control slices. The short-latency gamma-aminobutyric acid (GABA)A-mediated IPSP event appeared similar in null and control slices. When extracellular potassium was elevated and excitatory synaptic transmission was blocked, antidromic activation or short pulses of intracellular depolarizing current evoked voltage-dependent bursts of action potentials in the majority of cells recorded in Kcna1 null slices, but only single spikes in control slices. CONCLUSIONS: Lack of Kv1.1 potassium channel subunits in CA3 pyramidal cells leads to synaptic hyperexcitability, as reflected in the propensity of these cells to generate multiple action potentials. The action-potential burst did not appear to result from loss of GABAA receptor-mediated inhibition. This property of CA3 neurons, seen particularly when tissue conditions become abnormal (e.g., elevated extracellular potassium), helps to explain the high seizure susceptibility of Kcna1-null mice.     

Denervation-induced dendritic alterations in CA1 pyramidal cells following kainic acid hippocampal lesions in rats

Kainic acid (KA) lesions of the CA3 region of the hippocampus lead to denervation of ipsilateral CA1 neurons. To assess denervation-induced post-synaptic changes, intracellular physiological recordings were performed in the CA1 region in vitro, from both control and KA-treated tissue. The neurons were intracellularly stained with neurobiotin, reconstructed using a quantitative three-dimensional system and analyzed for morphometric and electrotonic parameters. Total dendritic length was slightly longer in the denervated CA1 cells and there was a selective and significant increase in both branches and terminals in the mid-stratum radiatum (300–500 μm from the soma using Sholl analysis) in the KA-treated rats compared to untreated controls, particularly for cells at 5 days post-lesion and later, which exhibited graded synaptically-evoked bursts. However, there was no significant difference in the basal dendritic arborization. Electrotonic modelling of the dendritic structure revealed specific membrane resistivity values of 33.4 kΩ·cm² for the normal CA1 cells and 29.8 KΩ-cm² for the KA-treated cells, assuming an internal resistivity of 200 Ω·cm², shrinkage correction of 1.57 and a spatial distribution of dendritic spines. The number of dendritic terminals of these denervated CA1 neurons at electrotonic distances between 0.5λ and 0.7λ also significantly increased in the cells from KA-treated animals. These findings indicate that there is a selective and specific increase in the number of apical terminals and dendritic branches following the unilateral kainic acid lesion. These apical branch changes may represent dendritic sprouting as a post-synaptic response to the denervation, which was particularly marked in neurons exhibiting graded synaptic bursting behavior.     

Post-ischemic potentiation of Schaffer collateral/CA1 pyramidal cell responses of the rat hippocampus in vivo: involvement of receptors

The present study examined the functional changes in the hippocampal CA1 pyramidal cell system in vivo occurring after 12-min forebrain ischemia in the rat. A population excitatory postsynaptic potential and orthodromic population spike of CA1 pyramidal cells to stimulation of the Schaffer collaterals were potentiated at 6–8 h post-ischemia. These changes were not associated with an increase in excitability of the CA1 pyramidal cells as evaluated from the antidromic population spike induced by alveus stimulation, suggesting the presence of an increased synaptic efficacy. The post-ischemic potentiation was prevented by pretreatment with the (NMDA) receptor antagonist, MK801, in a dose-dependent manner. These findings suggest that 12-min forebrain ischemia in the rat activates NMDA receptors, which results in an increase in synaptic efficacy to the CA1 pyramidal cells at 6–8 h post-ischemia.     

Voltage-clamp analysis of halothane effects on GABAA fast and GABAA slow inhibitory currents

Voltage-clamped GABA_{A fast} and GABA_{A slow} inhibitory postsynaptic currents (IPSCs) were selectively elicited in hippocampal area CA1 pyramidal neurons. Clinically relevant concentrations of halothane (1.2 vol.%) prolonged both GABA_{A fast} and GABA_{A slow} IPSC decay times approximately 2.5 fold, while having little to no effect on current amplitudes or rise times. Current–voltage analysis revealed that IPSC reversal potentials (−70 to −75 mV) remained constant in the presence of halothane. Under control conditions, GABA_{A slow} IPSC decay times increased linearly with membrane depolarization, and this IPSC decay time voltage dependence was not significantly altered by halothane. These results confirm the existence of separable GABA_{A fast} and GABA_{A slow} IPSCs in hippocampus, and further elucidate the effects of halothane on these currents.     

Dopamine decreases cell excitability in rat striatal neurons by pre- and postsynaptic mechanisms

The mechanism by which dopamine (DA) decreases the amplitude of the EPSP-IPSP sequences evoked by cortical stimulation was investigated by means of electrophysiological and biochemical methods. Intracellular recordings indicate that DA decreases the amplitude of the excitatory and inhibitory events by reducing the increase in membrane conductance measured at the peaks of the EPSP-IPSP. The non-synaptic input resistance was not modified. In addition the catecholamine (+50/+200 nA balanced current) was shown to decrease the action of glutamate (-30/-80 nA balanced current) and GABA (+40/+100 nA balanced current) when iontophoretically applied. These observations suggest that DA interferes with the excitatory (glutamatergic) and inhibitory (GABAergic) transmission at the postsynaptic site in striatal neurons. However, the depression of cellular excitability elicited by DA could not be ascribed only to its interaction with synaptic transmission at the postsynaptic level. In fact the catecholamine also inhibited spike frequency driven by depolarizing pulses and decreased the depolarization-induced release of glutamate at the presynaptic site, as shown by biochemical experiments with striatal synaptosomal preparations. A neuromodulatory role of DA in the depression of the excitability of striatal neurons by presynaptic and postsynaptic mechanisms is suggested.     

Guanosine phosphate analogs modulate ethanol potentiation of GABAA-mediated synaptic currents in hippocampal CA1 neurons

Recent studies have demonstrated that ethanol potentiation of GABA_A receptor function can be regulated by second-messenger-dependent processes. As a preliminary step to further characterize this interaction, we used the whole-cell patch-clamp technique to study the effects of guanosine phosphate analogs on ethanol potentiation of GABA_A-mediated synaptic transmission, in rat hippocampal CA1 neurons. Intracellular dialysis with 400 μM GDPβS, an analog that inhibits G-protein coupled events, significantly reduced ethanol, but not pentobarbital, potentiation of IPSCs. In contrast, dialysing neurons with 100 μM GTPγS, an irreversible G-protein activator, selectively facilitated ethanol potentiation of GABA_A IPSCs. These results suggest that one or more G-protein coupled events regulate the ethanol sensitivity of synaptic GABA_A receptors.     

Effect of nilvadipine on the voltage-dependent Ca channels in rat hippocampal CA1 pyramidal neurons

Effects of nilvadipine on the low- and high-voltage activated Ca²⁺ currents (LVA and HVA I_Ca, respectively) were compared with other organic Ca²⁺ antagonists in acutely dissociated rat hippocampal CA1 pyramidal neurons. The inhibitory effects of nilvadipine, amlodipine and flunarizine on LVA I_Ca were concentration- and use-dependent. The apparent half-maximum inhibitory concentrations (IC₅₀s) at every 1- and 30-s stimulation were 6.3×10⁻⁷ M and 1.8×10⁻⁶ M for flunarizine, 1.9×10⁻⁶ M and 7.6×10⁻⁶ M for nilvadipine, and 4.0×10⁻⁶ M and 8.0×10⁻⁶ M for amlodipine, respectively. Thus, the strength of the use-dependence was in the sequence of nilvadipine>flunarizine>amlodipine. Nilvadipine also inhibited the HVA I_Ca in a concentration-dependent manner with an IC₅₀ of 1.5×10⁻⁷ M. The hippocampal CA1 neurons were observed to have five pharmacologically distinct HVA Ca²⁺ channel subtypes consisting of L-, N-, P-, Q- and R-types. Nilvadipine selectively inhibited the L-type Ca²⁺ channel current which comprised 34% of the total HVA I_Ca. On the other hand, amlodipine non-selectively inhibited the HVA Ca²⁺ channel subtypes. These results suggest that the inhibitory effect of nilvadipine on the neuronal Ca²⁺ influx through both LVA and HVA L-type Ca²⁺ channels, in combination with the cerebral vasodilatory action, may prevent neuronal damage during ischemia.     

Effects of GABA on CA3 pyramidal cell dendrites in rabbit hippocampal slices

Using the in vitro rabbit hippocampal slice preparation, we have investigated the effects of gamma-aminobutyric acid (GABA) iontophoresis on CA3 pyramidal cell dendrites. The predominant response (70% of the cells tested) was a hyperpolarization associated with a 30% decrease in cell input resistance (Rm). These hyperpolarizations displayed a very pronounced voltage dependency: they were decreased by cell depolarization and flattened by hyperpolarization. Bicuculline methiodide (BMI, 50 microM) did not abolish this response, nor did intracellular iontophoresis of chloride ions. In 5% of the cells, an additional hyperpolarization was obtained with longer ejection times; it reversed close to the reversal potential of the early component of the IPSP. In 25% of the cells, dendritic GABA application produced a depolarization. This response was reversed with cell membrane depolarization and was associated with a large (80%) decrease in Rm. The depolarizations were abolished by BMI (50 microM) and greatly increased by increasing the intracellular chloride concentration. None of the responses to GABA were affected by blockade of synaptic transmission. We conclude that the predominant response of CA3 pyramidal cell dendrites to GABA application is a hyperpolarization mediated by GABAB receptors and probably carried by potassium ions. The depolarizing responses are mediated via GABAA receptors and depend on an increase in chloride permeability.