Intracellular acidosis and pH regulation in central respiratory chemoreceptors.

Dysfunctions of brainstem regions responsible for central CO2 chemoreception have been proposed as an underlying pathophysiology of Sudden Infant Death Syndrome (SIDS). We recorded respiratory motor output and intracellular pH (pHi) from chemosensitive neurons in an in vitro tadpole brainstem during normocapnia and hypercapnia. Flash photolysis of the H+ donor nitrobenzaldehyde was used to induce focal decreases in pHi alone. Hypercapnia and flash photolysis significantly decreased pHi from normocapnia. In addition, chemoreceptors did not regulate pHi during hypercapnia, but demonstrated significant pHi recovery when only pHi was reduced by flash photolysis. Respiration was stimulated by decreases in pHi (hypercapnia and flash photolysis) by decreases in burst cycle. These data represent our ability to load the brainstem with nitrobenzaldehyde without disrupting the respiration, to quantify changes in chemoreceptor pHi recovery, and to provide insights regarding mechanisms of human health conditions with racial/ethnic health disparities such as SIDS and Apnea of Prematurity (AOP).

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Optical recording of intracellular pH in respiratory chemoreceptors.

We studied the spontaneously active in vitro tadpole brainstem and recorded whole nerve respiratory activity while simultaneously visualizing intracellular pH (pHi) dynamics using the pH-sensitive dye, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF, AM). The isolated, superfused tadpole brainstem is well oxygenated and retains synaptic connectivity among respiratory central pattern generators, central respiratory chemoreceptors, and respiratory motor neurons. We generated a calibration curve to correlate the emitted fluorescence of BCECF to pHi. In addition, we demonstrated that the dye loading protocol that we established labeled an adequate number of cells and did not disrupt spontaneous respiratory rhythmogenesis or the respiratory response to central chemoreceptor stimulation. Validation of the use of the pH sensitive dye BCECF in this preparation will permit further characterization of the pH regulatory responses of central respiratory chemoreceptors and allow correlation between the changes in pHi in central chemoreceptors and respiratory motor output recorded from cranial nerves.

Serotonergic modulation of respiratory rhythmogenesis and central chemoreception.

In addition to evidence supporting serotonergic modulation of respiratory rhythmogenesis, serotonergic mechanisms play a role in central respiratory chemoreception. We examined the role of serotonin 5HT1A receptors in respiratory rhythmicity and central respiratory chemosensitivity in in vitro brainstem preparations of the bullfrog tadpole, Rana catesbeiana. Spontaneous respiratory motor output was recorded from cranial nerve 7 at control bath pH (7.8) and hypercapnic bath pH (7.4) as bath concentrations of a 5HT1A receptor agonist were steadily increased from 0.5 to 25 microM. Activation of the 5HT1A receptor significantly altered the respiratory burst cycle. Significant increases in both gill and lung burst cycle were observed in response to bath application of 8-OH-DPAT; gill burst cycle in response to 8-OH-DPAT was influenced by bath pH, as gill burst cycle at bath pH 7.8 was not significantly increased at 0.5 or 5.0 microM 8-OH-DPAT. However, when the pH was reduced to 7.4 gill burst cycle was significantly increased at these same bath concentrations of 8-OH-DPAT. Gill burst amplitude was not altered in response to bath application of 8-OH-DPAT; however, lung burst amplitude was significantly decreased at 25.0 microM 8-OH-DPAT at bath pH 7.8. These data indicate that 5HT1A receptors are involved in neural respiratory rhythmogenic and chemoreceptive circuits in the bullfrog tadpole, and support the hypothesis that abnormalities in serotonergic systems may be an underlying component of Sudden Infant Death Syndrome.

Tyrosine kinase phosphorylation of GABA(A) receptor alpha1, beta2 and gamma2 subunits following chronic intermittent ethanol (CIE) exposure of cultured cortical neurons of mice.

There is evidence that many of the GABA(A) receptor subunits contain consensus sequence for tyrosine kinase, and phosphorylation may play a key role in ethanol's regulation of GABA(A) receptors. Recently, we investigated the effect of chronic exposure of ethanol (CE) on tyrosine kinase phosphorylation and reported that there was an up-regulation in tyrosine kinase phosphorylation of the beta(2)- and gamma(2)- subunits and no effect on alpha(1)-subunit of the GABA(A) receptor in the cultured cortical neurons of mice. In the present study, we have further investigated the effect of chronic intermittent administration of ethanol (CIE) on tyrosine kinase phosphorylation of the GABA(A) receptor subunits (alpha(1), beta(2), and gamma(2)) in the mouse cultured cortical neurons by immunoprecipitation and Western blot techniques. We observed that there was an up-regulation in the tyrosine kinase phosphorylation of the GABA(A )receptor beta(2)- and gamma(2)-subunits following CIE exposure, and no effect on alpha(1)-subunit in the cultured cortical neurons of mice. These CIE changes, unlike CE, were not reverted back to the control level following ethanol withdrawal even after 7 days. Acute exposure of ethanol did not cause any change in the tyrosine kinase regulation of the GABA(A) receptor subunits. In conclusion, the CIE exposure, unlike chronic/acute ethanol exposure, regulates the tyrosine kinase phosphorylation of the selective population of GABA(A )receptors in a long lasting manner.

Electroporation as a tool to transfer the plasmid pRL489 in Oscillatoria MKU 277.

To establish the gene transfer system in the cyanobacteria, we successfully introduced the shuttle plasmid pRL489 into Oscillatoria MKU 277 by electroporation with transformation frequencies of up to 102 cfu/mug of plasmid DNA. These findings suggest that Oscillatoria MKU 277 can be used as an experimental tool for genetic maneuvering.

Methylation of NMDA receptor NR2B gene as a function of age in the mouse brain.

We have previously reported that there is an up-regulation of the NR2B gene expression in the adult cortex and cultured fetal cortical neurons of mice following chronic ethanol treatment due to demethylation of cytosine residues in the NR2B gene CpG island. In the present study, we investigated the methylation pattern of the NR2B CpG island as a function of the mouse age by digesting the cortex genomic DNA with HpaII enzyme, amplifying the interested regions by performing PCR and detecting the methylated regions by Southern hybridization so as to determine whether age affects the methylation process. We observed demethylation of various regions of NR2B gene (5227-5567), (5647-6003), (6091-6445), (6424-7024) of adult mouse cortex. Our results indicate that methylation of NR2B gene in the mouse brain is age-dependent phenomenon.