Youngnam Kang

Impairment of motor coordination, Purkinje cell synapse formation, and cerebellar long-term depression in GluRδ2 mutant mice

Of the six glutamate receptor (GluR) channel subunit families identified by molecular cloning, five have been shown to constitute either the AMPA, kainate, or NMDA receptor channel, whereas the function of the delta subunit family remains unknown. The selective localization of the delta 2 subunit of the GluR delta subfamily in cerebellar Purkinje cells prompted us to examine its possible physiological roles by the gene targeting technique. Analyses of the GluR delta 2 mutant mice reveal that the delta 2 subunit plays important roles in motor coordination, formation of parallel fiber-Purkinje cell synapses and climbing fiber-Purkinje cell synapses, and long-term depression of parallel fiber-Purkinje cell synaptic transmission. These results suggest a close relationship between synaptic plasticity and synapse formation in the cerebellum.

Immunohistochemical localization of voltage‐gated calcium channels in substantia nigra dopamine neurons

The rhythmic firing of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) is thought to be mediated by nifedipine‐sensitive Ca2+ channels, although an involvement of ω‐conotoxin‐sensitive Ca2+ channels is also suggested. In an attempt to localize such Ca2+ channels at both the regional and cellular levels, their expression and distribution patterns were immunohistochemically investigated in the rat SNc. The three distinct subtypes of voltage‐gated Ca2+ channels were tested: the class B N‐type α1 subunit (CNB1), the class C L‐type α1 subunit (CNC1) and the class D L‐type α1 subunit (CND1). A large number of SNc neurons showed intense immunoreactivity against CND1 and they were distributed throughout the entire extent. By contrast, many fewer neurons displayed less intense CNC1 immunoreactivity and many of them were located in the lateral aspect of the SNc. No immunoreactivity against CNB1 was detected in the SNc. Moreover, double immunofluorescence analysis in combination with tyrosine hydroxylase staining revealed that virtually all DA neurons were CND1‐immunoreactive whereas many DA neurons especially in the medial SNc exhibited only faint or no immunoreactivity against CNC1. Both CNC1 and CND1 were expressed in cell bodies and proximal dendrites of SNc DA neurons, whilst their distal dendrites that penetrated into the substantia nigra pars reticulata expressed CND1 alone. Thus, the ubiquitously and intensely expressed class D α1 subunit of L‐type Ca2+ channels that is sensitive to both nifedipine and ω‐conotoxin may be responsible for the pacemaker activity of SNc DA neurons.

Selectively targeting pain in the trigeminal system

&NA; We tested whether it is possible to selectively block pain signals in the orofacial area by delivering the permanently charged lidocaine derivative QX‐314 into nociceptors via TPRV1 channels. We examined the effects of co‐applied QX‐314 and capsaicin on nociceptive, proprioceptive, and motor function in the rat trigeminal system. QX‐314 alone failed to block voltage‐gated sodium channel currents (INa) and action potentials (APs) in trigeminal ganglion (TG) neurons. However, co‐application of QX‐314 and capsaicin blocked INa and APs in TRPV1‐positive TG and dental nociceptive neurons, but not in TRPV1‐negative TG neurons or in small neurons from TRPV1 knock‐out mice. Immunohistochemistry revealed that TRPV1 is not expressed by trigeminal motor and trigeminal mesencephalic neurons. Capsaicin had no effect on rat trigeminal motor and proprioceptive mesencephalic neurons and therefore should not allow QX‐314 to enter these cells. Co‐application of QX‐314 and capsaicin inhibited the jaw‐opening reflex evoked by noxious electrical stimulation of the tooth pulp when applied to a sensory but not a motor nerve, and produced long‐lasting analgesia in the orofacial area. These data show that selective block of pain signals can be achieved by co‐application of QX‐314 with TRPV1 agonists. This approach has potential utility in the trigeminal system for treating dental and facial pain.

Developmental profile of GABAA-mediated synaptic transmission in pyramidal cells of the somatosensory cortex

Inhibitory synaptic transmission mediated by γ‐aminobutyric acid (GABA)A receptors is involved in regulation of experience‐dependent cortical plasticity. However, little information is available on their presynaptic and postsynaptic developmental profiles. The present study aims to investigate the developmental changes of miniature and unitary inhibitory postsynaptic currents (mIPSCs and uIPSCs) in mouse barrel cortex. mIPSCs recorded from supragranular pyramidal neurons showed a gradual increase in frequency during postnatal days 6–15 (PD6–15) followed by a marked increase at PD16–20, and mIPSCs frequency reached a plateau at about PD21–30. The amplitude of mIPSCs showed a transient decrease at PD10–12 followed by an increase during PD13–30, and reached a plateau at about PD30. Their decay time constant progressively decreased during the first 30 days postnatally, and reached a steady level at about PD30. Paired recordings from interneurons and synaptically coupled target pyramidal cells revealed that uIPSC amplitude increased with age up to PD30. In contrast, failure rate and coefficient of variation decreased during PD7–15 and showed little change at a later stage. Short‐term depression induced by presynaptic stimulation at 33 Hz progressively decreased during PD6–20, and was stabilized at about PD21–30. Quantal analysis revealed that the number of release sites increased with age up to PD30, while the release probability increased during PD6–12 and then reached a plateau level. These results suggest that the number of release sites and release probability of GABA and GABAA‐mediated IPSC kinetics show distinct developmental profiles, which could play roles in regulating the onset and offset of critical periods for experience‐dependent cortical plasticity.

Differential Columnar Processing in Local Circuits of Barrel and Insular Cortices

The columnar organization is most apparent in the whisker barrel cortex but seems less apparent in the gustatory insular cortex. We addressed here whether there are any differences between the two cortices in columnar information processing by comparing the spatiotemporal patterns of excitation spread in the two cortices using voltage-sensitive dye imaging. In contrast to the well known excitation spread in the horizontal direction in layer II/III induced in the barrel cortex by layer IV stimulation, the excitation caused in the insular cortex by stimulation of layer IV spread bidirectionally in the vertical direction into layers II/III and V/VI, displaying a columnar image pattern. Bicuculline or picrotoxin markedly extended the horizontal excitation spread in layer II/III in the barrel cortex, leading to a generation of excitation in the underlying layer V/VI, whereas those markedly increased the amplitude of optical responses throughout the whole column in the insular cortex, subsequently widening the columnar image pattern. Such synchronous activities as revealed by the horizontal and vertical excitation spreads were consistently induced in the barrel and insular cortices, respectively, even by stimulation of different layers with varying intensities. Thus, a unique functional column existed in the insular cortex, in which intracolumnar communication between the superficial and deep layers was prominent, and GABAA action is involved in the inhibition of the intracolumnar communication in contrast to its involvement in intercolumnar lateral inhibition in the barrel cortex. These results suggest that the columnar information processing may not be universal across the different cortical areas.

A PHENYTOIN-SENSITIVE CATIONIC CURRENT PARTICIPATES IN GENERATING THE AFTERDEPOLARIZATION AND BURST AFTERDISCHARGE IN RAT NEOCORTICAL PYRAMIDAL CELLS

We report here on the ionic mechanisms underlying the depolarizing afterpotential (DAP) in neocortical pyramidal cells, with special interest in those underlying the burst afterdischarge. Injections of short depolarizing current pulses under whole‐cell current clamp with a CsCl‐based internal medium generated, in most pyramidal cells, a single action potential with a plateau phase (plateau‐AP), followed by a slowly decaying DAP both in the absence and presence of TTX. Under voltage‐clamp, the same cells displayed a slow tail current (tail‐I) at the offset of depolarization. When intracellular free Ca2+ was chelated with 10 mm BAPTA or when extracellular Ca2+ was replaced with equimolar Ba2+, neither the slow DAP nor the slow tail‐I was observed. Extracellular application of Co2+ or Cd2+ reduced Ca2+ currents and the slow tail‐I. Cation substitution experiments revealed that the channel generating the slow tail‐I was permeable to K+ and Cs+ more than to Na+ (PK≈PCs > PNa > PNMDG≈PTEA). The cationic slow tail‐I was not reduced by applying antagonists of the metabotropic glutamate receptor (MCPG, 1 mm) and the muscarinic receptor (atropine, 1–10 μm). Thus, the slow DAP was produced by activation of the cationic channel whose gating is solely dependent on [Ca2+]i. An increase in [K+]o from 3 to 6 or 9 mm enhanced the slow DAP, and resulted in a generation of burst afterdischarges. An anticonvulsant, phenytoin (PT; 1–10 μm) suppressed the slow DAP while enhancing the plateau‐AP in the presence of TTX, most likely by blocking the cationic channel.

Bidirectional Interactions between H-Channels and Na+–K+ Pumps in Mesencephalic Trigeminal Neurons

The Na+–K+ pump current (Ip) and the h-current (Ih) flowing through hyperpolarization-activated channels (h-channels) participate in generating the resting potential. These two currents are thought to be produced independently. We show here bidirectional interactions between Na+–K+ pumps and h-channels in mesencephalic trigeminal neurons. Activation of Ih leads to the generation of two types of ouabain-sensitive Ip with temporal profiles similar to those of instantaneous and slow components of Ih, presumably reflecting Na+ transients in a restricted cellular space. Moreover, the Ip activated by instantaneous Ih can facilitate the subsequent activation of slow Ih. Such counteractive and cooperative interactions were also disclosed by replacing extracellular Na+ with Li+, which is permeant through h-channels but does not stimulate the Na+–K+ pump as strongly as Na+ ions. These observations indicate that the interactions are bidirectional and mediated by Na+ ions. Also after substitution of extracellular Na+ with Li+, the tail Ih was reduced markedly despite an enhancement of Ih itself, attributable to a negative shift of the reversal potential for Ih presumably caused by intracellular accumulation of Li+ ions. This suggests the presence of a microdomain where the interactions can take place. Thus, the bidirectional interactions between Na+–K+ pumps and h-channels are likely to be mediated by Na+ microdomain. Consistent with these findings, hyperpolarization-activated and cyclic nucleotide-modulated subunits (HCN1/2) and the Na+–K+ pumpα3 isoform were colocalized in plasma membrane of mesencephalic trigeminal neurons having numerous spines.

Differential connections by intracortical axon collaterals among pyramidal tract cells in the cat motor cortex.

1. Recurrent EPSPs were produced in fast pyramidal tract (PT) cells in the cat motor cortex by stimulation of the medullary pyramid and/or by the glutamate‐induced activity of neighbouring PT cells using the spike‐triggered averaging (spike‐TA) method. 2. In fast PT cells located lateral to the end of the cruciate sulcus, predominantly the motor cortical representation area of the distal forelimb, two components (fast and slow) of recurrent EPSPs were produced by pyramid stimulation. 3. In response to pyramid stimulation, the appearance of the fast and slow components of recurrent EPSPs correlated with the appearance of N1 and N2 field potentials, respectively. 4. The monosynaptic nature of both the fast and slow components of recurrent EPSPs was demonstrated by a double shock test (interstimulus interval less than 5 ms) and high frequency repetitive stimulation (50‐100 Hz). 5. The generation of the fast and slow components of recurrent EPSPs was attributed to the synaptic action of recurrent collaterals of fast and slow PT cells, respectively. 6. The amplitude of the slow component of recurrent EPSPs markedly increased with an increase in the stimulus frequency whereas that of the fast component did not, despite the change in stimulus frequency. 7. Selected spike‐triggered averaging also revealed frequency facilitation of recurrent individual EPSPs produced in fast PT cells by the activity of single slow PT cells. 8. In fast PT cells located in the anterior and posterior lips of the cruciate sulcus, the motor cortical representation area of the proximal limb or trunk, only the slow component of recurrent EPSPs was produced by pyramid stimulation. 9. It is concluded that the pattern of recurrent connections between neighbouring PT cells differs depending on the motor cortical representation area, and that frequency facilitation of recurrent EPSPs is caused mainly by the input from axon collaterals of slow PT cells.

The role of Ca2+-dependent cationic current in generating gamma frequency rhythmic bursts: modeling study.

Fast rhythmic bursting pyramidal neuron or chattering neuron is a promising candidate for the pacemaker of coherent gamma-band (25-70 Hz) cortical oscillation. It, however, still remains to be clarified how the neuron generates such high-frequency bursts. Here, we demonstrate in a single-compartment model neuron that the fast rhythmic bursts (FRBs) can be achieved through Ca2+-activated channels in the entire gamma frequency range. In a previous in vitro study, a subset of rat cortical pyramidal cells displayed a long-lasting depolarizing afterpotential (DAP) following a plateau-type action potential when K+ conductances were suppressed with Cs+, and this DAP was found to be mediated by a Ca2+-dependent cationic current. This current appeared also suitable for producing a hump-like DAP, a characteristic of the chattering neurons, because of its reversal potential being approximately -40 mV. In the present theoretical study, we show that the enhancement of such a DAP leads to generation of doublet/triplet spikes seen during FRBs. The firing pattern during FRBs is primarily determined by a Ca2+-dependent cationic current and a small-conductance Ca2+-dependent potassium current, which are differentially activated by a biphasically decaying Ca2+ transient produced by fast buffering and a slow pump extrusion after each spike. With varying intensities of injected current pulses, the interburst frequencies of the FRBs range over the entire gamma frequency band (25-70 Hz) in our model, while the intraburst frequencies remain higher than 300 Hz. Our model suggests that FRBs are essentially generated in the soma, unlike the model based on a persistent sodium current, and that the alteration of Ca2+ sensitivity of Ca2+-dependent cationic current plays an essential role in controlling the FRB pattern.

Protein Kinase G Dynamically Modulates TASK1-Mediated Leak K+ Currents in Cholinergic Neurons of the Basal Forebrain

Leak K+ conductance generated by TASK1/3 channels is crucial for neuronal excitability. However, endogenous modulators activating TASK channels in neurons remained unknown. We previously reported that in the presumed cholinergic neurons of the basal forebrain (BF), activation of NO-cGMP-PKG (protein kinase G) pathway enhanced the TASK1-like leak K+ current (I-Kleak). As 8-Br-cGMP enhanced the I-Kleak mainly at pH 7.3 as if changing the I-Kleak from TASK1-like to TASK3-like current, such an enhancement of the I-Kleak would result either from an enhancement of hidden TASK3 component or from an acidic shift in the pH sensitivity profile of TASK1 component. In view of the report that protonation of TASK channel decreases its open probability, the present study was designed to examine whether the activation of PKG increases the conductance of TASK1 channels by reducing their binding affinity for H+, i.e., by increasing Kd for protonation, or not. We here demonstrate that PKG activation and inhibition respectively upregulate and downregulate TASK1 channels heterologously expressed in PKG-loaded HEK293 cells at physiological pH, by causing shifts in the Kd in the acidic and basic directions, respectively. Such PKG modulations of TASK1 channels were largely abolished by mutating pH sensor H98. In the BF neurons that were identified to express ChAT and TASK1 channels, similar dynamic modulations of TASK1-like pH sensitivity of I-Kleak were caused by PKG. It is strongly suggested that PKG activation and inhibition dynamically modulate TASK1 currents at physiological pH by bidirectionally changing Kd values for protonation of the extracellular pH sensors of TASK1 channels in cholinergic BF neurons.