Xuesi M. Shao

Electrostatic interactions of S4 voltage sensor in shaker K+ channel

The S4 segment comprises part of the voltage sensor in Shaker K+ channels. We have used a strategy similar to intragenic suppression, but without a genetic selection, to identify electrostatic interactions of the S4 segment that may be important in the mechanism of voltage-dependent activation. The S4 neutralization mutations K374Q and R377Q block maturation of the protein, suggesting that they prevent proper folding. K374Q is specifically and efficiently rescued by the second site mutations E293Q and D316N, located in putative transmembrane segments S2 and S3, respectively. These results suggest that K374, E293, and D316 form a network of strong, local, electrostatic interactions that stabilize the structure of the channel. Some other double mutant combinations result in inefficient suppression, identifying weak, presumably long-range electrostatic interactions. A simple structural hypothesis is proposed to account for the effects of the rescued double mutant combinations on the relative stabilities of open and closed channel conformations.

Silencing preBötzinger Complex somatostatin-expressing neurons induces persistent apnea in awake rat

Delineating neurons that underlie complex behaviors is of fundamental interest. Using adeno-associated virus 2, we expressed the Drosophila allatostatin receptor in somatostatin (Sst)-expressing neurons in the preBötzinger Complex (preBötC). Rapid silencing of these neurons in awake rats induced a persistent apnea without any respiratory movements to rescue their breathing. We hypothesize that breathing requires preBötC Sst neurons and that their sudden depression can lead to serious, even fatal, respiratory failure.

Dihydromyricetin As A Novel Anti-Alcohol Intoxication Medication

Alcohol use disorders (AUDs) constitute the most common form of substance abuse. The development of AUDs involves repeated alcohol use leading to tolerance, alcohol withdrawal syndrome, and physical and psychological dependence, with loss of ability to control excessive drinking. Currently there is no effective therapeutic agent for AUDs without major side effects. Dihydromyricetin (DHM; 1 mg/kg, i.p. injection), a flavonoid component of herbal medicines, counteracted acute alcohol (EtOH) intoxication, and also withdrawal signs in rats including tolerance, increased anxiety, and seizure susceptibility; DHM greatly reduced EtOH consumption in an intermittent voluntary EtOH intake paradigm in rats. GABAA receptors (GABAARs) are major targets of acute and chronic EtOH actions on the brain. At the cellular levels, DHM (1 μm) antagonized both acute EtOH-induced potentiation of GABAARs and EtOH exposure/withdrawal-induced GABAAR plasticity, including alterations in responsiveness of extrasynaptic and postsynaptic GABAARs to acute EtOH and, most importantly, increases in GABAAR α4 subunit expression in hippocampus and cultured neurons. DHM anti-alcohol effects on both behavior and CNS neurons were antagonized by flumazenil (10 mg/kg in vivo; 10 μm in vitro), the benzodiazepine (BZ) antagonist. DHM competitively inhibited BZ-site [3H]flunitrazepam binding (IC50, 4.36 μm), suggesting DHM interaction with EtOH involves the BZ sites on GABAARs. In summary, we determined DHM anti-alcoholic effects on animal models and determined a major molecular target and cellular mechanism of DHM for counteracting alcohol intoxication and dependence. We demonstrated pharmacological properties of DHM consistent with those expected to underlie successful medical treatment of AUDs; therefore DHM is a therapeutic candidate.

Ethanol Facilitates Glutamatergic Transmission to Dopamine Neurons in the Ventral Tegmental Area

The cellular mechanisms underlying alcohol addiction are poorly understood. In several brain areas, ethanol depresses glutamatergic excitatory transmission, but how it affects excitatory synapses on dopamine neurons of the ventral tegmental area (VTA), a crucial site for the development of drug addiction, is not known. We report here that in midbrain slices from rats, clinically relevant concentrations of ethanol (10–80 mM) increase the amplitude of evoked EPSCs and reduce their paired-pulse ratio in dopamine neurons in the VTA. The EPSCs were mediated by glutamate α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. In addition, ethanol increases the frequency but not the amplitude of spontaneous EPSCs. Furthermore, ethanol increases extracellular glutamate levels in the VTA of midbrain slices. The effects of ethanol are mimicked by SKF 38393, a dopamine D1 receptor agonist, and by GBR 12935, a dopamine reuptake inhibitor, and they are blocked by SKF 83566, a D1 antagonist, or by reserpine, which depletes dopamine stores. The enhancement of sEPSC frequency reaches a peak with 40 mM ethanol and declines with concentrations ⩾80 mM ethanol, which is quite likely a result of D2 receptor activation as raclopride, a D2 receptor blocker, significantly enhanced 80 mM ethanol-induced enhancement of sEPSCs. Finally, 6, 7-dinitroquinoxaline-2, 3-dione (DNQX), an AMPA receptor antagonist, attenuates ethanol-induced excitation of VTA DA neurons. We therefore conclude that, acting via presynaptic D1 receptors, ethanol at low concentrations increases glutamate release in the VTA, thus raising somatodendritic dopamine release, which further activates the presynaptic D1 receptors. Enhancement of this positive feedback loop may significantly contribute to the development of alcohol addiction.

The selectivity of the channel coupled to the 5-HT3 receptor.

The 5-HT3 receptor is unusual among receptors for biogenic amines in that it is directly coupled to an ion channel that is highly permeable to Na+ and K+. We have studied the permeation properties of this channel in order to achieve a more detailed understanding of its physiological function and to extend the comparison with other ligand gated channels. The 5-HT3 receptor channel is significantly permeable to the organic cations Tris, choline, and N-methyl-glucamine, with permeabilities decreasing with size. The permeability ratios for Tris and choline are similar to those determined for the nicotinic receptor; the permeability ratio for Tris is also similar to that of a non-N-methyl-D-aspartate (non-NMDA) excitatory amino acid receptor. This suggests that the diameters at the narrowest parts of these 3 channels are similar. The Ca2+ permeability of the 5-HT3 receptor channel is relatively low, with an upper bound to PCa/PNa estimated as 0.076. The single channel conductance, as determined by noise analysis, was also relatively low, with a value of 4.4 +/- 0.5 pS. Thus, both the Ca2+ permeability and single channel conductance are lower than those of the nicotinic receptor. In these respects, the 5-HT3 receptor is closer to non-NMDA excitatory amino acid receptors. These results are interpreted in terms of a model of the 5-HT3 receptor channel in which the interior has a lower polarizability, and possibly a greater length, in comparison with the nicotinic acetylcholine receptor channel.

Cholinergic neurotransmission in the preBÖtzinger Complex modulates excitability of inspiratory neurons and regulates respiratory rhythm

We investigated whether there is endogenous acetylcholine (ACh) release in the preBötzinger Complex (preBötC), a medullary region hypothesized to contain neurons generating respiratory rhythm, and how endogenous ACh modulates preBötCneuronal function and regulates respiratory pattern. Using a medullary slice preparation from neonatal rat, we recorded spontaneous respiratory-related rhythm from the hypoglossal nerve roots (XIIn) and patch-clamped preBötC inspiratory neurons. Unilateral microinjection of physostigmine, an acetylcholinesterase inhibitor, into the preBötC increased the frequency of respiratory-related rhythmic activity from XIIn to 116+/-13% (mean+/-S.D.) of control. Ipsilateral physostigmine injection into the hypoglossal nucleus (XII nucleus) induced tonic activity, increased the amplitude and duration of the integrated inspiratory bursts of XIIn to 122+/-17% and 117+/-22% of control respectively; but did not alter frequency. In preBötC inspiratory neurons, bath application of physostigmine (10 microM) induced an inward current of 6.3+/-10.6 pA, increased the membrane noise, decreased the amplitude of phasic inspiratory drive current to 79+/-16% of control, increased the frequency of spontaneous excitatory postsynaptic currents to 163+/-103% and decreased the whole cell input resistance to 73+/-22% of control without affecting the threshold for generation of action potentials. Bath application of physostigmine concurrently induced tonic activity, increased the frequency, amplitude and duration of inspiratory bursts of XIIn motor output. Bath application of 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 2 microM), a M3 muscarinic acetylcholine receptor (mAChR) selective antagonist, increased the input resistance of preBötC inspiratory neurons to 116+/-9% of control and blocked all of the effects of physostigmine except for the increase in respiratory frequency. Dihydro-beta-erythroidine (DH-beta-E; 0.2 microM), an alpha4beta2 nicotinic receptor (nAChR) selective antagonist, blocked all the effects of physostigmine except for the increase in inspiratory burst amplitude. In the presence of both 4-DAMP and DH-beta-E, physostigmine induced opposite effects, i.e. a decrease in frequency and amplitude of XIIn rhythmic activity. These results suggest that there is cholinergic neurotransmission in the preBötC which regulates respiratory frequency, and in XII nucleus which regulates tonic activity, and the amplitude and duration of inspiratory bursts of XIIn in neonatal rats. Physiologically relevant levels of ACh release, via mAChRs antagonized by 4-DAMP and nAChRs antagonized by DH-beta-E, modulate the excitability of inspiratory neurons and excitatory neurotransmission in the preBötC, consequently regulating respiratory rhythm.

Severe Neurologic Impairment in Mice with Targeted Disruption of the Electrogenic Sodium Bicarbonate Cotransporter NBCe2 (Slc4a5 Gene)

The choroid plexus lining the four ventricles in the brain is where the majority of cerebrospinal fluid (CSF) is produced. The secretory function of the choroid plexus is mediated by specific transport systems that allow the directional flux of nutrients and ions into the CSF and the removal of toxins. Normal CSF dynamics and chemistry ensure that the environment for neural function is optimal. Here, we report that targeted disruption of the Slc4a5 gene encoding the electrogenic sodium bicarbonate cotransporter NBCe2 results in significant remodeling of choroid plexus epithelial cells, including abnormal mitochondrial distribution, cytoskeletal protein expression, and ion transporter polarity. These changes are accompanied by very significant abnormalities in intracerebral ventricle volume, intracranial pressure, and CSF electrolyte levels. The Slc4a5−/− mice are significantly more resistant to induction of seizure behavior than wild-type controls. In the retina of Slc4a5−/− mice, loss of photoreceptors, ganglion cells, and retinal detachment results in visual impairment assessed by abnormal electroretinogram waveforms. Our findings are the first demonstration of the fundamental importance of NBCe2 in the biology of the nervous system.

Modulation of AMPA receptors by cAMP‐dependent protein kinase in PreBötzinger complex inspiratory neurons regulates respiratory rhythm in the rat

We hypothesize that phosphorylation of AMPA receptors or associated synaptic proteins modulates the excitability of respiratory neurons in the preBötzinger Complex (preBötC), affecting respiratory rhythm. Using neonatal rat medullary slices that spontaneously generate respiratory rhythm, we examined the role of the cAMP–PKA pathway (PKA:cAMP‐dependent protein kinase) in modulating glutamatergic synaptic transmission, the excitability of inspiratory neurons in the preBötC and respiratory rhythm. Microinjection of forskolin, an activator of adenylate cyclase, into the preBötC with or without the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX), decreased the period (increased the frequency) of respiratory‐related rhythmic motor output in the hypoglossal nerve (XIIn) to 84 % (without IBMX) and to 72% (with IBMX) of the pre‐injection baseline. In the presence of MK‐801, a non‐competitive NMDA receptor antagonist, microinjection of forskolin plus IBMX decreased the period to 66% of baseline levels. Microinjection of Rp‐adenosine 3′,5′‐cyclic monophosphothioate (Rp‐cAMPS), a PKA inhibitor, increased the period to 145% of baseline levels. Concurrent microinjection of Rp‐cAMPS and forskolin had no effect on the period. Bath application of 7β‐deacetyl‐7β‐[γ‐(morpholino)butyryl]‐forskolin hydrochloride (7Db‐forskolin, a water‐soluble derivative of forskolin): (1) decreased the period to 67% of baseline levels without affecting the amplitude of integrated XIIn inspiratory discharge, (2) induced a tonic inward current of 29 pA and enhanced inspiratory drive current (the amplitude increased to 183% and the integral increased to 184% of baseline) in voltage‐clamped (holding potential =−60 mV) preBötC inspiratory neurons and (3) increased the frequency to 195 % and amplitude to 118% of spontaneous excitatory postsynaptic currents (sEPSCs) during expiratory periods. Dideoxy‐forskolin did not have these effects. Intracellular perfusion with the catalytic subunit of PKA (cPKA) into preBötC inspiratory neurons progressively enhanced inspiratory drive currents and, in the presence of TTX, increased the inward currents induced by local ejection of AMPA; the latter currents were blocked by 1,2,3,4‐tetrahydro‐6‐nitro‐2,3‐dioxo‐benzo[f]quinoxaline‐7‐sulphonamide (NBQX, an AMPA/kainate receptor antagonist). The effects of cPKA were blocked by co‐application of PKA inhibitor (6–22) amide (PKI). These results suggest that phosphorylation of postsynaptic AMPA receptors through the cAMP–PKA pathway modulates both tonic and phasic excitatory amino acid synaptic transmission and excitability of inspiratory neurons in the preBötC and, therefore, regulates respiratory rhythm. Moreover, the basal level of endogenous PKA activity appears to be a determinant of resting respiratory frequency.

Central cholinergic regulation of respiration: nicotinic receptors.

Nicotinic acetylcholine receptors (nAChRs) are expressed in brainstem and spinal cord regions involved in the control of breathing. These receptors mediate central cholinergic regulation of respiration and effects of the exogenous ligand nicotine on respiratory pattern. Activation of α4* nAChRs in the preBötzinger Complex (preBötC), an essential site for normal respiratory rhythm generation in mammals, modulates excitatory glutamatergic neurotransmission and depolarizes preBötC inspiratory neurons, leading to increases in respiratory frequency. nAChRs are also present in motor nuclei innervating respiratory muscles. Activation of post- and/or extra-synaptic α4* nAChRs on hypoglossal (XII) motoneurons depolarizes these neurons, potentiating tonic and respiratory-related rhythmic activity. As perinatal nicotine exposure may contribute to the pathogenesis of sudden infant death syndrome (SIDS), we discuss the effects of perinatal nicotine exposure on development of the cholinergic and other neurotransmitter systems involved in control of breathing. Advances in understanding of the mechanisms underlying central cholinergic/nicotinic modulation of respiration provide a pharmacological basis for exploiting nAChRs as therapeutic targets for neurological disorders related to neural control of breathing such as sleep apnea and SIDS.

Micro-agar salt bridge in patch-clamp electrode holder stabilizes electrode potentials

Maintaining a stable electrode potential is critical for patch-clamp measurements. The electrode potential of conventional patch electrode-holder assembly, where an Ag/AgCl wire is in direct contact with the patch pipette filling solution, is subject to drift if the pipette solution contains a low concentration of chloride ions (Cl-). We developed an agar bridge of 3 M KCl filled in a polyimide microtubing which forms an electrical connection between an Ag/AgCl wire and the pipette solution. We examined the offset potentials of the micro-agar salt bridge electrode assembly in parallel with a conventional electrode assembly in generic recording conditions (the pipette solution contained 5 mM NaCl). The junction potential between the Ag/AgCl wire and the pipette filling solution in the conventional electrode contributed to most of the offset potential drift observed during the course of 30 min recordings. The drift was up to 27.3 mV after several changes of the glass pipette. In contrast, the micro-agar salt bridge stabilized the electrode potential within typically 2 mV without affecting the patch electrode resistance, capacitance or noise level. Numerical simulations showed that Cl- diffusion from the agar bridge to the tip caused a negligible 0.4 microM Cl- concentration change at the pipette tip within 30 min. This method is easy to implement and provides long-term recording stability. The micro-agar salt bridge can fit in most commercial patch electrode holders and can be conveniently maintained.