Development and Function of the Voltage-Gated Sodium Current in Immature Mammalian Cochlear Inner Hair Cells

Inner hair cells (IHCs), the primary sensory receptors of the mammalian cochlea, fire spontaneous Ca²⁺ action potentials before the onset of hearing. Although this firing activity is mainly sustained by a depolarizing L-type (Ca_V1.3) Ca²⁺ current (I _Ca), IHCs also transiently express a large Na⁺ current (I _Na). We aimed to investigate the specific contribution of I _Na to the action potentials, the nature of the channels carrying the current and whether the biophysical properties of I _Na differ between low- and high-frequency IHCs. We show that I _Na is highly temperature-dependent and activates at around −60 mV, close to the action potential threshold. Its size was larger in apical than in basal IHCs and between 5% and 20% should be available at around the resting membrane potential (−55 mV/−60 mV). However, in vivo the availability of I _Na could potentially increase to >60% during inhibitory postsynaptic potential activity, which transiently hyperpolarize IHCs down to as far as −70 mV. When IHCs were held at −60 mV and I _Na elicited using a simulated action potential as a voltage command, we found that I _Na contributed to the subthreshold depolarization and upstroke of an action potential. We also found that I _Na is likely to be carried by the TTX-sensitive channel subunits Na_V1.1 and Na_V1.6 in both apical and basal IHCs. The results provide insight into how the biophysical properties of I _Na in mammalian cochlear IHCs could contribute to the spontaneous physiological activity during cochlear maturation in vivo.     

脂多糖对大鼠多巴胺能神经元毒性作用的研究

目的　建立新的帕金森病 (Parkinson’sdisease ,PD)动物模型 ,探讨其发病机制。方法　在大鼠脑黑质(substantianigra ,SN)内注射脂多糖 (Lipopolysaccharide ,LPS)后 ,按大鼠不同存活期用高效液相色谱 (HPLC)来测定脑内多巴胺 (Dopamine,DA)及其代谢产物的含量 ;用免疫组化法观察酪氨酸羟化酶 (Tyrosinehydroxylase ,TH)阳性神经细胞、小胶质细胞的形态及数量变化。结果　DA及其代谢产物的含量在LPS注射侧随时间不同有不同程度下降 ,于第 14天达到最低 (P <0 0 1) ;注射侧黑质TH阳性神经元可以达到全部消失 ,该处可见大量被激活并有形态改变的小胶质细胞。结论　LPS可导致大鼠黑质多巴胺能神经元的损害     

Pyrethroids Differentially Alter Voltage-Gated Sodium Channels from the Honeybee Central Olfactory Neurons

The sensitivity of neurons from the honey bee olfactory system to pyrethroid insecticides was studied using the patch-clamp technique on central ‘antennal lobe neurons’ (ALNs) in cell culture. In these neurons, the voltage-dependent sodium currents are characterized by negative potential for activation, fast kinetics of activation and inactivation, and the presence of cumulative inactivation during train of depolarizations. Perfusion of pyrethroids on these ALN neurons submitted to repetitive stimulations induced (1) an acceleration of cumulative inactivation, and (2) a marked slowing of the tail current recorded upon repolarization. Cypermethrin and permethrin accelerated cumulative inactivation of the sodium current peak in a similar manner and tetramethrin was even more effective. The slow-down of channel deactivation was markedly dependent on the type of pyrethroid. With cypermethrin, a progressive increase of the tail current amplitude along with successive stimulations reveals a traditionally described use-dependent recruitment of modified sodium channels. However, an unexpected decrease in this tail current was revealed with tetramethrin. If one considers the calculated percentage of modified channels as an index of pyrethroids effects, ALNs are significantly more susceptible to tetramethrin than to permethrin or cypermethrin for a single depolarization, but this difference attenuates with repetitive activity. Further comparison with peripheral neurons from antennae suggest that these modifications are neuron type specific. Modeling the sodium channel as a multi-state channel with fast and slow inactivation allows to underline the effects of pyrethroids on a set of rate constants connecting open and inactivated conformations, and give some insights to their specificity. Altogether, our results revealed a differential sensitivity of central olfactory neurons to pyrethroids that emphasize the ability for these compounds to impair detection and processing of information at several levels of the bees olfactory pathway.     

c-Fos Expression by Dopaminergic Receptor Activation in Rat Hippocampal Neurons

While dopamine is likely to modulate hippocampal synaptic plasticity, there has been little information about how dopamine affects synaptic transmission in the hippocampus. The expression of IEGs including c-fos has been associated with late phase LTP in the CA1 region of the hippocampus. The induction of c-fos by dopaminergic receptor activation in the rat hippocampus was investigated by using semiquantitative RT-PCR and immuno-cytochemistry. The hippocampal slices which were not treated with dopamine showed little expression of c-fos mRNA. However, the induction of c-fos mRNA was detected as early as 5 min after dopamine treatment, peaked at 60 min, and remained elevated 5 h after treatment. Temporal profiles of increases in c-fos mRNA by R(+)-SKF-38393 (50 M) and forskolin (50 M) were similar to that of dopamine. An increase in [cAMP] was observed in dopamine-, SKF-, or forskolin-treated hippocampal slices. By immunocytochemical studies, control hippocampal cells showed little expression of c-Fos immunoreactivity. However, when cells were treated with dopamine, an increase in the expression of c-Fos immunoreactivity was observed after treatment for 2 h. The treatment of hippocampal neurons with R(+)-SKF38393 (50 M) or forskolin (50 M) also induced a significant increase in c-Fos expression. These results indicate that the dopamine D1 receptor-mediated cAMP dependant pathway is associated with the expression of c-Fos in the hippocampal neurons. These data are consistent with the possible role of endogenous dopamine on synaptic plasticity via the regulation of gene expression. Furthermore, these results imply that dopamine might control the process of memory storage in the hippocampus through gene expression.     

Mapping the Effects of SI Cortex Stimulation on Somatosensory Relay Neurons in the Rat Thalamus: Direct Responses and Afferent Modulation

We have used single-unit recording techniques to map the spatial distribution of the primary somatosensory (SI) cortical influences on thalamic somatosensory relay nuclei in the rat. A total of 193 microelectrode penetrations were made to record single neurons in tracks through the medial and lateral ventroposterior (VPL and VPM), ventrolateral (VL), posterior (Po), and reticular (nRt) thalamic nuclei. Single units were classified according to their (1) location within the nuclei, (2) receptive fields, and (3) response to standardized microstimulation in deep layers of the SI cortical forepaw areas. The SI stimulation produced short-latency (1- to 7-msec) excitatory responses in different percentages of neurons recorded in the following thalamic nuclei: VPL, 42.0%; Po, 25.0%; nRt, 16.4%; VL, 13.6%; and VPM, 9.9%. Within the VPL, the highest proportion of responsive neurons was found in the anterior region. Although most of the VL region was unresponsive, the caudal subregion bordering the rostral VPL showed some responsiveness (13.6% of neurons). In general, the spatial pattern of corticothalamic influences appeared to reciprocate the known thalamocortical connection patterns, but with a heterogeneity that was unpredicted.

The same parameters of SI cortical stimulation were used in studies of corticofugal modulation of afferent transmission through the VPL thalamus. A condition—test (C-T) paradigm was implemented in which the cortical stimulation (C) was delivered at a range of time intervals before test (T) mechanical vibratory stimulation was applied to digit 4 of the contralateral forepaw. The time course of cortical effects was analyzed by measuring the averaged evoked unit responses of thalamic neurons to the T stimuli, and plotting them as a function of C-T intervals from 5 to 50 msec. Of the 20 VPL neurons tested during SI stimulation, the average response to T stimulation was decreased a mean of 36%, with the suppression peaking (at 49% inhibition of the afferent response) about 15 msec after the C stimulus. Considerable rostrocaudal variation was observed, however. Whereas neurons in the rostral VPL (near VL) were strongly inhibited (-69%), neurons in the middle and caudal VPL exhibited facilitations at long and short C-T intervals, respectively. This study establishes a specific projection system from the forepaw region of SI cortex to different subregions of the VPL thalamus, producing specific temporal patterns of sensory modulation.     

Postnatal Development of the Light Response of the Dopaminergic Neurons in the Rat Retina

The effect of light on retinal dopamine (DA) synthesis and content in dark-adapted rats was assessed 15 h and 2, 4, 7 and 16 days after eye opening (13 to 14 days after birth). The accumulation of dihydroxyphenylalanine (DOPA) following inhibition of its decarboxylation was used to estimate DA synthesis. At 7 and 16 days, but not earlier, light significantly augmented DOPA formation. These increases were as dramatic as those reported for adult rats. DA in dark-adapted retinas ranged from 0 (undetectable) at 15 h to 83% of adult levels at 16 days, but were only 36% of that of adult retinas at 7 days. Light produced a significant decline in DA at 16 days but not at any other time point. These results indicate that the dopaminergic neurons synthesize transmitter and respond to light at a time when the neuronal pools of DA are not yet mature.     

Effects of Hypoxia on the Activities of Noradrenergic and Dopaminergic Neurons in the Rat Brain

The effects of hypoxia (10% O2, 90% N2) on the content, biosynthesis, and turnover of noradrenaline (NA) and 3,4-dihydroxyphenylethylamine (dopamine, DA) in the rat brain were examined. Up to 24 h following exposure to hypoxia, NA content in the whole brain was decreased, whereas DA content remained unchanged. The accumulation of 3,4-dihydroxyphenylalanine (DOPA) after central decarboxylase inhibition was decreased. The turnover rate of DA after synthesis inhibition was markedly decreased up to 8 h and returned to the control level within 24 h. In contrast, the turnover rate of NA was all but unchanged, except for a 4-h exposure. The 2-h exposure to the hypoxic environment resulted in a significant decrease in NA content and DOPA accumulation in all brain regions tested, but no significant change was observed in DA content. The turnover rate of DA was remarkably decreased in all brain regions tested, whereas the rate of NA was slightly decreased only in the cerebral cortex and hippocampus. These results suggest that although hypoxia decreases the biosynthesis of both NA and DA, the effects of oxygen depletion on the functional activities of NA neurons differ considerably from those of DA neurons: Only in the cerebral cortex and hippocampus are the NA neurons slightly sensitive to hypoxia, whereas the DA neurons are most sensitive in all brain regions.     

The Distribution of Afferent Neurons in the Trigeminal Mesencephalic Nucleus and the Central Projection of Afferent Fibers of the Mylohyoid Nerve in the Rat

Horseradish peroxidase conjugated to wheatgerm agglutinin (HRP:WGA) was injected into the proximal cut ends of three branches of the mylohyoid nerve in rats: the branch to the mylohyoid muscle (BrMh), the branch to the anterior belly of the digastricus muscle (BrDg), and the cutaneous branch (BrCu). HRP-labeled cells were detected in the ipsilateral caudal portion of the trigeminal mesencephalic nucleus (Vmes) and the ipsilateral ventromedial division of the trigeminal motor nucleus, except when HRP:WGA was applied to the BrCu. Morphologically, all labeled Vmes cells were of the pseudounipolar type.

Projections of the primary afferents of the BrMh were observed in the ipsilateral trigeminal nucleus caudalis, the upper cervical dorsal horns of laminae I -III, and the dorsolateral recticular formation (Rf), whereas the primary afferents of the BrDg terminated in the ipsilateral trigeminal nucleus principalis and Rf. These observations suggest that the role of the afferent inputs of the mylohyoid muscle differs from that of those of the anterior belly of the digastricus muscle in terms of several functions associated with jaw-closing and infrahyoid muscles.     

Post-Treatment with Voltage-Gated Na+ Channel Blocker Attenuates Kainic Acid-Induced Apoptosis in Rat Primary Hippocampal Neurons

Injection of rats with kainic acid (KA), a non-N-methyl-D-aspartate (NMDA) type glutamate receptor agonist, induces recurrent (delayed) convulsive seizures and subsequently hippocampal neurodegeneration, which is reminiscent of human epilepsy. The protective effect of anti-epileptic drugs on seizure-induced neuronal injury is well known; however, molecular basis of this protective effect has not yet been elucidated. In this study, we investigated the effect and signaling mediators of voltage-gated Na(+) channel blockers (Lamotrigine, Rufinamide, Oxcarbazepine, Valproic Acid, and Zonisamide) on KA-induced apoptosis in rat primary hippocampal neurons. Exposure of hippocampal neurons to 10 μM KA for 24 h caused significant increases in morphological and biochemical features of apoptosis, as determined by Wright staining and ApopTag assay, respectively. Analyses showed increases in expression and activity of cysteine proteases, production of reactive oxygen species (ROS), intracellular free [Ca(2+)], and Bax:Bcl-2 ratio during apoptosis. Cells exposed to KA for 15 min were then treated with Lamotrigine, Rufinamide, Oxcarbazepine, Valproic Acid, or Zonisamide. Post-treatment with one of these anti-epileptic drugs (500 nM) attenuated production of ROS and prevented apoptosis in hippocampal neurons. Lamotrigine, Rufinamide, and Oxcarbazepine appeared to be less protective when compared with Valproic Acid or Zonisamide. This difference may be due to blockade of T-type Ca(2+) channels also by Valproic Acid and Zonisamide. Our findings thus suggest that the anti-epileptic drugs that block both Na(+) channels and Ca(2+) channels are significantly more effective than agents that block only Na(+) channels for attenuating seizure-induced hippocampal neurodegeneration.     

Nitric Oxide Participates in the Stimulatory and Neurotoxic Action of Endothelin on Rat Striatal Dopaminergic Neurons

1. Our method of real-time monitoring of dopamine release from rat striatal slices revealed that endothelin (ET)-3-induced dopamine release was inhibited by N ^G-methyl-L-arginine (L-NMMA; 1 mM), an inhibitor of nitric oxide (NO) synthase, while N ^G-methyl-D-arginine (D-NMMA; 1 mM), an inactive isomer of L-NMMA, had no effect.2. The inhibition of L-NMMA (0.1 mM) became apparent when tissues were pretreated with tetrodotoxin (1 M) for 30 min and subsequently exposed to ET-3 (4 M).3. L-NMMA (0.1 and 1 mM) dose dependently protected against ET-3-triggered hypoxic/hypoglycemic impairment of striatal responses to high K⁺.4. Thus, NO may work as a promoter in mediation of the stimulatory and neurotoxic action of ET-3 on the striatal dopaminergic system, presumably by interacting with interneurons in the striatum.     

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.     

Sodium Metabisulfite Modulation of Potassium Channels in Pain-Sensing Dorsal Root Ganglion Neurons

The effects of sodium metabisulfite (SMB), a general food preservative, on potassium currents in rat dorsal root ganglion (DRG) neurons were investigated using the whole-cell patch-clamp technique. SMB increased the amplitudes of both transient outward potassium currents and delayed rectifier potassium current in concentration- and voltage-dependent manner. The transient outward potassium currents (TOCs) include a fast inactivating (A-current or I _A) current and a slow inactivating (D-current or I _D) current. SMB majorly increased I_A, and I_D was little affected. SMB did not affect the activation process of transient outward currents (TOCs), but the inactivation curve of TOCs was shifted to more positive potentials. The inactivation time constants of TOCs were also increased by SMB. For delayed rectifier potassium current (I _K), SMB shifted the activation curve to hyperpolarizing direction. SMB differently affected TOCs and I _K, its effects major on A-type K⁺ channels, which play a role in adjusting pain sensitivity in response to peripheral redox conditions. SMB did not increase TOCs and I _K when adding DTT in pipette solution. These results suggested that SMB might oxidize potassium channels, which relate to adjusting pain sensitivity in pain-sensing DRG neurons.     

Modulation of Voltage-Gated Ca2+ Channels by G Protein-Coupled Receptors in Celiac-Mesenteric Ganglion Neurons of Septic Rats

Septic shock, the most severe complication associated with sepsis, is manifested by tissue hypoperfusion due, in part, to cardiovascular and autonomic dysfunction. In many cases, the splanchnic circulation becomes vasoplegic. The celiac-superior mesenteric ganglion (CSMG) sympathetic neurons provide the main autonomic input to these vessels. We used the cecal ligation puncture (CLP) model, which closely mimics the hemodynamic and metabolic disturbances observed in septic patients, to examine the properties and modulation of Ca²⁺ channels by G protein-coupled receptors in acutely dissociated rat CSMG neurons. Voltage-clamp studies 48 hr post-sepsis revealed that the Ca²⁺ current density in CMSG neurons from septic rats was significantly lower than those isolated from sham control rats. This reduction coincided with a significant increase in membrane surface area and a negligible increase in Ca²⁺ current amplitude. Possible explanations for these findings include either cell swelling or neurite outgrowth enhancement of CSMG neurons from septic rats. Additionally, a significant rightward shift of the concentration-response relationship for the norepinephrine (NE)-mediated Ca²⁺ current inhibition was observed in CSMG neurons from septic rats. Testing for the presence of opioid receptor subtypes in CSMG neurons, showed that mu opioid receptors were present in ~70% of CSMG, while NOP opioid receptors were found in all CSMG neurons tested. The pharmacological profile for both opioid receptor subtypes was not significantly affected by sepsis. Further, the Ca²⁺ current modulation by propionate, an agonist for the free fatty acid receptors GPR41 and GPR43, was not altered by sepsis. Overall, our findings suggest that CSMG function is affected by sepsis via changes in cell size and α2-adrenergic receptor-mediated Ca²⁺ channel modulation.     

Structural Pharmacology of Voltage-Gated Sodium Channels

Voltage-gated sodium (Na_V) channels initiate and propagate action potentials in excitable tissues to mediate key physiological processes including heart contraction and nervous system function. Accordingly, Na_V channels are major targets for drugs, toxins and disease-causing mutations. Recent breakthroughs in cryo-electron microscopy have led to the visualization of human Na_V1.1, Na_V1.2, Na_V1.4, Na_V1.5 and Na_V1.7 channel subtypes at high-resolution. These landmark studies have greatly advanced our structural understanding of channel architecture, ion selectivity, voltage-sensing, electromechanical coupling, fast inactivation, and the molecular basis underlying Na_V channelopathies. Na_V channel structures have also been increasingly determined in complex with toxin and small molecule modulators that target either the pore module or voltage sensor domains. These structural studies have provided new insights into the mechanisms of pharmacological action and opportunities for subtype-selective Na_V channel drug design. This review will highlight the structural pharmacology of human Na_V channels as well as the potential use of engineered and chimeric channels in future drug discovery efforts.     

Modulation of the Acetylcholine-Induced Input Current by Noopept in Helix Lucorum Neurons

Biophysics - Abstract—Possible causes of the positive modulating effect of noopept (in a concentration range of 0.1 to 10&nbsp;nM) on the amplitude of the acetylcholine-induced input...     

Toxic Effects of Iron for Cultured Mesencephalic Dopaminergic Neurons Derived from Rat Embryonic Brains

Iron, a transition metal possibly involved in the pathogenesis of Parkinson's disease, was tested for its toxic effects toward cultures of dissociated rat mesencephalic cells. When cultures were switched for 24 h to serum-free conditions, the effective concentrations of ferrous iron (Fe2+) producing a loss of 50% of dopaminergic neurons, as quantified by tyrosine hydroxylase (TH) immunocytochemistry, TH mRNA in situ hybridization, and measurement of TH activity, were on the order of 200 microM. High-affinity dopamine (DA) uptake, which reflects integrity and function of dopaminergic nerve terminals, was impaired at significantly lower concentrations (EC50 = 67 microM). Toxic effects were not restricted to dopaminergic neurons inasmuch as trypan blue dye exclusion index and gamma-aminobutyric acid uptake, two parameters used to assess survival of other types of cells present in these cultures, were also affected. Protection against iron cytotoxicity was afforded by desferrioxamine and apotransferrin, two ferric iron-chelating agents. Normal supplementation of the culture medium by serum proteins during treatment was also effective, presumably via nonspecific sequestration. Potential interactions with DA were also investigated. Fe2+ at subtoxic concentrations and desferrioxamine in the absence of exogenous iron added to the cultures failed to potentiate or reduce DA cytotoxicity for mesencephalic cells, respectively. Transferrin, the glycoprotein responsible for intracellular delivery of iron, was ineffective in initiating selective cytotoxic effects toward dopaminergic neurons preloaded with DA. Altogether, these results suggest (a) that ferrous iron is a potent neurotoxin for dopaminergic neurons as well as for other cell types in dissociated mesencephalic cultures, acting likely via autoxidation into its ferric form, and (b) that the presence of intra- and extracellular DA is not required for the observed toxic effects.