Excitatory effect of thiamin on CA1 pyramidal neurones in rat hippocampal slices in vitro

Clinically, acute thiamin deficiency may lead to Wernicke encephalopathy and fulminant cardial beriberi. Both diseases respond to high parenteral doses of thiamin. The cofactor role of intracellular thiamin diphosphate has been thoroughly investigated, but an additional acute effect of unphosphorylated extracellular thiamin has been postulated but not elucidated. In order to investigate the role of thiamin at the membrane level in the central nervous system, a study using a well-established in vitro rat hippocampal slice model was designed. Hippocampal slices were perfused with 0.5, 0.75 and 1 mM thiamin solutions for 30 min and the pre-synaptic volley, field excitatory post-synaptic potential and population spike amplitudes were recorded continuously. The results showed an acute, excitatory effect of high-dose thiamin on hippocampal neurones by significantly increasing the number of repetitive afterdischarges. Additional experiments with low concentrations of the potassium channel blocker 4-aminopyridine showed similar findings. The results support previous evidence of thiamin affecting membrane ion channel activity, probably involving potassium channels, although the precise mechanisms of action are still unclear.     

Postsynaptic blockade of inhibitory postsynaptic currents by plasmin in CA1 pyramidal cells of rat hippocampus

We have shown previously that plasmin facilitated the generation of long-term potentiation (LTP) in CA1 and dentate region of rat hippocampus. In the present study, we investigated the effects of plasmin on postsynaptic currents in CA1 pyramidal neurons of rat hippocampal slices. Plasmin (100 nM) had no effect on NMDA nor on non-NMDA receptor-mediated excitatory postsynaptic currents. However, plasmin significantly decreased GABA_A receptor-mediated inhibitory postsynaptic currents. This effect of plasmin disappeared when intracellular Ca²⁺ was strongly chelated with BAPTA. Furthermore, plasmin attenuated the GABA-induced currents in CA1 pyramidal cells. These results suggest that the STP-enhancing effect of plasmin is due to a blockade of postsynaptic GABA_A responses and that an increase in intracellular Ca²⁺ by plasmin may be involved in its mechanism.     

Action of vasopressin on CA1 pyramidal neurons in rat hippocampal slices

The effects of arginine⁸-vasopressin (AVP) on the excitability of 47 pyramidal cells of the CA1 region of the hippocampus were determined by using intracellular recording techniques in a submerged slice preparation. Addition of 10⁻⁶ M AVP to the bathing medium evoked an increase in spike discharge which was slow in onset and only gradually reversible. The discharge was accompanied by an increase in excitatory postsynaptic potentials without significant change of the resting input resistance. AVP-induced excitation was found in 81% of ventral and 29% of dorsal hippocampal CA1 pyramidal cells. In low Ca²⁺, high Mg²⁺ solution this excitatory action by AVP was blocked. Microiontophoretic application of AVP onto apical or basal dendrites or the cell body did not result in excitation. These observations suggest that the action of AVP on CA1 pyramidal cells is transsynaptic and is more pronounced in ventral than dorsal CA1.     

High-threshold calcium channel activity in rat hippocampal neurones during hypoxia

Whole-cell patch clamp recordings in combination with direct control and measurements of O2 tension (pO2) in bath solution were used to determine the sensitivity of Ca2+ channels of cultured hippocampal neurones to hypoxia in glucose free solution. In all tested neurones, a lowering of pO2 to 4/50 mmHg did not induce changes either in magnitude, kinetics or voltage-current relations of total Ca2+ currents, which composed mainly from two types, L-type (64%) and N-type (31%) components. Hypoxia only induced a delay of Ca2+ current run-down about 27.5% and 39% at 50 and 4 mmHg pO2 respectively that presumably depended on changes in cytoplasmic channel-modulatory metabolites. The obtained results demonstrate that Ca2+ channel molecules in cultured hippocampal neurones are themselves insensitive to short-lasting (10-20 min) oxygen and glucose deprivation, and that they are not a principal target for hypoxic influences on hippocampal function.     

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.     

Diminished calcium currents in aged hippocampal dentate gyrus granule neurones

Voltage-dependent calcium currents in hippocampal dentate gyrus granule neurons of young adult (4-6 months) and aged (24-26 months) Fischer 344 rats have been examined using single-electrode voltage-clamp techniques. Calcium currents, especially a high threshold slowly inactivating L-type current, were significantly depressed in neurons obtained from aged animals. Furthermore, these age-dependent changes could be reversed by intracellular injection of the calcium chelator, ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). These results are in agreement with the hypothesis that aging results in a persistent increase in the free cytoplasmic calcium concentration in mammalian central neurons.     

A physiological test for electrotonic coupling between CA1 pyramidal cells in rat hippocampal slices

Antidromic stimulation in slices of rat hippocampal cortex was used to test for electrotonic coupling between CA1 pyramidal neurons. The slices were incubated in low Ca2+ medium containing Mn2+, in order to block chemical synapses. In a small percentage of intracellular recordings, short-latency depolarizations could be observed after chemical transmission had been blocked. Preceding somatic action potentials did not block the depolarizations. This indirect test provides electrophysiological evidence for coupling between some CA1 pyramidal cells.     

Two distinct types of transient outward currents in area postrema neurons in rat brain slices

We investigated the electrophysiological properties of the area postrema neurons in acutely prepared rat brain slices using the whole-cell patch-clamp technique. Two different types of transient outward potassium current (I(to)), fast and slow, were found in the area postrema. Both the decay time constant and rise time were significantly faster in the fast I(to) than in the slow I(to). Both current-clamp and voltage-clamp recordings revealed that the activation of fast and slow I(to) contributes to generation of the different spiking patterns, late spiking and interrupted spiking, respectively. The activation and inactivation of both I(to) were strongly voltage-dependent. Curve fitting by the Boltzmann equation revealed no significant difference in the activation and inactivation curves for each I(to) except that the slope factor of inactivation was larger for fast I(to). Both I(to) were suppressed dose-dependently by application of 4-aminopyridine. Each spiking pattern was enhanced when cells were held at a more hyperpolarized membrane potential, i.e. a longer latency of the first spike or longer interspike interval between the first and second spikes. The voltage-dependent modulation of the spiking pattern was consistent with the voltage-dependent activation of I(to). The present study shows significant subdivisions of the area postrema neurons distinguished by a difference in the kinetics of I(to) and spiking patterns. We discuss the role of I(to) as the ionic current underlying neuronal excitability.     

Efects of corticosterone on the electrophysiology of hippocampal CA1 pyramidal Cells in vitro

Modulation of CA1 field potential amplitudes by normal and stress concentrations of corticosterone (CT) was observed in hippocampal slice preparations from adrenalectomized rats. Slices exposed to CT levels characteristic of a morning (4 nM) or evening (7 nM) resting state showed increased population spike amplitudes in the CA1 pyramidal cell field within 10 min. A stress concentration (15 nM) also increased spike amplitudes, but only at the higher stimulus intensities. The effects of these doses were essentially the same 10 and 60 min after administration. The hormone facilitated responding more in morning resting concentrations than in concentrations characteristic of the evening resting state. This occurred, however, only for relatively low intensity stimuli. The data provide some support for the suggestion that circadian fluctuations in magnitude of long-term potentiation result from corresponding changes in CT level. The rapid onset of the observed changes is difficult to account for in terms of generally accepted mechanisms of receptor binding.     

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.     

Autofluorescence as a confound in the determination of calcium levels in hippocampal slices using fura-2AM dye

Recent publications have reported calcium level determinations in slices of brain using imaging techniques and the dye fura-2AM. In general these studies ignore or deal only perfunctorily with the problem of autofluorescence in slices. This confound, which is a result of the pyridine nucleotides that are normally present in tissue, has been previously reported to interfere with Ca²⁺ measurements in slices. Because these pyridine compounds are involved in cell metabolism, the fluorescence intensity is labile over time following experimental manipulations. We were studying Ca²⁺ levels in hippocampal slices using standard imaging techniques. We found significant and variable autofluorescence at the wavelengths used for calcium determination which interfered with data interpretation in fura-treated slices. The intensity of this autofluorescence is an additive effect and is not large enough to be observed when imaging monolayers. In this paper we present a method for conducting experiments and analyzing data that decreases interference from autofluorescence. Experiments were carried out on both slices bulk loaded with fura-2AM and slices loaded with control buffer. A point to point subtraction of the control slice values gave representative calcium fluorescence values. Hippocampal slices were challenged with sodium cyanide or kainic acid. The metabolic response, seen in the fura-free slices, and the calcium response varied within and between these two treatments. Regional differences in the hippocampal sub fields were also demonstrated in response to the two treatments. These corresponded to known regional vulnerabilities to cyanide and kainate. We conclude that autofluorescence in slices need be considered when determining calcium concentrations using fura-2AM.     

Kindling-induced epilepsy alters calcium currents in granule cells of rat hippocampal slices

Single electrode voltage-clamp recordings were obtained from dentate gyrus granule cells (GCs) in hippocampal slices of control and commissurally kindled rats. Two types of calcium currents, a transient and a sustained current, were studied in control and kindled neurons. The threshold of the transient calcium current was lowered in kindled GCs. The sustained calcium current was absent in kindled neurons but it could be restored by the intracellular administration of the calcium chelator EGTA. Our findings are consistent with the hypothesis that the loss of an intraneuronal calcium binding protein (Calbindin-D28K; CaBP) reduces the intraneuronal calcium buffering capacity in kindled neurons and results in the enhanced calcium-dependent inactivation of sustained calcium currents.     

Extracellular calcium alters frequency modulation of [H]acetylcholine release from rat hippocampal slices

The concentration of extracellular Ca²⁺ has been shown to enhance or attenuate [³H]acetylcholine (ACh) release subsequent to a conditioning stimulus in rat brain hippocampal slices. Slices were incubated in vitro in [³H]choline solution. Subsequently the slices were subjected to two consecutive electrical stimulations separated by 15 or 30 min at 0.25, 1, 4 and 16 Hz and [³H]ACh release was assessed. It was found that a conditioning stimulus may reduce [³H]ACh release during a second stimulation. This phenomenon is frequency related and disappears when the two stimulations are 30 min apart. High extracellular Ca²⁺ (4.0 mM) further attenuated [³H]ACh release during the second stimulation whereas low Ca²⁺ (0.32 mM) abolished the decrease in [³H]ACh release following the second stimulation in all frequencies tested.     

Delayed potassium current and non-specific outward current in pyramidal neurones acutely dissociated from rat hippocampus

The electrical property of delayed K+ currents (IKD) was studied in pyramidal neurones freshly isolated from the rat hippocampal CA1 region. The IKD was separated pharmacologically from other membrane currents. Activation and inactivation processes of the IKD were highly voltage-dependent in the potential range between -30 and +20 mV. The steady-state inactivation of IKD was observed at -100 mV or more positive potentials. The potential for half steady-state inactivation was -65 mV. The IKD was fully inactivated around -20 mV. Reactivation of IKD consisted of two exponential components. After pharmacological suppression of IKD, the small amount of residual voltage-dependent outward current (one-fifteenth to one-twentieth of IKD amplitude) was observed. The current kinetics was similar to that of IKD and greatly reduced by substitution of internal K+ with N-methyl-D-glucamine+. It was concluded that the properties of IKD was basically similar to those of IKD in other excitable tissues and that the residual current might be non-specific outward current.     

Repeated administration of pentylenetetrazol alters susceptibility of rat hippocampus to primed-burst stimulation: evidence from in vitro study on CA1 of hippocampal slices

The effectiveness of θ pattern primed-bursts (PBs) on development of primed-burst (PB) potentiation was investigated in hippocampal CA1 of pentylenetetrazol-kindled rats. Experiments were carried out in the hippocampal slices from control and kindled rats at two post-kindling periods, i.e. 48–144 h (early phase) and 30–33 days (long-lasting phase). Field potentials (population excitatory post-synaptic potential, pEPSP) were recorded at stratum radiatum following stimulation of the stratum fibers. θ pattern primed-bursts were delivered to stratum radiatum and PB potentiation was assessed. The results showed that 48–144 h after kindling, PB potentiation in CA1 of kindled slices is significantly greater than control slices. In contrast, 30–33 days after kindling PB potentiation was not observed and the pEPSP slope was depressed after PBs delivery, which lasted at least 60 min. Our results suggest that shortly after kindling, PB potentiation can be more readily induced while one month later, it is more difficult to elicit. These findings may help to explain the behavioral deficits seen with the kindling model of epilepsy.     

Transient depression of excitatory synaptic transmission induced by adenosine uptake inhibition in rat hippocampal slices

The transient property of the dipyridamole-induced depression of excitatory synaptic transmission was analyzed using field EPSPs (fEPSPs) recorded from the CA1 region in rat hippocampal slices. The fEPSPs were depressed by 1 microM dipyridamole and then gradually recovered to the control level. The depression was antagonized by aminophylline or DPCPX, although it was not significantly affected by DMPX. The results suggest that the fEPSP depression is induced by a mechanism through the A(1) receptor.     

Interleukin-1β depresses calcium currents in CA1 hippocampal neurons at pathophysiological concentrations

Interleukin-1 is present in the central nervous system (CNS) during acute and chronic pathological processes. In the present study, we examined the interaction between recombinant human interleukin-1β (rhIL-1β) and the voltage-dependent calcium (Ca²⁺) current using the whole-cell patch clamp technique. RhIL-1β depressed the voltage-gated Ca²⁺ current in acutely dissociated guinea pig hippocampal CA1 neurons. This depression is rapid and is observed at pathophysiological concentrations ( 1.97 pg/10 μl)- Concomitant application of rhIL-1β and rhIL-1 receptor antagonist had no effect indicating neuroactive specificity of rhIL-1β. The depression of the inward Ca²⁺ current by IL-1β may play a role in:

1. 1) the regulation of neuronal excitability;

2. 2) the induction of neurological manifestations during disease; and

3. 3) in the induction and/or progression of neurodegenerative processes.

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.