Rat Hippocampal CA1 Pyramidal Neurons Research Articles

Evaluation of mechanisms involved in regulation of intrinsic excitability by extracellular calcium in CA1 pyramidal neurons of rat.

It is well recognized that changes in the extracellular concentration of calcium ions influence the excitability of neurons, yet what mechanism(s) mediate these effects is still a matter of debate. Using patch-clamp recordings from rat hippocampal CA1 pyramidal neurons, we examined the contribution of G-proteins and intracellular calcium-dependent signaling mechanisms to changes in intrinsic excitability evoked by altering the extracellular calcium concentration from physiological (1.2 mM) to a commonly used experimental (2 mM) level. We find that the inhibitory effect on intrinsic excitability of calcium ions is mainly expressed as an increased threshold for action potential firing (with no significant effect on resting membrane potential) that is not blocked by either the G-protein inhibitor GDPβS or the calcium chelator BAPTA. Our results therefore argue that in the concentration range studied, G-protein coupled calcium-sensing receptors, non-selective cation conductances, and intracellular calcium signaling pathways are not involved in mediating the effect of extracellular calcium ions on intrinsic excitability. Analysis of the derivative of the action potential, dV/dt versus membrane potential, indicates a current shift towards more depolarized membrane potentials at the higher calcium concentration. Our results are thus consistent with a mechanism in which extracellular calcium ions act directly on the voltage-gated sodium channels by neutralizing negative charges on the extracellular surface of these channels to modulate the threshold for action potential activation.

Mechanisms of NMDA Receptor Inhibition by Sepimostat-Comparison with Nafamostat and Diarylamidine Compounds.

N-methyl-D-aspartate (NMDA) receptors are inhibited by many amidine and guanidine compounds. In this work, we studied the mechanisms of their inhibition by sepimostat-an amidine-containing serine protease inhibitor with neuroprotective properties. Sepimostat inhibited native NMDA receptors in rat hippocampal CA1 pyramidal neurons with IC50 of 3.5 ± 0.3 µM at -80 mV holding voltage. It demonstrated complex voltage dependence with voltage-independent and voltage-dependent components, suggesting the presence of shallow and deep binding sites. At -80 mV holding voltage, the voltage-dependent component dominates, and we observed pronounced tail currents and overshoots evidencing a "foot-in-the-door" open channel block. At depolarized voltages, the voltage-independent inhibition by sepimostat was significantly attenuated by the increase of agonist concentration. However, the voltage-independent inhibition was non-competitive. We further compared the mechanisms of the action of sepimostat with those of structurally-related amidine and guanidine compounds-nafamostat, gabexate, furamidine, pentamidine, diminazene, and DAPI-investigated previously. The action of all these compounds can be described by the two-component mechanism. All compounds demonstrated similar affinity to the shallow site, which is responsible for the voltage-independent inhibition, with binding constants in the range of 3-30 µM. In contrast, affinities to the deep site differed dramatically, with nafamostat, furamidine, and pentamidine being much more active.

Bi-directional modulation of hyperpolarization-activated cation currents (Ih) by ethanol in rat hippocampal CA3 pyramidal neurons

It is widely acknowledged that ethanol (EtOH) can alter many neuronal functions, including synaptic signaling, firing discharge, and membrane excitability, through its interaction with multiple membrane proteins and intracellular pathways. Previous work has demonstrated that EtOH enhances the firing rate of hippocampal GABAergic interneurons and thus the presynaptic GABA release at CA1 and CA3 inhibitory synapses through a positive modulation of the hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels. Activation of HCN channels produce an inward current, commonly called Ih, which plays an essential role in generating/regulating specific neuronal activities in GABAergic interneurons and principal glutamatergic pyramidal neurons such as those in the CA3 subregion. Since the direct effect of EtOH on HCN channels expressed in CA3 pyramidal neurons was not thoroughly elucidated, we investigated the possible interaction between EtOH and HCN channels and the impact on excitability and postsynaptic integration of these neurons. Patch-clamp recordings were performed in single CA3 pyramidal neurons from acute male rat coronal hippocampal slices. Our results show that EtOH modulates HCN-mediated Ih in a concentration-dependent and bi-directional manner, with a positive modulation at lower (20 mM) and an inhibitory action at higher (60–80 mM) concentrations. The modulation of Ih by EtOH was mimicked by forskolin, antagonized by different drugs that selectively interfere with the AC/cAMP/PKA intracellular pathway, as well as by the selective HCN inhibitor ZD7288. Altogether, these data further support the evidence that HCN channels may represent an important molecular target through which EtOH may regulate neuronal activity.

Cordycepin suppresses glutamatergic and GABAergic synaptic transmission through activation of A1 adenosine receptor in rat hippocampal CA1 pyramidal neurons.

Cordycepin (known as 3-deoxyadenosine, CRD), a natural product from the valuable traditional Chinese medicine Cordyceps militaris, has been reported to improve cognitive function and modulate neuroprotective effects on the central nervous system (CNS). However, the modulating mechanisms of cordycepin on information processing in hippocampal CA1 pyramidal neurons are not fully understood. To clarify how cordycepin modulates synaptic responses of pyramidal neurons in rat hippocampal CA1 region, we conducted an electrophysiological experiment using whole-cell patch-clamp technique. The spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs, respectively) and the spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs, respectively) recorded by this technique evaluated pure single or multi-synapse responses and enabled us to accurately quantify how cordycepin influenced the pre and postsynaptic aspects of synaptic transmission. The present results showed that cordycepin significantly decreased the frequency of both glutamatergic and GABAergic postsynaptic currents without affecting the amplitude, while these inhibitory effects were antagonized by the A1 adenosine receptor antagonist (DPCPX), but not the A2A (ZM 241385), A2B (MRS1754) and A3 (MRS1191) adenosine receptor antagonists. Taken together, our results suggested that cordycepin had a clear presynaptic effect on glutamatergic and GABAergic transmission, and provided novel evidence that cordycepin suppresses the synaptic transmission through the activation of A1AR.

Tapentadol Suppresses Glutamatergic Transmission and Neuronal Firing in Rat Hippocampal CA3 Pyramidal Neurons

Background: Tapentadol, a centrally acting oral analgesic, activates µ-opioid receptor and inhibits norepinephrine reuptake. Given that glutamate plays a crucial role in mediating pain, this study investigated the influence of tapentadol on spontaneous glutamatergic synaptic transmission and evoked neuronal excitability in rat hippocampal CA3 pyramidal neurons, which has been suggested to be involved in nociceptive perception. Methods: We used electrophysiological technique to determine the effect of tapentadol on spontaneous excitatory postsynaptic currents (sEPSC), glutamate-activated currents, and neuronal excitability in CA3 pyramidal neurons in rat hippocampal slices. We also used isolated nerve terminals (synaptosomes) prepared from the rat hippocampus to examine the effect of tapentadol on glutamate release. Results: Whole-cell patch clamp recordings revealed that tapentadol effectively decreased the frequencies of sEPSCs and miniature EPSCs (mEPSCs) without changing their amplitudes in hippocampal CA3 pyramidal neurons. However, glutamate-evoked inward currents were not affected by tapentadol. Further, tapentadol decreased 4-aminopyridine-induced glutamate release from hippocampal synaptosomes, and this effect was prevented by chelating the extracellular Ca²⁺ ions and blocking the N- and P/Q-type Ca²⁺ channels. In addition, burst firing induced by 4-aminopyridine and tonic repetitive firing induced by depolarizing pulses were attenuated by tapentadol. Conclusions: We conclude that tapentadol inhibits glutamatergic synaptic transmission, without modifying postsynaptic receptor sensitivity, and that this decline of excitation consequently suppresses neuronal hyperexcitability in the hippocampal CA3 area.

Interferon-γ potentiates GABAA receptor-mediated inhibitory currents in rat hippocampal CA1 pyramidal neurons

The neural transmission and plasticity can be differentially modulated by various elements of the immune system. Interferon-γ (IFN-γ) is a “pro-inflammatory” cytokine mainly produced by T lymphocytes, activates its corresponding receptor and plays important roles under both homeostatic and inflammatory conditions. However, the impact of IFN-γ on the γ-aminobutyric acid (GABA)-mediated currents in the hippocampus, a major brain region involved in the cognitive function, has not been investigated. Here we detected abundant expression of both IFN-γ receptor subunit gene transcripts (Ifngr1 and Ifngr2) in the rat hippocampus by quantitative PCR. In addition, we pre-incubated rat hippocampal slices with IFN-γ (100 ng/ml) and recorded GABA-activated spontaneous and miniature postsynaptic inhibitory currents (sIPSCs and mIPSCs) and tonic currents in hippocampal CA1 pyramidal neurons by the whole-cell patch-clamp method. The pre-incubation with IFN-γ increased the frequency but not the mean amplitude, rise time or decay time of both sIPSCs and mIPSCs in hippocampal CA1 pyramidal neurons, suggesting a presynaptic effect of IFN-γ. Moreover, the GABA-activated tonic currents were enhanced by IFN-γ. In conclusion, the potentiation of GABAergic currents in hippocampal neurons by IFN-γ may contribute to the disturbed neuronal excitability and cognitive dysfunction during neuroinflammation.

P: 74 Cholestasis Decreases Dendritic Spine Density in a Rat Hippocampal Organotypic Culture

BACKGROUND: Executive functioning impairment in children with cholestatic liver disease is increasingly recognized. Injury to developing neuronal networks could be an underlying mechanism. Given that the CSF of rat pups following bile duct ligation is highly concentrated in ammonia, murocholic and taurocholic acids, we hypothesized that cholestasis may significantly alter density of synaptic contacts onto pyramidal neurons, known to be essential in the development of executive functioning. METHODS: Transverse hippocampal organotypic slice (400 µm thick) were harvested from 6–7-day-old Wistar rats. They were maintained for 15 days in a CO2-incubator (33°C). pc-DNA3.1-EGFP plasmid biolistic transfection was performed 7 days after harvesting. 3 days following transfection, control medium or experimental medium containing 100 µM α-murocholic-acid, 200 µM taurocholic-acid and 2.5 mM ammonium was added to the culture (day 0 of exposure, MIX condition). Confocal microscopy using imageJ was used for to manually count CA1 pyramidal neuron dendritic spines as proxies for excitatory synapses. Static analysis quantified dendritic spines density each day. Dynamic analysis quantified dendritic spines turnover (loss and neo-formation) for each 24 h time-window. Statistical analysis was conducted using PRISM software for multiple t-test or mixed-effect ANOVA. RESULTS: Static analysis showed a biphasic profile in MIX condition. During early phase (first 3 days of exposure), we observed >50% decrease in dendritic spine density compared to control (cf Figure 1, P < 0.001). On days 3 to 4), spine density recovered to reach control value. Dynamic analysis showed 15% loss in dendritic spines stability during the early phase of exposure to MIX condition, compared to controls, with comparable low rates of spine turnover. During the late phase of MIX exposure, spine turnover increased significantly in favor of spine neo-formation: spine neo-formation was 10 times higher (0.280 vs 0.023 spines.µm-1, P = 0.033) than controls, while spine loss was 6 times higher in neurons exposed to MIX (0.154 vs 0.026 spines.µm-1, P = 0.0026) than controls. CONCLUSIONS: We demonstrate here that mimicking cholestasis ex vivo leads to a biphasic response in spine density of rat hippocampal CA1 pyramidal neurons. Spine density decreases during the first 3-days of exposure to a mix of bile acids and ammonium chloride-owing to decreased in spine stability. This is followed by increased spine turnover with spine neo-formation in excess of spine loss, yielding a final spine density similar to control animals. Whether these new spines are functionally equivalent to controls remains to be determined.

β3-Adrenergic receptor-dependent modulation of the medium afterhyperpolarization in rat hippocampal CA1 pyramidal neurons.

Action potential firing in hippocampal pyramidal neurons is regulated by generation of an afterhyperpolarization (AHP). Three phases of AHP are recognized, with the fast AHP regulating action potential firing at the onset of a burst and the medium and slow AHPs supressing action potential firing over hundreds of milliseconds and seconds, respectively. Activation of β-adrenergic receptors suppresses the slow AHP by a protein kinase A-dependent pathway. However, little is known regarding modulation of the medium AHP. Application of the selective β-adrenergic receptor agonist isoproterenol suppressed both the medium and slow AHPs evoked in rat CA1 hippocampal pyramidal neurons recorded from slices maintained in organotypic culture. Suppression of the slow AHP was mimicked by intracellular application of cAMP, with the suppression of the medium AHP by isoproterenol still being evident in cAMP-dialyzed cells. Suppression of both the medium and slow AHPs was antagonized by the β-adrenergic receptor antagonist propranolol. The effect of isoproterenol to suppress the medium AHP was mimicked by two β3-adrenergic receptor agonists, BRL37344 and SR58611A. The medium AHP was mediated by activation of small-conductance calcium-activated K+ channels and deactivation of H channels at the resting membrane potential. Suppression of the medium AHP by isoproterenol was reduced by pretreating cells with the H-channel blocker ZD7288. These data suggest that activation of β3-adrenergic receptors inhibits H channels, which suppresses the medium AHP in CA1 hippocampal neurons by utilizing a pathway that is independent of a rise in intracellular cAMP. This finding highlights a potential new target in modulating H-channel activity and thereby neuronal excitability. NEW & NOTEWORTHY The noradrenergic input into the hippocampus is involved in modulating long-term synaptic plasticity and is implicated in learning and memory. We demonstrate that activation of functional β3-adrenergic receptors suppresses the medium afterhyperpolarization in hippocampal pyramidal neurons. This finding provides an additional mechanism to increase action potential firing frequency, where neuronal excitability is likely to be crucial in cognition and memory.

GABAergic inhibition reduces the impact of synaptic excitation on somatic excitation

The effect of excitatory synaptic input on the excitation of the cell body is believed to vary depending on where and when the synaptic activation occurs in dendritic trees and the spatiotemporal modulation by inhibitory synaptic input. However, few studies have examined how individual synaptic inputs influence the excitability of the cell body in spontaneously active neuronal networks mainly because of the lack of an appropriate method. We developed a calcium imaging technique that monitors synaptic inputs to hundreds of spines from a single neuron with millisecond resolution in combination with whole-cell patch-clamp recordings of somatic excitation. In rat hippocampal CA3 pyramidal neurons ex vivo, a fraction of the excitatory synaptic inputs were not detectable in the cell body against background noise. These synaptic inputs partially restored their somatic impact when a GABAA receptor blocker was intracellularly perfused. Thus, GABAergic inhibition reduces the influence of some excitatory synaptic inputs on the somatic excitability. Numerical simulation using a single neuron model demonstrates that the timing and locus of a dendritic GABAergic input are critical to exert this effect. Moreover, logistic regression analyses suggest that the GABAergic inputs sectionalize spine activity; that is, only some subsets of synchronous synaptic activity seemed to be preferably passed to the cell body. Thus, dendrites actively sift inputs from specific presynaptic cell assemblies.

Thermal effect induced by low-frequency magnetic field on physiological characteristics in hippocampal CA1 region of rat

The aim of the present study is to analyze the influences of low-frequency magnetic stimulation on spontaneous excitatory postsynaptic currents (sEPSC) and action potentials (AP) of rat hippocampal CA1pyramidal neurons. Although this is a very common subject of neuroscience and electrophysiology, the thermal effects of the applied magnetic fields were always neglected in most previous studies, which might lead to improper conclusions. Therefore, we preformed a series of experiments to investigate the possible influences of these thermal effects. According to our previous simulation results, the temperature will not be higher than 4 °C even with the heating of a very high intensity applied magnetic field. So we do a series of whole-cell clamp experiments under 25 °C, 30 °C, 35 °C, and 40 °C, respectively. Using whole-cell patch-clamp technique, sEPSC and AP measurements were acquired for flux densities of 0 (thermal effect only), 7, 14, and 23 mT at temperatures of 25 °C, 30 °C, 35 °C, and 40 °C. The results show that the amplitudes of sEPSC and AP would both decrease with the temperature regardless of the intensity of the magnetic field. As is known, the sEPSC and AP will both affect the functions of neuron network, so we could reach to the conclusion that the temperature increase originating from the thermal heating effects of the applied magnetic field will also have influences on the functions of neurons or synapses. Furthermore, according the results, the thermal sensitivity of sEPSC and AP might be an inherent characteristic of neurons, and was not affected by the strength of the electromagnetic field.

Liraglutide modulates GABAergic signaling in rat hippocampal CA3 pyramidal neurons predominantly by presynaptic mechanism

Backgroundγ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain where it regulates activity of neuronal networks. The receptor for glucagon-like peptide-1 (GLP-1) is expressed in the hippocampus, which is the center for memory and learning. In this study we examined effects of liraglutide, a GLP-1 analog, on GABA signaling in CA3 hippocampal pyramidal neurons.MethodsWe used patch-clamp electrophysiology to record synaptic and tonic GABA-activated currents in CA3 pyramidal neurons in rat hippocampal brain slices.ResultsWe examined the effects of liraglutide on the neurons at concentrations ranging from one nM to one μM. Significant changes of the spontaneous inhibitory postsynaptic currents (sIPSCs) were only recorded with 100 nM liraglutide and then in just ≈50% of the neurons tested at this concentration. In neurons affected by liraglutide both the sIPSC frequency and the most probable amplitudes increased. When the action potential firing was inhibited by tetrodotoxin (TTX) the frequency and amplitude of IPSCs in TTX and in TTX plus 100 nM liraglutide were similar.ConclusionsThe results demonstrate that liraglutide regulation of GABA signaling of CA3 pyramidal neurons is predominantly presynaptic and more limited than has been observed for GLP-1 and exendin-4 in hippocampal neurons.

Resveratrol Attenuates Aβ-Induced Early Hippocampal Neuron Excitability Impairment via Recovery of Function of Potassium Channels.

Alzheimer's disease (AD) is an age-related neurodegenerative disease. Amyloid-β (Aβ) is not only the morphological hallmark but also the initiator of the pathology process of AD. As a natural compound found in grapes, resveratrol shows a protective effect on the pathophysiology of AD, but the underlying mechanism is not very clear. This study was to investigate whether resveratrol could attenuate Aβ-induced early impairment in hippocampal neuron excitability and the underlying mechanism. The excitability and voltage-gated potassium currents were examined in rat hippocampal CA1 pyramidal neurons by using whole-cell patch-clamp technique. It was found that Aβ25-35 increased the excitability of neurons. Resveratrol could reverse the Aβ25-35-induced increase in the frequency of repetitive firing and the spike half-width of action potential (AP). Moreover, resveratrol can attenuate Aβ25-35-induced decreases in transient potassium channel (I A ) and delay rectifier potassium channel (I K(DR)) of neurons. It was also found that resveratrol could decline the increase of protein kinase A (PKA) and inhibit the activation of PI3K/Akt signaling pathway induced by Aβ25-35. The results suggest that resveratrol alleviates Aβ25-35-induced dysfunction in hippocampal CA1 pyramidal neurons via recovery of the function of I A and I K(DR) by inhibiting the increase of PKA and the activation of PI3K/Akt signaling pathway.

Modulation Effects of Cordycepin on Voltage-Gated Sodium Channels in Rat Hippocampal CA1 Pyramidal Neurons in the Presence/Absence of Oxygen.

Our previous study revealed that cordycepin features important neuroprotective effects against hypoxic insult by improvement of neuronal electrophysiological function. Modulation on voltage-gated sodium channel (VGSC) in CA1 neurons is the initial event during hypoxia/ischemia. However, no study comprehensively investigated cordycepin on VGSC. Hence, this study investigated modulation effects of cordycepin on VGSC not only in oxygen physiological conditions but also in acute oxygen deprivation injury conditions. Results revealed that cordycepin (80 μM) reduced the amplitude of VGSC currents (INa) (77.6% of control, p < 0.01) within 1 min of drug exposure coupled with a negative shift in steady-state inactivation and prolonged recovery time course from inactivation. Additionally, this mild reduction on the peak of INa induced by the pretreatment with cordycepin can attenuate and delay the following hypoxia causing rapid dramatic decrease in INa with no additive change in the voltage dependence of inactivation. As modulation on VGSC in CA1 neurons represents the initial event during ischemia, we propose that suppression effect of cordycepin on VGSC is an important neuronal protective mechanism that may enhance neuronal tolerance to acute oxygen deprivation and delay hypoxia-induced neuronal injuries.

Effect of phenytoin on sodium conductances in rat hippocampal CA1 pyramidal neurons.

The antiepileptic drug phenytoin (PHT) is thought to reduce the excitability of neural tissue by stabilizing sodium channels (NaV) in inactivated states. It has been suggested the fast-inactivated state (IF) is the main target, although slow inactivation (IS) has also been implicated. Other studies on local anesthetics with similar effects on sodium channels have implicated the NaV voltage sensor interactions. In this study, we reexamined the effect of PHT in both equilibrium and dynamic transitions between fast and slower forms of inactivation in rat hippocampal CA1 pyramidal neurons. The effects of PHT were observed on fast and slow inactivation processes, as well as on another identified "intermediate" inactivation process. The effect of enzymatic removal of IF was also studied, as well as effects on the residual persistent sodium current (INaP). A computational model based on a gating charge interaction was derived that reproduced a range of PHT effects on NaV equilibrium and state transitions. No effect of PHT on IF was observed; rather, PHT appeared to facilitate the occupancy of other closed states, either through enhancement of slow inactivation or through formation of analogous drug-bound states. The overall significance of these observations is that our data are inconsistent with the commonly held view that the archetypal NaV channel inhibitor PHT stabilizes fast inactivation states, and we demonstrate that conventional slow activation "IS" and the more recently identified intermediate-duration inactivation process "II" are the primary functional targets of PHT. In addition, we show that the traditional explanatory frameworks based on the "modulated receptor hypothesis" can be substituted by simple, physiologically plausible interactions with voltage sensors. Additionally, INaP was not preferentially inhibited compared with peak INa at short latencies (50 ms) by PHT.

Regulation of GABA Equilibrium Potential by mGluRs in Rat Hippocampal CA1 Neurons.

The equilibrium potential for GABA-A receptor mediated currents (EGABA) in neonatal central neurons is set at a relatively depolarized level, which is suggested to be caused by a low expression of K+/Cl- co-transporter (KCC2) but a relatively high expression of Na+-K+-Cl- cotransporter (NKCC1). Theta-burst stimulation (TBS) in stratum radiatum induces a negative shift in EGABA in juvenile hippocampal CA1 pyramidal neurons. In the current study, the effects of TBS on EGABA in neonatal and juvenile hippocampal CA1 neurons and the underlying mechanisms were examined. Metabotropic glutamate receptors (mGluRs) are suggested to modulate KCC2 and NKCC1 levels in cortical neurons. Therefore, the involvement of mGluRs in the regulation of KCC2 or NKCC1 activity, and thus EGABA, following TBS was also investigated. Whole-cell patch recordings were made from Wistar rat hippocampal CA1 pyramidal neurons, in a slice preparation. In neonates, TBS induces a positive shift in EGABA, which was prevented by NKCC1 antisense but not NKCC1 sense mRNA. (RS)-a-Methyl-4-carboxyphenylglycine (MCPG), a group I and II mGluR antagonist, blocked TBS-induced shifts in both juvenile and neonatal hippocampal neurons. While blockade of mGluR1 or mGluR5 alone could interfere with TBS-induced shifts in EGABA in neonates, only a combined blockade could do the same in juveniles. These results indicate that TBS induces a negative shift in EGABA in juvenile hippocampal neurons but a positive shift in neonatal hippocampal neurons via corresponding changes in KCC2 and NKCC1 expressions, respectively. mGluR activation seems to be necessary for both shifts to occur while the specific receptor subtype involved seems to vary.

Tetrodotoxin abruptly blocks excitatory neurotransmission in mammalian CNS

The present study utilised a 'synaptic bouton' preparation of mechanically isolated rat hippocampal CA3 pyramidal neurons, which permits direct physiological and pharmacological quantitative analyses at the excitatory and inhibitory single synapse level. Evoked excitatory and inhibitory postsynaptic currents (eEPSCs and eIPSCs) were generated by focal paired-pulse electrical stimulation of single boutons. The sensitivity of eEPSC to tetrodotoxin (TTX) was higher than that of the voltage-dependent Na(+) channel whole-cell current (INa) in the postsynaptic CA3 soma membrane. The synaptic transmission was strongly inhibited by 3 nM TTX, at which concentration the INa was hardly suppressed. The IC50 values of eEPSC and INa for TTX were 2.8 and 37.9 nM, respectively, and complete inhibition was 3-10 nM for eEPSC and 1000 nM for INa. On the other hand, both eEPSC and eIPSC were equally and gradually inhibited by decreasing the external Na(+) concentration ([Na]o), which decreases the Na(+)gradient across the cell membrane. The results indicate that TTX at 3-10 nM could block most of voltage-dependent Na(+) channels on presynaptic nerve terminal, resulting in abruptly inhibition of action potential dependent excitatory neurotransmission.

The GLP-1 Receptor Agonist Exendin-4 and Diazepam Differentially Regulate GABAA Receptor-Mediated Tonic Currents in Rat Hippocampal CA3 Pyramidal Neurons.

Glucagon-like peptide-1 (GLP-1) is a metabolic hormone that is secreted in a glucose-dependent manner and enhances insulin secretion. GLP-1 receptors are also found in the brain where their signalling affects neuronal activity. We have previously shown that the GLP-1 receptor agonists, GLP-1 and exendin-4 enhanced GABA-activated synaptic and tonic currents in rat hippocampal CA3 pyramidal neurons. The hippocampus is the centre for memory and learning and is important for cognition. Here we examined if exendin-4 similarly enhanced the GABA-activated currents in the presence of the benzodiazepine diazepam. In whole-cell recordings in rat brain slices, diazepam (1 μM), an allosteric positive modulator of GABAA receptors, alone enhanced the spontaneous inhibitory postsynaptic current (sIPSC) amplitude and frequency by a factor of 1.3 and 1.6, respectively, and doubled the tonic GABAA current normally recorded in the CA3 pyramidal cells. Importantly, in the presence of exendin-4 (10 nM) plus diazepam (1 μM), only the tonic but not the sIPSC currents transiently increased as compared to currents recorded in the presence of diazepam alone. The results suggest that exendin-4 potentiates a subpopulation of extrasynaptic GABAA receptors in the CA3 pyramidal neurons.

Repeated transcranial magnetic stimulation prevents kindling-induced changes in electrophysiological properties of rat hippocampal CA1 pyramidal neurons

The mechanisms underlying antiepileptic or antiepileptogenic effects of repeated transcranial magnetic stimulation (rTMS) are poorly understood. In this study, we investigated the effect of rTMS applied during rapid amygdala kindling on some electrophysiological properties of hippocampal CA1 pyramidal neurons. Male Wistar rats were kindled by daily electrical stimulation of the basolateral amygdala in a semi-rapid manner (12 stimulations/day) until they achieved stage-5 seizure. One group (kindled+rTMS (KrTMS)) of animals received rTMS (1Hz for 4min) 5min after termination of daily kindling stimulations. Twenty four hours following the last kindling stimulation electrophysiological properties of hippocampal CA1 pyramidal neurons were investigated using whole-cell patch-clamp technique. Amygdala kindling significantly depolarized the resting membrane potential and increased the input resistance, spontaneous firing activity, number of evoked spikes and half-width of the first evoked spike. Kindling also decreased the first-spike latency and amplitude significantly. Application of rTMS during kindling somehow prevented the development of seizures and protected CA1 pyramidal neurons of hippocampus against deleterious effect of kindling on both passive and active neuronal electrophysiological properties. Interestingly, application of rTMS alone enhanced the excitability of CA1 pyramidal neurons significantly. Based on the results of our study, it may be suggested that rTMS exerts its anticonvulsant effect, in part, through preventing the amygdala kindling-induced changes in electrophysiological properties of hippocampal CA1 pyramidal neurons. It seems that rTMS exerts protective effects on the neural circuits involved in spreading the seizures from the focus to other parts of the brain.

GLP-1 and exendin-4 transiently enhance GABAA receptor-mediated synaptic and tonic currents in rat hippocampal CA3 pyramidal neurons.

Glucagon-like peptide-1 (GLP-1) is a hormone that stimulates insulin secretion. Receptors for GLP-1 are also found in the brain, including the hippocampus, the center for memory and learning. Diabetes is a risk factor for decreased memory functions. We studied effects of GLP-1 and exendin-4, a GLP-1 receptor agonist, on γ-aminobutyric acid (GABA) signaling in hippocampal CA3 pyramidal neurons. GABA is the main inhibitory neurotransmitter and decreases neuronal excitability. GLP-1 (0.01-1 nmol/L) transiently enhanced synaptic and tonic currents, and the effects were blocked by exendin (9-39). Ten pmol/L GLP-1 increased both the spontaneous inhibitory postsynaptic current (sIPSC) amplitudes and frequency by a factor of 1.8. In 0.1, 1 nmol/L GLP-1 or 10, 50, or 100 nmol/L exendin-4, only the sIPSC frequency increased. The tonic current was enhanced by 0.01-1 nmol/L GLP-1 and by 0.5-100 nmol/L exendin-4. When action potentials were inhibited by tetrodotoxin (TTX), inhibitory postsynaptic currents decreased and currents were no longer potentiated by GLP-1 or exendin-4. In contrast, although the tonic current decreased in TTX, it was still enhanced by GLP-1 or exendin-4. The results demonstrate GLP-1 receptor regulation of hippocampal function and are consistent with GLP-1 receptor agonists enhancing GABAA signaling by pre- and postsynaptic mechanisms.

Intracellular calcium elevation during plateau potentials mediated by extrasynaptic NMDA receptor activation in rat hippocampal CA1 pyramidal neurons is primarily due to calcium entry through voltage‐gated calcium channels

We reported previously that plateau potentials mediated by extrasynaptic N-methyl-d-aspartate receptors (NMDARs) can be induced either by synaptic stimulation in the presence of glutamate transporter antagonist or by iontophoresis of NMDA in rat hippocampal CA1 pyramidal neurons. To examine whether the plateau potentials are accompanied by an elevation of intracellular Ca2+ and to determine the source of Ca2+ elevation, we performed Ca2+ imaging during the plateau potential. Neurons were loaded with Ca2+ indicator fluo-4, and the plateau potentials were generated either synaptically in the presence of glutamate transporter antagonist or by iontophoretically applying NMDA. We have found that a transient elevation in intracellular Ca2+ accompanies the plateau potential. The synaptically induced plateau potential and the Ca2+ elevation were blocked by 5,7-dichlorokynurenic acid (5,7-dCK), an antagonist for the glycine-binding sites of NMDAR. A mixture of Cd2+ and tetrodotoxin did not block NMDA-induced plateau potentials, but completely abolished the accompanying Ca2+ elevation in both the presence and absence of Mg2+ ions in the bathing solution. The NMDA-induced plateau potential was blocked by further adding 5,7-dCK. Our results show that the NMDAR-mediated plateau potential is accompanied by elevation of intracellular Ca2+ that is primarily caused by the influx of Ca2+ through voltage-gated Ca2+ channels.