L-bupivacaine Inhibition of Nociceptive Transmission in Rat Peripheral and Dorsal Horn Neurons.

BACKGROUND: Although the widely used single L-enantiomers of local anesthetics have less toxic effects on the cardiovascular and central nervous systems, the mechanisms mediating their antinociceptive actions are not well understood. The authors hypothesized that significant differences in the ion channel blocking abilities of the enantiomers of bupivacaine would be identified. METHODS: The authors performed electrophysiologic analysis on rat dorsal root ganglion neurons in vitro and on spinal transmissions in vivo. RESULTS: In the dorsal root ganglion, these anesthetics decreased the amplitudes of action potentials. The half-maximum inhibitory concentrations of D-enantiomer D-bupivacaine were almost equal for Aß (29.5 µM), Aδ (29.7µM), and C (29.8 µM) neurons. However, the half-maximum inhibitory concentrations of L-bupivacaine was lower for Aδ (19.35 µM) and C (19.5 µM) neurons than for A ß (79.4 µM) neurons. Moreover, D-bupivacaine almost equally inhibited tetrodotoxin-resistant (mean ± SD: 15.8 ± 10.9% of the control, n = 14, P < 0.001) and tetrodotoxin-sensitive (15.4 ± 15.6% of the control, n = 11, P = 0.004) sodium currents. In contrast, L-bupivacaine suppressed tetrodotoxin-resistant sodium currents (26.1 ± 19.5% of the control, n = 18, P < 0.001) but not tetrodotoxin-sensitive sodium currents (74.5 ± 18.2% of the control, n = 11, P = 0.477). In the spinal dorsal horn, L-bupivacaine decreased the area of pinch-evoked excitatory postsynaptic currents (39.4 ± 11.3% of the control, n = 7, P < 0.001) but not touch-evoked responses (84.2 ± 14.5% of the control, n = 6, P = 0.826). In contrast, D-bupivacaine equally decreased pinch- and touch-evoked responses (38.8 ± 9.5% of the control, n = 6, P = 0.001, 42.9 ± 11.8% of the control, n = 6, P = 0.013, respectively). CONCLUSIONS: These results suggest that the L-enantiomer of bupivacaine (L-bupivacaine) effectively inhibits noxious transmission to the spinal dorsal horn by blocking action potential conduction through C and Aδ afferent fibers.

Synaptic Multivesicular Release in the Cerebellar Cortex: Its Mechanism and Role in Neural Encoding and Processing.

The number of synaptic vesicles released during fast release plays a major role in determining the strength of postsynaptic response. However, it remains unresolved how the number of vesicles released in response to action potentials is controlled at a single synapse. Recent findings suggest that the Cav2.1 subtype (P/Q-type) of voltage-gated calcium channels is responsible for inducing presynaptic multivesicular release (MVR) at rat cerebellar glutamatergic synapses from granule cells to molecular layer interneurons. The topographical distance from Cav2.1 channels to exocytotic Ca(2+) sensors is a critical determinant of MVR. In physiological trains of presynaptic neurons, MVR significantly impacts the excitability of postsynaptic neurons, not only by increasing peak amplitude but also by prolonging decay time of the postsynaptic currents. Therefore, MVR contributes additional complexity to neural encoding and processing in the cerebellar cortex.

Cav2.1 channels control multivesicular release by relying on their distance from exocytotic Ca2+ sensors at rat cerebellar granule cells.

The concomitant release of multiple numbers of synaptic vesicles [multivesicular release (MVR)] in response to a single presynaptic action potential enhances the flexibility of synaptic transmission. However, the molecular mechanisms underlying MVR at a single CNS synapse remain unclear. Here, we show that the Cav2.1 subtype (P/Q-type) of the voltage-gated calcium channel is specifically responsible for the induction of MVR. In the rat cerebellar cortex, paired-pulse activation of granule cell (GC) ascending fibers leads not only to a facilitation of the peak amplitude (PPFamp) but also to a prolongation of the decay time (PPPdecay) of the EPSCs recorded from molecular layer interneurons. PPFamp is elicited by a transient increase in the number of released vesicles. PPPdecay is highly dependent on MVR and is caused by dual mechanisms: (1) a delayed release and (2) an extrasynaptic spillover of the GC transmitter glutamate and subsequent pooling of the glutamate among active synapses. PPPdecay was specifically suppressed by the Cav2.1 channel blocker ω-agatoxin IVA, while PPFamp responded to Cav2.2/Cav2.3 (N-type/R-type) channel blockers. The membrane-permeable slow Ca(2+) chelator EGTA-AM profoundly reduced the decay time constant (τdecay) of the second EPSC; however, it only had a negligible impact on that of the first, thereby eliminating PPPdecay. These results suggest that the distance between presynaptic Cav2.1 channels and exocytotic Ca(2+) sensors is a key determinant of MVR. By transducing presynaptic action potential firings into unique Ca(2+) signals and vesicle release profiles, Cav2.1 channels contribute to the encoding and processing of neural information.

Large-scale somatotopic refinement via functional synapse elimination in the sensory thalamus of developing mice.

Functional synapse elimination and strengthening are crucial developmental processes in the formation of precise neuronal circuits in the somatosensory system, but the underlying alterations in topographical organization are not yet fully understood. To address this issue, we generated transgenic mice in which afferent fibers originating from the whisker-related brain region, called the maxillary principal trigeminal nucleus (PrV2), were selectively visualized with genetically expressed fluorescent protein. We found that functional synapse elimination drove and established large-scale somatotopic refinement even after the thalamic barreloid architecture was formed. Before functional synapse elimination, the whisker sensory thalamus was innervated by afferent fibers not only from the PrV2, but also from the brainstem nuclei representing other body parts. Most notably, only afferent fibers from PrV2 onto a whisker sensory thalamic neuron selectively survived and were strengthened, whereas other afferent fibers were preferentially eliminated via their functional synapse elimination. This large-scale somatotopic refinement was at least partially dependent on somatosensory experience. These novel results uncovered a previously unrecognized role of developmental synapse elimination in the large-scale, instead of the fine-scale, somatotopic refinement even after the initial segregation of the barreloid map.

Compromised maturation of GABAergic inhibition underlies abnormal network activity in the hippocampus of epileptic Ca2+ channel mutant mice, tottering.

Cholinergically induced network activity is a useful analogue of theta rhythms involved in memory processing or epileptiform activity in the hippocampus, providing a powerful tool to elucidate the mechanisms of synchrony in neuronal networks. In absence epilepsy, although its association with cognitive impairments has been reported, the mechanisms underlying hippocampal synchrony remain poorly investigated. Here we simultaneously recorded electrical activities from 64 sites in hippocampal slices of CaV2.1 Ca(2+) channel mutant tottering (tg) mice, a well-established mouse model of spontaneous absence epilepsy, to analyze the spatiotemporal pattern of cholinergically induced hippocampal network activity. The cholinergic agonist carbachol induced oscillatory discharges originating from the CA3 region. In tg/tg mice, this hippocampal network activity was characterized by enhanced occupancy of discharges of relatively high frequency (6-10 Hz) compared to the wild type. Pharmacological analyses of slices, patch clamp electrophysiological characterization of isolated neurons, and altered patterns of hippocampal GABAA receptor subunit and Cl(-) transporter messenger RNA (mRNA) transcript levels revealed that this abnormality is attributable to a developmental retardation of GABAergic inhibition caused by immature intracellular Cl(-) regulation. These results suggest that the inherited CaV2.1 Ca(2+) channel mutation leads to developmental abnormalities in Cl(-) transporter expression and GABAA receptor compositions in hippocampal neurons and that compromised maturation of GABAergic inhibition contributes to the abnormal synchrony in the hippocampus of tg absence epileptic mice.

Novel KCNQ1 Q234K variant, identified in patients with long QT syndrome and epileptiform activity, induces both gain- and loss-of-function of slowly activating delayed rectifier potassium currents.

Introduction: KCNQ1 and KCNE1 form slowly activating delayed rectifier potassium currents (IKs). Loss-of-function of IKs by KCNQ1 variants causes type-1 long QT syndrome (LQTS). Also, some KCNQ1 variants are reported to cause epilepsy. Segment 4 (S4) of voltage-gated potassium channels has several positively-charged amino acids that are periodically aligned, and acts as a voltage-sensor. Intriguingly, KCNQ1 has a neutral-charge glutamine at the third position (Q3) in the S4 (Q234 position in KCNQ1), which suggests that the Q3 (Q234) may play an important role in the gating properties of IKs. We identified a novel KCNQ1 Q234K (substituted for a positively-charged lysine) variant in patients (a girl and her mother) with LQTS and epileptiform activity on electroencephalogram. The mother had been diagnosed with epilepsy. Therefore, we sought to elucidate the effects of the KCNQ1 Q234K on gating properties of IKs. Methods: Wild-type (WT)-KCNQ1 and/or Q234K-KCNQ1 were transiently expressed in tsA201-cells with KCNE1 (E1) (WT + E1-channels, Q234K + E1-channels, and WT + Q234K + E1-channels), and membrane currents were recorded using whole-cell patch-clamp techniques. Results: At 8-s depolarization, current density (CD) of the Q234K + E1-channels or WT + Q234K + E1-channels was significantly larger than the WT + E1-channels (WT + E1: 701 ± 59 pA/pF; Q234K + E1: 912 ± 50 pA/pF, p < 0.01; WT + Q234K + E1: 867 ± 48 pA/pF, p < 0.05). Voltage dependence of activation (VDA) of the Q234K + E1-channels or WT + Q234K + E1-channels was slightly but significantly shifted to depolarizing potentials in comparison to the WT + E1-channels ([V1/2] WT + E1: 25.6 ± 2.6 mV; Q234K + E1: 31.8 ± 1.7 mV, p < 0.05; WT + Q234K + E1: 32.3 ± 1.9 mV, p < 0.05). Activation rate of the Q234K + E1-channels or WT + Q234K + E1-channels was significantly delayed in comparison to the WT + E1-channels ([half activation time] WT + E1: 664 ± 37 ms; Q234K + E1: 1,417 ± 60 ms, p < 0.01; WT + Q234K + E1: 1,177 ± 71 ms, p < 0.01). At 400-ms depolarization, CD of the Q234K + E1-channels or WT + Q234K + E1-channels was significantly decreased in comparison to the WT + E1-channels (WT + E1: 392 ± 42 pA/pF; Q234K + E1: 143 ± 12 pA/pF, p < 0.01; WT + Q234K + E1: 209 ± 24 pA/pF, p < 0.01) due to delayed activation rate and depolarizing shift of VDA. Conclusion: The KCNQ1 Q234K induced IKs gain-of-function during long (8-s)-depolarization, while loss of-function during short (400-ms)-depolarization, which indicates that the variant causes LQTS, and raises a possibility that the variant may also cause epilepsy. Our data provide novel insights into the functional consequences of charge addition on the Q3 in the S4 of KCNQ1.

Rewiring of afferent fibers in the somatosensory thalamus of mice caused by peripheral sensory nerve transection.

The remodeling of neural circuitry and changes in synaptic efficacy after peripheral sensory nerve injury are considered the basis for functional reorganization in the brain, including changes in receptive fields. However, when or how the remodeling occurs is largely unknown. Here we show the rapid rewiring of afferent fibers in the mature ventral posteromedial thalamic nucleus of mice after transection of the peripheral whisker sensory nerve, using the whole-cell voltage-clamp technique. Transection induced the recruitment of afferent fibers to a thalamic relay neuron within 5-6 d of injury. The rewiring was pathway specific, but not sensory experience dependent or peripheral nerve activity dependent. The newly recruited fibers mediated small EPSCs, and postsynaptic GluA2-containing AMPA receptors were selectively upregulated at the new synapses. This rapid and pathway-specific remodeling of thalamic circuitry may be an initial step in the massive axonal reorganization at supraspinal levels, which occurs months or years after peripheral sensory nerve injury.

Enhanced inhibitory neurotransmission in the cerebellar cortex of Atp1a3-deficient heterozygous mice.

Dystonia is characterized by excessive involuntary and prolonged simultaneous contractions of both agonist and antagonist muscles. Although the basal ganglia have long been proposed as the primary region, recent studies indicated that the cerebellum also plays a key role in the expression of dystonia. One hereditary form of dystonia, rapid-onset dystonia with parkinsonism (RDP), is caused by loss of function mutations of the gene for the Na pump α3 subunit (ATP1A3). Little information is available on the affected brain regions and mechanism for dystonia by the mutations in RDP. The Na pump is composed of α and ß subunits and maintains ionic gradients of Na(+) and K(+) across the cell membrane. The gradients are utilized for neurotransmitter reuptake and their alteration modulates neural excitability. To provide insight into the molecular aetiology of RDP, we generated and analysed knockout heterozygous mice (Atp1a3(+/-)). Atp1a3(+/-) showed increased symptoms of dystonia that is induced by kainate injection into the cerebellar vermis. Atp1a3 mRNA was highly expressed in Purkinje cells and molecular-layer interneurons, and its product was concentrated at Purkinje cell soma, the site of abundant vesicular γ-aminobutyric acid transporter (VGAT) signal, suggesting the presynaptic localization of the α3 subunit in the inhibitory synapse. Electrophysiological studies showed that the inhibitory neurotransmission at molecular-layer interneuron-Purkinje cell synapses was enhanced in Atp1a3(+/-) cerebellar cortex, and that the enhancement originated via a presynaptic mechanism. Our results shed light on the role of Atp1a3 in the inhibitory synapse, and potential involvement of inhibitory synaptic dysfunction for the pathophysiology of dystonia.

Paired-pulse facilitation of multivesicular release and intersynaptic spillover of glutamate at rat cerebellar granule cell-interneurone synapses.

A simple form of presynaptic plasticity, paired-pulse facilitation (PPF), has been explained as a transient increase in the probability of vesicular release. Using the whole-cell patch-clamp technique to record synaptic activity in rat cerebellar slices, we found different forms of presynaptically originated short-term plasticity during glutamatergic excitatory neurotransmission from granule cells (GCs) to molecular-layer interneurones (INs). Paired-pulse activation of GC axons at short intervals (30-100 ms) elicited not only a facilitation in the peak amplitude (PPF(amp)), but also a prolongation in the decay-time constant (PPP(decay)) of the EPSCs recorded from INs. The results of pharmacological tests and kinetics analyses suggest that the mechanisms underlying the respective types of short-term plasticity were different. PPF(amp) was elicited by a transient increase in the number of released vesicles. On the other hand, PPP(decay) was caused not only by delayed release as has been reported but also by extrasynaptic spillover of the GC transmitter and the subsequent intersynaptic pooling. Both PPF(amp) and PPP(decay) closely rely on repetitive-activation-induced multivesicular release. Using a dynamic clamp technique, we further examined the physiological significance of different presynaptic plasticity, and found that PPF(amp) and PPP(decay) can differentially encode and process neuronal information by influencing the total synaptic charge transferred to postsynaptic INs to reflect activation frequency of the presynaptic GCs.

In vivo patch-clamp recording from locus coeruleus neurones in the rat brainstem.

Locus coeruleus (LC) neurones extend noradrenergic projections throughout the neuroaxis and are involved in homeostatic functions such as pain modulation, arousal and cardio-respiratory control. To address the cellular mechanisms underlying pain modulation we have developed a patch-clamp recording technique from LC neurones in anaesthetized rats. These recordings showed LC discharge in vivo to be driven by both spontaneous membrane potential oscillations and CNQX-sensitive EPSCs opposed by bicuculine-sensitive IPSCs. Hindlimb pinch evoked a biphasic action potential response underpinned by a slow monophasic excitatory current. This approach allows detailed characterisation of the synaptic and integrative mechanisms of LC responses to naturalistic stimulation.

Roles of the subthalamic nucleus and subthalamic HCN channels in absence seizures.

Absence seizures consist of a brief and sudden impairment of consciousness. They are characterized by bilaterally synchronized spike and wave discharges (SWDs), which reflect abnormal oscillations in the thalamocortical loops. Recent studies have suggested that the basal ganglia are involved in generation of the SWDs, but their roles are poorly understood at the molecular and cellular levels. Here we studied the pathophysiological roles of the basal ganglia, using in vivo and in vitro measurements of tottering mice, a well-established model of absence epilepsy. We found that the membrane excitability in subthalamic nucleus (STN) neurons was enhanced in tottering mice, which resulted from reduced hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity. Pharmacological blockade and activation of HCN channel activity in vitro bidirectionally altered the membrane excitability of the STN neurons. Furthermore, these pharmacological modulations of HCN channel activity in the STN in vivo bidirectionally altered the mean SWD duration. In addition, STN deep brain stimulation modulated SWDs in a frequency-dependent manner. These results indicate that STN is involved in the rhythm maintenance system of absence seizures.

Hippocampus-related cognitive disorders develop in the absence of epilepsy and ataxia in the heterozygous Cacna1a mutant mice tottering.

CACNA1A-associated epilepsy and ataxia frequently accompany cognitive impairments as devastating co-morbidities. However, it is unclear whether the cognitive deficits are consequences secondary to the neurological symptoms elicited by CACNA1A mutations. To address this issue, Cacna1a mutant mice tottering (tg), and in particular tg/+ heterozygotes, serve as a suitable model system, given that tg/+ heterozygotes fail to display spontaneous absence epilepsy and ataxia typically observed in tg/tg homozygotes. Here, we examined hippocampus-dependent behaviors and hippocampal learning-related synaptic plasticity in tg mice. In behavioral analyses of tg/+ and tg/tg, acquisition and retention of spatial reference memory were characteristically impaired in the Morris water maze task, while working memory was intact in the eight-arm radial maze and T-maze tasks. tg/+ heterozygotes showed normal motor function in contrast to tg/tg homozygotes. In electrophysiological analyses, Schaffer collateral-CA1 synapses showed a deficit in the maintenance of long-term potentiation in tg/+ and tg/tg mice and an increased paired-pulse facilitation induced by paired pulses with 100 ms in tg/tg mice. Our results indicate that the tg mutation causes a dominant disorder of the hippocampus-related memory and synaptic plasticity, raising the possibility that in CACNA1A-associated human diseases, functionally aberrant CaV2.1 Ca2+ channels actively induce the observed cognitive deficits independently of the neurological symptoms.

Kinase-dead knock-in mouse reveals an essential role of kinase activity of Ca2+/calmodulin-dependent protein kinase IIalpha in dendritic spine enlargement, long-term potentiation, and learning.

Ca2+/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha) is an essential mediator of activity-dependent synaptic plasticity that possesses multiple protein functions. So far, the autophosphorylation site-mutant mice targeted at T286 and at T305/306 have demonstrated the importance of the autonomous activity and Ca2+/calmodulin-binding capacity of CaMKIIalpha, respectively, in the induction of long-term potentiation (LTP) and hippocampus-dependent learning. However, kinase activity of CaMKIIalpha, the most essential enzymatic function, has not been genetically dissected yet. Here, we generated a novel CaMKIIalpha knock-in mouse that completely lacks its kinase activity by introducing K42R mutation and examined the effects on hippocampal synaptic plasticity and behavioral learning. In homozygous CaMKIIalpha (K42R) mice, kinase activity was reduced to the same level as in CaMKIIalpha-null mice, whereas CaMKII protein expression was well preserved. Tetanic stimulation failed to induce not only LTP but also sustained dendritic spine enlargement, a structural basis for LTP, at the Schaffer collateral-CA1 synapse, whereas activity-dependent postsynaptic translocation of CaMKIIalpha was preserved. In addition, CaMKIIalpha (K42R) mice showed a severe impairment in inhibitory avoidance learning, a form of memory that is dependent on the hippocampus. These results demonstrate that kinase activity of CaMKIIalpha is a common critical gate controlling structural, functional, and behavioral expression of synaptic memory.

Bone cancer induces a unique central sensitization through synaptic changes in a wide area of the spinal cord.

BACKGROUND: Chronic bone cancer pain is thought to be partly due to central sensitization. Although murine models of bone cancer pain revealed significant neurochemical changes in the spinal cord, it is not known whether this produces functional alterations in spinal sensory synaptic transmission. In this study, we examined excitatory synaptic responses evoked in substantia gelatinosa (SG, lamina II) neurons in spinal cord slices of adult mice bearing bone cancer, using whole-cell voltage-clamp recording techniques. RESULTS: Mice at 14 to 21 days after sarcoma implantation into the femur exhibited hyperalgesia to mechanical stimuli applied to the skin of the ipsilateral hind paw, as well as showing spontaneous and movement evoked pain-related behaviors. SG neurons exhibited spontaneous excitatory postsynaptic currents (EPSCs). The amplitudes of spontaneous EPSCs were significantly larger in cancer-bearing than control mice without any changes in passive membrane properties of SG neurons. In the presence of TTX, the amplitude of miniature EPSCs in SG neurons was increased in cancer-bearing mice and this was observed for cells sampled across a wide range of lumbar segmental levels. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor- and N-methyl-D-aspartate (NMDA) receptor-mediated EPSCs evoked by focal stimulation were also enhanced in cancer-bearing mice. Dorsal root stimulation elicited mono- and/or polysynaptic EPSCs that were caused by the activation of Adelta and/or C afferent fibers in SG neurons from both groups of animals. The number of cells receiving monosynaptic inputs from Adelta and C fibers was not different between the two groups. However, the amplitude of the monosynaptic C fiber-evoked EPSCs and the number of SG neurons receiving polysynaptic inputs from Adelta and C fibers were increased in cancer-bearing mice. CONCLUSIONS: These results show that spinal synaptic transmission mediated through Adelta and C fibers is enhanced in the SG across a wide area of lumbar levels following sarcoma implantation in the femur. This widespread spinal sensitization may be one of the underlying mechanisms for the development of chronic bone cancer pain.

Glutamate transporter EAAT4 in Purkinje cells controls intersynaptic diffusion of climbing fiber transmitter mediating inhibition of GABA release from interneurons.

Neurotransmitters diffuse out of the synaptic cleft and act on adjacent synapses to exert concerted control of the synaptic strength within neural pathways that converge on single target neurons. The excitatory transmitter released from climbing fibers (CFs), presumably glutamate, is shown to inhibit γ-aminobutyric acid (GABA) release at basket cell (BC)-Purkinje cell (PC) synapses in the rat cerebellar cortex through its extrasynaptic diffusion and activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors on BC axon terminals. This study aimed at examining how the CF transmitter-diffusion-mediated presynaptic inhibition is controlled by glutamate transporters. Pharmacological blockade of the PC-selective neuronal transporter EAAT4 markedly enhanced CF-induced inhibition of GABAergic transmission. Tetanic CF-stimulation elicited long-term potentiation of glutamate transporters in PCs, and thereby attenuated the CF-induced inhibition. Combined use of electrophysiology and immunohistochemistry revealed a significant inverse relationship between the level of EAAT4 expression and the inhibitory action of CF-stimulation on the GABA release at different cerebellar lobules - the CF-induced inhibition was profound in lobule III, where the EAAT4 expression level was low, whereas it was minimal in lobule X, where EAAT4 was abundant. The findings clearly demonstrate that the neuronal glutamate transporter EAAT4 in PCs plays a critical role in the extrasynaptic diffusion of CF transmitter - it appears not only to retrogradely determine the degree of CF-mediated inhibition of GABAergic inputs to the PC by controlling the glutamate concentration for intersynaptic diffusion, but also regulate synaptic information processing in the cerebellar cortex depending on its differential regional distribution as well as use-dependent plasticity of uptake efficacy.

TRPA1-expressing primary afferents synapse with a morphologically identified subclass of substantia gelatinosa neurons in the adult rat spinal cord.

The TRPA1 channel has been proposed to be a molecular transducer of cold and inflammatory nociceptive signals. It is expressed on a subset of small primary afferent neurons both in the peripheral terminals, where it serves as a sensor, and on the central nerve endings in the dorsal horn. The substantia gelatinosa (SG) of the spinal cord is a key site for integration of noxious inputs. The SG neurons are morphologically and functionally heterogeneous and the precise synaptic circuits of the SG are poorly understood. We examined how activation of TRPA1 channels affects synaptic transmission onto SG neurons using whole-cell patch-clamp recordings and morphological analyses in adult rat spinal cord slices. Cinnamaldehyde (TRPA1 agonist) elicited a barrage of excitatory postsynaptic currents (EPSCs) in a subset of the SG neurons that responded to allyl isothiocyanate (less specific TRPA1 agonist) and capsaicin (TRPV1 agonist). Cinnamaldehyde evoked EPSCs in vertical and radial but not islet or central SG cells. Notably, cinnamaldehyde produced no change in inhibitory postsynaptic currents and nor did it produce direct postsynaptic effects. In the presence of tetrodotoxin, cinnamaldehyde increased the frequency but not amplitude of miniature EPSCs. Intriguingly, cinnamaldehyde had a selective inhibitory action on monosynaptic C- (but not Adelta-) fiber-evoked EPSCs. These results indicate that activation of spinal TRPA1 presynaptically facilitates miniature excitatory synaptic transmission from primary afferents onto vertical and radial cells to initiate action potentials. The presence of TRPA1 channels on the central terminals raises the possibility of bidirectional modulatory action in morphologically identified subclasses of SG neurons.

A computational analysis of the effect of sevoflurane in a human ventricular cell model of long QT syndrome: Importance of repolarization reserve in the QT-prolonging effect of sevoflurane.

The slowly and rapidly activating delayed rectifier K+ channels (IKs and IKr, respectively) contribute to the repolarization of ventricular action potential in human heart and thereby determine QT interval on an electrocardiogram. Loss-of-function mutations in genes encoding IKs and IKr cause type 1 and type 2 long QT syndrome (LQT1 and LQT2, respectively), accompanied by a high risk of malignant ventricular arrhythmias and sudden cardiac death. This study was designed to investigate which cardiac electrophysiological conditions exaggerate QT-prolonging and arrhythmogenic effects of sevoflurane. We used the O'Hara-Rudy dynamic model to reconstruct human ventricular action potential and a pseudo-electrocardiogram, and simulated LQT1 and LQT2 phenotypes by decreasing conductances of IKs and IKr, respectively. Sevoflurane, but not propofol, prolonged ventricular action potential duration and QT interval in wild-type, LQT1 and LQT2 models. The QT-prolonging effect of sevoflurane was more profound in LQT2 than in wild-type and LQT1 models. The potent inhibitory effect of sevoflurane on IKs was primarily responsible for its QT-prolonging effect. In LQT2 model, IKs was considerably enhanced during excessive prolongation of ventricular action potential duration by reduction of IKr and relative contribution of IKs to ventricular repolarization was markedly elevated, which appears to underlie more pronounced QT-prolonging effect of sevoflurane in LQT2 model, compared with wild-type and LQT1 models. This simulation study clearly elucidates the electrophysiological basis underlying the difference in QT-prolonging effect of sevoflurane among wild-type, LQT1 and LQT2 models, and may provide important information for developing anesthetic strategies for patients with long QT syndrome in clinical settings.

Contrary roles of kainate receptors in transmitter release at corticothalamic synapses onto thalamic relay and reticular neurons.

Corticothalamic fibres, which originate from layer VI pyramidal neurons in the cerebral cortex, provide excitatory synaptic inputs to both thalamic relay neurons and reticular neurons; reticular neurons in turn supply inhibitory inputs to thalamic relay neurons. Pyramidal cells in layer VI in the mouse somatosensory cortex highly express mRNA encoding kainate receptors, which facilitate or depress transmitter release at several synapses in the central nervous system. We report here that contrary modulation of transmitter release from corticothalamic fibres onto thalamic relay and reticular neurons is mediated by activation of kainate receptors in mouse thalamic ventrobasal complex and thalamic reticular nucleus. Exogenous kainate presynaptically depresses the synaptic transmission at corticothalamic synapses onto thalamic relay neurons, but facilitates it at corticothalamic synapses onto reticular neurons. Meanwhile, the lemniscal synaptic transmission, which sends primary somatosensory inputs to relay neurons, is not affected by kainate. In addition, GluR5-containing kainate receptors are involved in the depression of corticothalamic synaptic transmission onto relay neurons, but not onto reticular neurons. Furthermore, synaptically activated kainate receptors mimic these effects; high-frequency stimulation of corticothalamic fibres depresses synaptic transmission onto relay neurons, but facilitates it onto reticular neurons. Our results suggest that the opposite sensitivity of kainate receptors at the two corticothalamic synapses is governed by cortical activity and regulates the balance of excitatory and inhibitory inputs to thalamic relay neurons and therefore their excitability.

Differential Involvement of Kinase Activity of Ca2+/Calmodulin-Dependent Protein Kinase IIα in Hippocampus- and Amygdala-Dependent Memory Revealed by Kinase-Dead Knock-In Mouse.

Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) is a key mediator of activity-dependent neuronal modifications and has been implicated in the molecular mechanisms of learning and memory. Indeed, several types of CaMKIIα knock-in (KI) and knock-out (KO) mice revealed impairments in hippocampal synaptic plasticity and behavioral learning. On the other hand, a similar role for CaMKIIα has been implicated in amygdala-dependent memory, but detailed analyses have not much been performed yet. To better understand its involvement in amygdala-dependent memory as compared to hippocampus-dependent memory, here we performed biochemical analyses and behavioral memory tests using the kinase-dead CaMKIIα (K42R)-KI mouse. In the Morris water maze tasks, homozygous mutants performed well in the visible platform trials, while they failed to form spatial memory in the hippocampus-dependent hidden platform trials. In fear conditioning, these mice were impaired but showed a certain level of amygdala-dependent cued fear memory, which lasted four weeks, while they showed virtually no hippocampus-dependent context discrimination. Neither stronger stimulation nor repetitive stimulation compensated for their memory deficits. The differential outcome of hippocampus- and amygdala-dependent memory in the mutant mouse was not due to differential expression of CaMKIIα between the hippocampus and the amygdala, because biochemical analyses revealed that both kinase activity and protein levels of CaMKII were indistinguishable between the two brain regions. These results indicate that kinase activity of CaMKIIα is indispensable for hippocampus-dependent memory, but not necessarily for amygdala-dependent memory. There may be a secondary, CaMKIIα activity-independent pathway, in addition to the CaMKIIα activity-dependent pathway, in the acquisition of amygdala-dependent memory.

Impaired feedforward inhibition of the thalamocortical projection in epileptic Ca2+ channel mutant mice, tottering.

The tottering (tg) mice have a mutation in the CaV2.1 (P/Q-type) voltage-dependent Ca2+ channel alpha(1)2.1 subunit gene. tg mice show not only cerebellar ataxia but also absence epilepsy, which begins at approximately 3 weeks of age and persists throughout life. Similarities in EEG and sensitivity to antiepileptic drugs suggest that tg mice are a good model for human absence epilepsy. Although imbalance between excitatory and inhibitory activity in the thalamocortical network is thought to contribute to the pathogenesis of absence epilepsy, the effect of the mutation on thalamocortical synaptic responses remains unknown. Here we showed imbalanced impairment of inhibitory synaptic responses in tg mice using brain slice preparations. Somatosensory thalamocortical projection makes not only monosynaptic glutamatergic connections but also disynaptic GABAergic connections, which mediate feedforward inhibition, onto layer IV neurons. In tg mice, IPSC amplitudes recorded from layer IV pyramidal cells of the somatosensory cortex in response to thalamic stimulation became disproportionately reduced compared with EPSC amplitudes at later developmental stages (postnatal days 21-30). Similar results were obtained by local stimulation of layer IV pyramidal neurons. However, IPSC reduction was not seen in layer V pyramidal neurons of epileptic tg mice or in layer IV pyramidal neurons of younger tg mice before the onset of epilepsy (postnatal days 14-16). These results showed that the feedforward inhibition from the thalamus to layer IV neurons of the somatosensory cortex was severely impaired in tg mice and that the impairment of the inhibitory synaptic transmission was correlated to the onset of absence epilepsy.