Biomedical subjects

Xuesi M Shao

Publications and source records attributed to Xuesi M Shao.

5 recordsLinked to original sources

PubMed2006

Efficient measurement of endogenous neurotransmitters in small localized regions of central nervous systems in vitro with HPLC.

High performance liquid chromatography (HPLC) is widely used to determine neurotransmitter concentrations in the central nervous system (CNS). Finding the optimal methods to sample from CNS tissue poses a challenge for neuroscientists. Here, we describe a method that allows assay of neurotransmitters (or other chemicals) in small regions (down to 180mum in diameter) in in vitro preparations concurrently with electrophysiological recordings. The efficiency for measuring small amounts of chemicals is enhanced by a sample collecting pipette with filter paper at the tip that makes close contact with the target region in CNS tissue. With a wire plunger in the calibrated pipette controlled by a microsyringe pump, there is virtually no dead volume. Samples in a volume of 10muL (taken, e.g., at 2muL/min over 5min) can be injected into a HPLC machine with microbore columns. We demonstrate the effectiveness of this method by measuring acetylcholine (ACh) in the ventral horn and its surrounding areas of the spinal cord in en bloc brainstem-spinal cord preparations. In control conditions, endogenous ACh levels in these regions were detectable. Application of neostigmine (an inhibitor of acetylcholinesterases (AChEs)) increased ACh concentrations, and at the same time, induced tonic/seizure-like activity in efferent motor output recorded from cervical ventral nerve roots. Higher ACh concentrations in the ventral horn were differentiated from nearby regions: the lateral and midline aspects of the ventral spinal cord. In addition, ACh in the preBötzinger Complex (preBötC) and the hypoglossal nucleus in medullary slice preparations can also be measured. Our results indicate that the method proposed in this study can be used to measure neurotransmitters in small and localized CNS regions. Correlation between changes in neurotransmitters in target regions and the neuronal activities can be revealed in vitro. Our data also suggest that there is endogenous ACh release in spinal ventral motor columns at fourth cervical (C4) level that regulates the respiratory-related motor activity.

Acetylcholine↗

PubMed2006

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.

Agar↗

PubMed2006

Golli protein negatively regulates store depletion-induced calcium influx in T cells.

Calcium influx is crucial for T cell activation and differentiation. The detailed regulation of this process remains unclear. We report here that golli protein, an alternatively spliced product of the myelin basic protein gene, plays a critical role in regulating calcium influx in T cells. Golli-deficient T cells were hyperproliferative and showed enhanced calcium entry upon T cell receptor stimulation. We further found that golli regulates calcium influx in T cells through the inhibition of the store depletion-induced calcium influx. Mutation of the myristoylation site on golli disrupted its association with the plasma membrane and reversed its inhibitory action on Ca2+ influx, indicating that membrane association of golli was essential for its inhibitory action. These results indicate that golli functions in a unique way to regulate T cell activation through a mechanism involving the modulation of the calcium homeostasis.

Animals↗

PubMed2003

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 7beta-deacetyl-7beta-[gamma-(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.

Animals↗

PubMed2002

Pharmacology of nicotinic receptors in preBötzinger complex that mediate modulation of respiratory pattern.

Nicotine regulates respiratory pattern by modulating excitatory neurotransmission affecting inspiratory neurons within the preBötzinger Complex (preBötC). The nicotinic acetylcholine receptor (nAChR) subtypes mediating these effects are unknown. Using a medullary slice preparation from neonatal rat, we recorded spontaneous respiratory-related rhythm from the hypoglossal nerve (XIIn) and patch-clamped inspiratory neurons in the preBötC simultaneously. The alpha7 nAChR antagonists alpha-bungarotoxin or methyllycaconitine (MLA) had little effect on the actions of low concentrations of nicotine (0.5 microM), which included an increase in respiratory frequency; a decrease in amplitude of XIIn inspiratory bursts; a tonic inward current associated with an increase in membrane noise; an increase in the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs), and; a decrease in the amplitude of inspiratory drive current in voltage-clamped preBötC inspiratory neurons. These nicotinic actions were completely reversed by dihydro-beta-erythroidine (DH-beta-E) or hexamethonium and reduced by D-tubocurarine. Comparable concentrations of RJR-2403 (0.5-1 microM), an agonist selective for alpha4beta2 nAChRs, increased respiratory frequency to 186% and decreased the amplitude of XIIn inspiratory bursts to 83% of baseline. In voltage-clamped preBötC inspiratory (including pacemaker) neurons, RJR-2403 induced a tonic inward current of -15.2 pA associated with an increase in membrane noise, increased the frequency to 157% and amplitude to 106% of spontaneous EPSCs, and decreased the amplitude of inspiratory drive current to 80% of baseline. MLA had little effect on RJR-2403 actions, while DH-beta-E completely reversed them. These results suggest that the predominant subtype of nAChRs in preBötC in neonatal rats that mediates the modulation of respiratory pattern by low concentrations of nicotine is an alpha4beta2 combination and not an alpha7 subunit homomer. We do not exclude the possibility that co-assembly of alpha4beta2 with other subunits or other nAChR subtypes are also expressed in preBötC neurons. The parallel changes in the cellular and systems level responses induced by different nicotinic agonists and antagonists support the idea that modulation of excitatory neurotransmission affecting preBötC inspiratory neurons is a mechanism underlying the cholinergic regulation of respiratory pattern (). This study provides a useful model system for evaluating potential therapeutic cholinergic agents for their respiratory effects and side effects.

Animals↗