Cloning and characterization of voltage-gated calcium channel alpha1 subunits in Xenopus laevis during development.

Voltage-gated calcium channels play a critical role in regulating the Ca2+ activity that mediates many aspects of neural development, including neural induction, neurotransmitter phenotype specification, and neurite outgrowth. Using Xenopus laevis embryos, we describe the spatial and temporal expression patterns during development of the 10 pore-forming alpha1 subunits that define the channels' kinetic properties. In situ hybridization indicates that CaV1.2, CaV2.1, CaV2.2, and CaV3.2 are expressed during neurula stages throughout the neural tube. These, along with CaV1.3 and CaV2.3, beginning at early tail bud stages, and CaV3.1 at late tail bud stages, are detected in complex patterns within the brain and spinal cord through swimming tadpole stages. Additional expression of various alpha1 subunits was observed in the cranial ganglia, retina, olfactory epithelium, pineal gland, and heart. The unique expression patterns for the different alpha1 subunits suggests they are under precise spatial and temporal regulation and are serving specific functions during embryonic development.

Expression patterns of glycine transporters (xGlyT1, xGlyT2, and xVIAAT) in Xenopus laevis during early development.

Glycine, a major inhibitory neurotransmitter in the vertebrate nervous system, not only functions in synaptic signaling, but has also been implicated in regulating neuronal differentiation, neuronal proliferation, synaptic modeling, and neural network stability. Elements of the glycinergic phenotype include the membrane-bound glycine transporters (GLYT1 and GLYT2), which remove glycine from the synaptic cleft, and the vesicular inhibitory amino acid transporter (VIAAT or VGAT), which sequesters both glycine and GABA into synaptic vesicles. Here, we describe the spatial and temporal expression patterns of xGlyT1, xGlyT2, and xVIAAT during early developmental stages of Xenopus laevis. In situ hybridization reveals that xGlyT1 is first expressed in early tailbud stages in the midbrain, hindbrain, and anterior spinal cord; it extends posteriorly through the spinal cord and appears in the forebrain, retina, between the somites, and in the blood islands by swimming tadpole stages. xGlyT2 and xVIAAT initially appear in late neurula stages in the anterior spinal cord. By swimming tadpole stages, the expression of these genes appears in the forebrain, midbrain, and hindbrain and extends posteriorly through the spinal cord; xVIAAT is also expressed in the retina. Confocal analysis of multiplex fluorescent in situ hybridization signal in the spinal cord reveals that xGlyT1 and xGlyT2 share little cellular colocalization. While there is significant coexpression between xVIAAT and xGlyT2, xVIAAT and the GABAergic marker glutamic acid decarboxylase (xGAD67), and xGlyT2 and xGAD67, each gene also appears to have discrete, non-colocalized areas of expression.

Hindbrain chemical mediators of reflex-induced inhibition of gastric tone produced by esophageal distension and intravenous nicotine.

The purpose of this study was to activate a vagovagal reflex by using esophageal distension and nicotine and test whether hindbrain nitric oxide and norepinephrine are involved in this reflex function. We used double-labeling immunocytochemical methods to determine whether esophageal distension (and nicotine) activates c-Fos expression in nitrergic and noradrenergic neurons in the nucleus tractus solitarii (NTS). We also studied c-Fos expression in the dorsal motor nucleus of the vagus (DMV) neurons projecting to the periphery. Esophageal distension caused 19.7 +/- 2.3% of the noradrenergic NTS neurons located 0.60 mm rostral to the calamus scriptorius (CS) to be activated but had little effect on c-Fos in DMV neurons. Intravenous administration of nicotine caused 19.7 +/- 4.2% of the noradrenergic NTS neurons 0.90 mm rostral to CS to be activated and, as reported previously, had no effect on c-Fos expression in DMV neurons. To determine whether norepinephrine and nitric oxide were central mediators of esophageal distension-induced decrease in intragastric pressure (balloon recording), N(G)-nitro-L-arginine methyl ester microinjected into the NTS (n = 5), but not into the DMV, blocked the vagovagal reflex. Conversely, alpha2-adrenergic blockers microinjected into the DMV (n = 7), but not into the NTS, blocked the vagovagal reflex. These data, in combination with our earlier pharmacological microinjection data with nicotine, indicate that both esophageal distension and nicotine produce nitric oxide in the NTS, which then activates noradrenergic neurons that terminate on and inhibit DMV neurons.