Cell‐specific regulation of neuronal activity by endogenous production of nitric oxide

Nitric oxide (NO) is a key regulator of neuronal excitability in the nervous system. While most studies have investigated its role as an intercellular messenger/modulator, less is known about potential physiological roles played by NO within NO‐producing neurons. We showed previously that intrinsic production of NO within B5 neurons of the pond snail Helisoma trivolvis increased neuronal excitability by acting on three ionic conductances. Here we demonstrate that intrinsically produced NO affected two of the same conductances in another buccal neuron, B19, where it had the opposite, namely inhibitory, effect on neuronal activity. Using single‐cell RT‐PCR, we show that B19 neurons express NO synthase (NOS) mRNA. The inhibition of intrinsic NO production with NOS inhibitors caused membrane potential depolarization, transient spiking and an increase in input resistance. Inhibition of the main intracellular receptor of NO, soluble guanylyl cyclase, had similar effects on the parameters mentioned above. An investigation of the effects of NO on ion channels revealed that intrinsic NO mediated neuronal hyperpolarization by activating voltage‐gated calcium channels that in turn caused the tonic opening of apamin‐sensitive calcium‐activated potassium channels. The analysis of action potentials in B5 and B19 neurons suggested that the opposite effects on neuronal excitability elicited by intrinsic NO were probably determined by differences in the ionic conductances that shape their action potentials. In summary, we describe a mechanism by which B19 neurons utilise intrinsically produced NO in a cell‐type‐specific fashion to decrease their neuronal activity, highlighting an important physiological role of NO within NO‐producing neurons.     

细胞外钙离子浓度对海马神经元兴奋性的影响

目的 在不同的生理、病理情况下,中枢神经系统细胞外钙离子浓度([Ca2 ]o)下降,导致神经元兴奋性增高.本研究的主要目的是探讨低钙条件下神经元兴奋性增高的机制,从而为临床治疗神经元过度兴奋性疾病探索新的治疗途径.方法 应用穿孔膜片钳及细胞培养技术,记录不同细胞外钙离子浓度对海马神经元兴奋性的影响.结果 低钙环境使神经元兴奋性显著增高,阈电位水平显著降低,动作电位幅度显著增高.并且出乎意料的是,mAHP拮抗剂apamin及sAHP拮抗剂Iso对海马神经元兴奋性的影响没有统计学的意义.作为INaP拮抗剂,低浓度的河豚毒(TTX)虽然阻断了低钙环境中神经元兴奋性的增加,但同时也阻断了正常钙离子浓度下的动作电位的发放.结论 低钙环境中海马神经元阈电位的显著下降可能是导致神经元兴奋性增高的主要原因.     

Development of resurgent and persistent sodium currents in mesencephalic trigeminal neurons

Sodium channels play multiple roles in the formation of neural membrane properties in mesencephalic trigeminal (Mes V) neurons and in other neural systems. Mes V neurons exhibit conditional robust high‐frequency spike discharges. As previously reported, resurgent and persistent sodium currents (I_NaR and I_NaP, respectively) may carry small currents at subthreshold voltages that contribute to generation of spike firing. These currents play an important role in maintaining and allowing high‐frequency spike discharge during a burst. In the present study, we investigated the developmental changes in tetrodotoxin‐sensitive I_NaR and I_NaP underlying high‐frequency spike discharges in Mes V neurons. Whole‐cell patch‐clamp recordings showed that both current densities increased one and a half times from postnatal day (P) 0–6 neurons to P7–14 neurons. Although these neurons do not exhibit subthreshold oscillations or burst discharges with high‐frequency firing, I_NaR and I_NaP do exist in Mes V neurons at P0–6. When the spike frequency at rheobase was examined in firing Mes V neurons, the developmental change in firing frequency among P7–14 neurons was significant. I_NaR and I_NaP density at ?40 mV also increased significantly among P7–14 neurons. The change to an increase in excitability in the P7–14 group could result from this quantitative change in I_NaP. In neurons older than P7 that exhibit repetitive firing, quantitative increases in I_NaR and I_NaP density may be major factors that facilitate and promote high‐frequency firing as a function of age in Mes V neurons.     

Sodium and calcium mechanisms of rhythmic bursting in excitatory neural networks of the pre‐Bötzinger complex: a computational modelling study

The neural mechanisms generating rhythmic bursting activity in the mammalian brainstem, particularly in the pre‐Bötzinger complex (pre‐BötC), which is involved in respiratory rhythm generation, and in the spinal cord (e.g. locomotor rhythmic activity) that persist after blockade of synaptic inhibition remain poorly understood. Experimental studies in rodent medullary slices containing the pre‐BötC identified two mechanisms that could potentially contribute to the generation of rhythmic bursting: one based on the persistent Na⁺ current (I_NaP), and the other involving the voltage‐gated Ca²⁺ current (I_Ca) and the Ca²⁺‐activated nonspecific cation current (I_CAN), activated by intracellular Ca²⁺ accumulated from extracellular and intracellular sources. However, the involvement and relative roles of these mechanisms in rhythmic bursting are still under debate. In this theoretical/modelling study, we investigated Na⁺‐dependent and Ca²⁺‐dependent bursting generated in single cells and heterogeneous populations of synaptically interconnected excitatory neurons with I_NaP and I_Ca randomly distributed within populations. We analysed the possible roles of network connections, ionotropic and metabotropic synaptic mechanisms, intracellular Ca²⁺ release, and the Na⁺/K⁺ pump in rhythmic bursting generated under different conditions. We show that a heterogeneous population of excitatory neurons can operate in different oscillatory regimes with bursting dependent on I_NaP and/or I_CAN, or independent of both. We demonstrate that the operating bursting mechanism may depend on neuronal excitation, synaptic interactions within the network, and the relative expression of particular ionic currents. The existence of multiple oscillatory regimes and their state dependence demonstrated in our models may explain different rhythmic activities observed in the pre‐BötC and other brainstem/spinal cord circuits under different experimental conditions.     

Activation of KCNN3/SK3/KCa2.3 channels attenuates enhanced calcium influx and inflammatory cytokine production in activated microglia

In neurons, small‐conductance calcium‐activated potassium (KCNN/SK/K_Ca2) channels maintain calcium homeostasis after N‐methyl‐D ‐aspartate (NMDA) receptor activation, thereby preventing excitotoxic neuronal death. So far, little is known about the function of KCNN/SK/K_Ca2 channels in non‐neuronal cells, such as microglial cells. In this study, we addressed the question whether KCNN/SK/K_Ca2 channels activation affected inflammatory responses of primary mouse microglial cells upon lipopolysaccharide (LPS) stimulation. We found that N‐cyclohexyl‐N‐[2‐(3,5‐dimethyl‐pyrazol‐1‐yl)‐6‐methyl‐4‐pyrimidinamine (CyPPA), a positive pharmacological activator of KCNN/SK/K_Ca2 channels, significantly reduced LPS‐stimulated activation of microglia in a concentration‐dependent manner. The general KCNN/SK/K_Ca2 channel blocker apamin reverted these effects of CyPPA on microglial proliferation. Since calcium plays a central role in microglial activation, we further addressed whether KCNN/SK/K_Ca2 channel activation affected the changes of intracellular calcium levels, [Ca²⁺]_i,, in microglial cells. Our data show that LPS‐induced elevation of [Ca²⁺]_i was attenuated following activation of KCNN2/3/K_Ca2.2/K_Ca2.3 channels by CyPPA. Furthermore, CyPPA reduced downstream events including tumor necrosis factor alpha and interleukin 6 cytokine production and nitric oxide release in activated microglia. Further, we applied specific peptide inhibitors of the KCNN/SK/K_Ca2 channel subtypes to identify which particular channel subtype mediated the observed anti‐inflammatory effects. Only inhibitory peptides targeting KCNN3/SK3/K_Ca2.3 channels, but not KCNN2/SK2/K_Ca2.2 channel inhibition, reversed the CyPPA‐effects on LPS‐induced microglial proliferation. These findings revealed that KCNN3/SK3/K_Ca2.3 channels can modulate the LPS‐induced inflammatory responses in microglial cells. Thus, KCNN3/SK3/K_Ca2.3 channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in the central nervous system. © 2012 Wiley Periodicals, Inc.     

Dorsal root ganglion neurons of embryonic chicks contain nitric oxide synthase and respond to nitric oxide

We investigated the function of nitric oxide (NO) in dorsal root ganglion (DRG) neurons from 10 day embryonic chicks and adult birds. NADPH-diaphorase activity, a histochemical marker for nitric ooxide synthase (NOS) in paraformaldehyde-fixed neurons, and NOS-like immunoreactivity were localized in all neurons in thoracic and lumbar ganglia from embros. However, only a subset of neurons from adults contained NOS-like immunoreactivity and NADPH-diaophorase activity. Thus, embryonic chick DRG neurons have the potential to synthesize NO in response to elevated cytoplasmic Ca²⁺. We also investigated the ability of dissociated embryonic chick DRG neurons to respond to NO by examining the effects of NO donors and 8-bromoguasonine 3′,5′-cyclic monophosphate (8-Br-cGMP) on Ca²⁺ current (I_Ca) using the amphotericin-permeabilized patch-clamp technique: sodium nitroprusside (5 μM) reduced I_Ca to 0.68 ± 0.06 (mean±S.D., n = 5) of control, S-nitroso-N-acetylpenicillamine) (1 μM) reduced I_Cato0.44 ± 0.06 (n = 4) of control, while 8-Br-cGMP (1 mM) reduced I_{Cato0.58 ± 0.22 (n = 5)} of control. I_Ca was reduced in every neuron tested and this effect was partially reversed after ≈ 10min of washing. Thus, I_Ca of embryonic chick DRG neurons is inhibited by NO, possibly by a cGMP-dependent mechanism. These results indicate that all DRG neurons in embryonic chicks contain NOS-like immunoreactivity and respond to NO. Further, the percentage of NADPH-diaphorase positive neurons is reduced during development.     

Long‐term high‐intensity sound stimulation inhibits h current (Ih) in CA1 pyramidal neurons

Afferent neurotransmission to hippocampal pyramidal cells can lead to long‐term changes to their intrinsic membrane properties and affect many ion currents. One of the most plastic neuronal currents is the hyperpolarization‐activated cationic current (I_h), which changes in CA1 pyramidal cells in response to many types of physiological and pathological processes, including auditory stimulation. Recently, we demonstrated that long‐term potentiation (LTP) in rat hippocampal Schaffer‐CA1 synapses is depressed by high‐intensity sound stimulation. Here, we investigated whether a long‐term high‐intensity sound stimulation could affect intrinsic membrane properties of rat CA1 pyramidal neurons. Our results showed that I_h is depressed by long‐term high‐intensity sound exposure (1 min of 110 dB sound, applied two times per day for 10 days). This resulted in a decreased resting membrane potential, increased membrane input resistance and time constant, and decreased action potential threshold. In addition, CA1 pyramidal neurons from sound‐exposed animals fired more action potentials than neurons from control animals; however, this effect was not caused by a decreased I_h. On the other hand, a single episode (1 min) of 110 dB sound stimulation which also inhibits hippocampal LTP did not affect I_h and firing in pyramidal neurons, suggesting that effects on I_h are long‐term responses to high‐intensity sound exposure. Our results show that prolonged exposure to high‐intensity sound affects intrinsic membrane properties of hippocampal pyramidal neurons, mainly by decreasing the amplitude of I_h.     

Subcellular Distribution of Persistent Sodium Conductance in Cortical Pyramidal Neurons

Cortical pyramidal neurons possess a persistent Na⁺ current (I_NaP), which, in contrast to the larger transient current, does not undergo rapid inactivation. Although relatively quite small, I_NaP is active at subthreshold voltages and therefore plays an important role in neuronal input–output processing. The subcellular distribution of channels responsible for I_NaP and the mechanisms that render them persistent are not known. Using high-speed fluorescence Na⁺ imaging and whole-cell recordings in brain slices obtained from mice of either sex, we reconstructed the I_NaP elicited by slow voltage ramps in soma and processes of cortical pyramidal neurons. We found that in all neuronal compartments, the relationship between persistent Na⁺ conductance and membrane voltage has the shape of a Boltzmann function. Although the density of channels underlying I_NaP was about twofold lower in the axon initial segment (AIS) than in the soma, the axonal channels were activated by ∼10 mV less depolarization than were somatic channels. This difference in voltage dependence explains why, at functionally critical subthreshold voltages, most I_NaP originates in the AIS. Finally, we show that endogenous polyamines constrain I_NaP availability in both somatodendritic and axonal compartments of nondialyzed cortical neurons.SIGNIFICANCE STATEMENT The most salient characteristic of neuronal sodium channels is fast inactivation. However, a fraction of the sodium current does not inactivate. In cortical neurons, persistent current (I_NaP) plays a prominent role in many important functions. Its subcellular distribution and generation mechanisms are, however, elusive. Using high-speed fluorescence Na⁺ imaging and electrical recordings, we reconstructed the I_NaP in soma and processes of cortical pyramidal neurons. We found that at near-threshold voltages I_NaP originates predominately from the axon, because of the distinctive voltage dependence of the underlying channels and not because of their high density. Finally, we show that the presence of endogenous polyamines significantly constrains I_NaP availability in all compartments of nondialyzed cortical neurons.     

Mechanism of the medium‐duration afterhyperpolarization in rat serotonergic neurons

Most serotonergic neurons display a prominent medium‐duration afterhyperpolarization (mAHP), which is mediated by small‐conductance Ca²⁺‐activated K⁺ (SK) channels. Recent ex vivo and in vivo experiments have suggested that SK channel blockade increases the firing rate and/or bursting in these neurons. The purpose of this study was therefore to characterize the source of Ca²⁺ which activates the mAHP channels in serotonergic neurons. In voltage‐clamp experiments, an outward current was recorded at ?60 mV after a depolarizing pulse to +100 mV. A supramaximal concentration of the SK channel blockers apamin or (‐)‐bicuculline methiodide blocked this outward current. This current was also sensitive to the broad Ca²⁺ channel blocker Co²⁺ and was partially blocked by both ω‐conotoxin and mibefradil, which are blockers of N‐type and T‐type Ca²⁺ channels, respectively. Neither blockers of other voltage‐gated Ca²⁺ channels nor DBHQ, an inhibitor of Ca²⁺‐induced Ca²⁺ release, had any effect on the SK current. In current‐clamp experiments, mAHPs following action potentials were only blocked by ω‐conotoxin and were unaffected by mibefradil. This was observed in slices from both juvenile and adult rats. Finally, when these neurons were induced to fire in an in vivo‐like pacemaker rate, only ω‐conotoxin was able to increase their firing rate (by ~30%), an effect identical to the one previously reported for apamin. Our results demonstrate that N‐type Ca²⁺ channels are the only source of Ca²⁺ which activates the SK channels underlying the mAHP. T‐type Ca²⁺ channels may also activate SK channels under different circumstances.     

Activation of ryanodine receptors is required for PKA‐mediated downregulation of A‐type K+ channels in rat hippocampal neurons

A‐type K⁺ channels (I_A channels) contribute to learning and memory mechanisms by regulating neuronal excitabilities in the CNS, and their expression level is targeted by Ca²⁺ influx via synaptic NMDA receptors (NMDARs) during long‐term potentiation (LTP). However, it is not clear how local synaptic Ca²⁺ changes induce I_A downregulation throughout the neuron, extending from the active synapse to the soma. In this study, we tested if two major receptors of endoplasmic reticulum (ER), ryanodine (RyRs), and IP₃ (IP₃R) receptors, are involved in Ca²⁺‐mediated I_A downregulation in cultured hippocampal neurons of rats. The downregulation of I_A channels was induced by doubling the Ca²⁺ concentration in culture media (3.6 mM for 24 hrs) or treating with glycine (200 μM for 3 min) to induce chemical LTP (cLTP), and the changes in I_A peaks were measured electrophysiologically by a whole‐cell patch. We confirmed that Ca²⁺ or glycine treatment significantly reduced I_A peaks and that their effects were abolished by blocking NMDARs or voltage‐dependent Ca²⁺ channels (VDCCs). In this cellular processing, blocking RyRs (by ryanodine, 10 μM) but not IP₃Rs (by 2APB, 100 μM) completely abolished I_A downregulation, and the LTP observed in hippocampal slices was more diminished by ryanodine rather than 2APB. Furthermore, blocking RyRs also reduced Ca²⁺‐mediated PKA activation, indicating that sequential signaling cascades, including the ER and PKA, are involved in regulating I_A downregulation. These results strongly suggest a possibility that RyR contribution and mediated I_A downregulation are required to regulate membrane excitability as well as synaptic plasticity in CA3‐CA1 connections of the hippocampus. © 2017 Wiley Periodicals, Inc.     

Neonatal mouse cortical but not isogenic human astrocyte feeder layers enhance the functional maturation of induced pluripotent stem cell‐derived neurons in culture

Human induced pluripotent stem (iPS) cell‐derived neurons and astrocytes are attractive cellular tools for nervous system disease modeling and drug screening. Optimal utilization of these tools requires differentiation protocols that efficiently generate functional cell phenotypes in vitro. As nervous system function is dependent on networked neuronal activity involving both neuronal and astrocytic synaptic functions, we examined astrocyte effects on the functional maturation of neurons from human iPS cell‐derived neural stem cells (NSCs). We first demonstrate human iPS cell‐derived NSCs can be rapidly differentiated in culture to either neurons or astrocytes with characteristic cellular, molecular and physiological features. Although differentiated neurons were capable of firing multiple action potentials (APs), few cells developed spontaneous electrical activity in culture. We show spontaneous electrical activity was significantly increased by neuronal differentiation of human NSCs on feeder layers of neonatal mouse cortical astrocytes. In contrast, co‐culture on feeder layers of isogenic human iPS cell‐derived astrocytes had no positive effect on spontaneous neuronal activity. Spontaneous electrical activity was dependent on glutamate receptor‐channel function and occurred without changes in I_Na, I_K, V_m, and AP properties of iPS cell‐derived neurons. These data demonstrate co‐culture with neonatal mouse cortical astrocytes but not human isogenic iPS cell‐derived astrocytes stimulates glutamatergic synaptic transmission between iPS cell‐derived neurons in culture. We present RNA‐sequencing data for an immature, fetal‐like status of our human iPS cell‐derived astrocytes as one possible explanation for their failure to enhance synaptic activity in our co‐culture system.     

Enhancement of neuronal outward delayed rectifier K+ current by human monocyte‐derived macrophages

Macrophages are critical cells in mediating the pathology of neurodegenerative disorders and enhancement of neuronal outward potassium (K⁺) current has implicated in neuronal apoptosis. To understand how activated macrophages induce neuronal dysfunction and injury, we studied the effects of lipopolysaccharide (LPS)‐stimulated human monocytes‐derived macrophage (MDM) on neuronal outward delayed rectifier K⁺ current (I_K) and resultant change on neuronal viability in primary rat hippocampal neuronal culture. Bath application of LPS‐stimulated MDM‐conditioned media (MCM) enhanced neuronal I_K in a concentration‐dependentmanner, whereas non‐stimulated MCM failed to alter neuronal I_K. The enhancement of neuronal I_K was repeated in a macrophage‐neuronal co‐culture system. The link of stimulated MCM (MCM(+))‐associated enhancement of I_K to MCM(+)‐induced neuronal injury, as detected by PI/DAPI (propidium iodide/4′,6‐diamidino‐2‐phenylindol) staining and MTT assay, was demonstrated by experimental results showing that addition of I_K blocker tetraethylammonium to the culture protected hippocampal neurons from MCM(+)‐associated challenge. Further investigation revealed elevated levels of K_v 1.3 and K_v 1.5 channel expression in hippocampal neurons after addition of MCM(+) to the culture. These results suggest that during brain inflammation macrophages, through their capacity of releasing bioactive molecules, induce neuronal injury by enhancing neuronal I_K and that modulation of K_v channels is a new approach to neuroprotection. © 2009 Wiley‐Liss, Inc.     

Adenosine modulates the excitability of layer II stellate neurons in entorhinal cortex through A1 receptors

Stellate neurons in layer II entorhinal cortex (EC) provide the main output from the EC to the hippocampus. It is believed that adenosine plays a crucial role in neuronal excitability and synaptic transmission in the CNS, however, the function of adenosine in the EC is still elusive. Here, the data reported showed that adenosine hyperpolarized stellate neurons in a concentration‐dependent manner, accompanied by a decrease in firing frequency. This effect corresponded to the inhibition of the hyperpolarization‐activated, cation nonselective (HCN) channels. Surprisingly, the adenosine‐induced inhibition was blocked by 3 μM 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), a selective A₁ receptor antagonists, but not by 10 μM 3,7‐dimethyl‐1‐propargylxanthine (DMPX), a selective A₂ receptor antagonists, indicating that activation of adenosine A₁ receptors were responsible for the direct inhibition. In addition, adenosine reduced the frequency but not the amplitude of miniature EPSCs and IPSCs, suggesting that the global depression of glutamatergic and GABAergic transmission is mediated by a decrease in glutamate and GABA release, respectively. Again the presynaptic site of action was mediated by adenosine A₁ receptors. Furthermore, inhibition of spontaneous glutamate and GABA release by adenosine A₁ receptor activation was mediated by voltage‐dependent Ca²⁺ channels and extracellular Ca²⁺. Therefore, these findings revealed direct and indirect mechanisms by which activation of adenosine A₁ receptors on the cell bodies of stellate neurons and on the presynaptic terminals could regulate the excitability of these neurons. © 2010 Wiley‐Liss, Inc.     

Phenotype‐dependent Ca2+ dynamics in single boutons of various anatomically identified GABAergic interneurons in the rat hippocampus

Interneurons (INs) of the hippocampus exert versatile inhibition on pyramidal cells by silencing the network at different oscillation frequencies. Although IN discharge can phase‐lock to various rhythms in the hippocampus, under high‐frequency axon firing, the boutons may not be able to follow the fast activity. Here, we studied Ca²⁺ responses to action potentials (APs) in single boutons using combined two‐photon microscopy and patch clamp electrophysiology in three types of INs: non‐fast‐spiking (NFS) neurons showing cannabinoid 1 receptor labelling and dendrite targeting, fast‐spiking partially parvalbumin‐positive cells synapsing with dendrites (DFS), and parvalbumin‐positive cells with perisomatic innervation (PFS). The increase in [Ca²⁺]_i from AP trains was substantially higher in NFS boutons than in DFS or PFS boutons. The decay of bouton Ca²⁺ responses was markedly faster in DFS and PFS cells compared with NFS neurons. The bouton‐to‐bouton variability of AP‐evoked Ca²⁺ transients in the same axon was surprisingly low in each cell type. Importantly, local responses were saturated after shorter trains of APs in NFS cells than in PFS cells. This feature of fast‐spiking neurons might allow them to follow higher‐frequency gamma oscillations for a longer time than NFS cells. The function of NFS boutons may better support asynchronous GABA release. In conclusion, we demonstrate several neuron‐specific Ca²⁺ transients in boutons of NFS, PFS and DFS neurons, which may serve differential functions in hippocampal networks.     

Human cerebrospinal fluid promotes spontaneous gamma oscillations in the hippocampus in vitro

Gamma oscillations (30–80 Hz) are fast network activity patterns frequently linked to cognition. They are commonly studied in hippocampal brain slices in vitro, where they can be evoked via pharmacological activation of various receptor families. One limitation of this approach is that neuronal activity is studied in a highly artificial extracellular fluid environment, as provided by artificial cerebrospinal fluid (aCSF). Here, we examine the influence of human cerebrospinal fluid (hCSF) on kainate‐evoked and spontaneous gamma oscillations in mouse hippocampus. We show that hCSF, as compared to aCSF of matched electrolyte and glucose composition, increases the power of kainate‐evoked gamma oscillations and induces spontaneous gamma activity in areas CA3 and CA1 that is reversed by washout. Bath application of atropine entirely abolished hCSF‐induced gamma oscillations, indicating critical contribution from muscarinic acetylcholine receptor‐mediated signaling. In separate whole‐cell patch clamp recordings from rat hippocampus, hCSF increased theta resonance frequency and strength in pyramidal cells along with enhancement of h‐current (I_h) amplitude. We found no evidence of intrinsic gamma frequency resonance at baseline (aCSF) among fast‐spiking interneurons, and this was not altered by hCSF. However, hCSF increased the excitability of fast‐spiking interneurons, which likely contributed to gamma rhythmogenesis. Our findings show that hCSF promotes network gamma oscillations in the hippocampus in vitro and suggest that neuromodulators distributed in CSF could have significant influence on neuronal network activity in vivo.     

Postnatal development of temporal integration,spike timing and spike threshold regulation by a dendrotoxin‐sensitive K+ current in rat CA1 hippocampal cells

Spike timing and network synchronization are important for plasticity, development and maturation of brain circuits. Spike delays and timing can be strongly modulated by a low‐threshold, slowly inactivating, voltage‐gated potassium current called D‐current (I_D). I_D can delay the onset of spiking, cause temporal integration of multiple inputs, and regulate spike threshold and network synchrony. Recent data indicate that I_D can also undergo activity‐dependent, homeostatic regulation. Therefore, we have studied the postnatal development of I_D‐dependent mechanisms in CA1 pyramidal cells in hippocampal slices from young rats (P7–27), using somatic whole‐cell recordings. At P21–27, these neurons showed long spike delays and pronounced temporal integration in response to a series of brief depolarizing current pulses or a single long pulse, whereas younger cells (P7–20) showed shorter discharge delays and weak temporal integration, although the spike threshold became increasingly negative with maturation. Application of α‐dendrotoxin (α‐DTX), which blocks I_D, reduced the spiking latency and temporal integration most strongly in mature cells, while shifting the spike threshold most strongly in a depolarizing direction in these cells. Voltage‐clamp analysis revealed an α‐DTX‐sensitive outward current (I_D) that increased in amplitude during development. In contrast to P21–23, I_D in the youngest group (P7–9) showed smaller peri‐threshold amplitude. This may explain why long discharge delays and robust temporal integration only appear later, 3 weeks postnatally. We conclude that I_D properties and I_D‐dependent functions develop postnatally in rat CA1 pyramidal cells, and I_D may modulate network activity and plasticity through its effects on synaptic integration, spike threshold, timing and synchrony.     

Transient cerebral ischemia increases CA1 pyramidal neuron excitability

In human and experimental animals, the hippocampal CA1 region is one of the most vulnerable areas of the brain to ischemia. Pyramidal neurons in this region die 2–3 days after transient cerebral ischemia whereas other neurons in the same region remain intact. The mechanisms underlying the selective and delayed neuronal death are unclear. We tested the hypothesis that there is an increase in post-synaptic intrinsic excitability of CA1 pyramidal neurons after ischemia that exacerbates glutamatergic excitotoxicity. We performed whole-cell patch-clamp recordings in brain slices obtained 24 h after in vivo transient cerebral ischemia. We found that the input resistance and membrane time constant of the CA1 pyramidal neurons were significantly increased after ischemia, indicating an increase in neuronal excitability. This increase was associated with a decrease in voltage sag, suggesting a reduction of the hyperpolarization-activated non-selective cationic current (I_h). Moreover, after blocking I_h with ZD7288, the input resistance of the control neurons increased to that of the post-ischemia neurons, suggesting that a decrease in I_h contributes to increased excitability after ischemia. Finally, when lamotrigine, an enhancer of dendritic I_h, was applied immediately after ischemia, there was a significant attenuation of CA1 cell loss. These data suggest that an increase in CA1 pyramidal neuron excitability after ischemia may exacerbate cell loss. Moreover, this dendritic channelopathy may be amenable to treatment.     

Enhancement in activities of large conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia

It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization (fAHP) increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance Ca²⁺-activated potassium (BK_Ca) channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by using inside-out configuration of patch clamp techniques. (1) The unitary conductance of BK_Ca channels in post-ischemic neurons (295 pS) was higher than that in control neurons (245 pS) in symmetrical 140/140 mM K⁺ in inside-out patch; (2) the membrane depolarization for an e-fold increase in open probability (P_o) showed no significant differences between two groups while the membrane potential required to produce one-half of the maximum P_o was more negative after ischemia, indicating no obvious changes in channel voltage dependence; (3) the [Ca²⁺]_i required to half activate BK_Ca channels was only 1 μM in post-ischemic whereas 2 μM in control neurons, indicating an increase in [Ca²⁺]_i sensitivity after ischemia; and (4) BK_Ca channels had a longer open time and a shorter closed time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BK_Ca channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal excitability and increase in fAHP.