Cytoplasmic polyadenylation element binding protein 2 (CPEB2) is an RNA-binding protein and translational regulator. To understand the physiological function of CPEB2, we generated CPEB2 knock-out (KO) mice and found that most died within 3 d after birth. CPEB2 is highly expressed in the brainstem, which controls vital functions, such as breathing. Whole-body plethysmography revealed that KO neonates had aberrant respiration with frequent apnea. Nevertheless, the morphology and function of the respiratory rhythm generator and diaphragm neuromuscular junctions appeared normal. We found that upregulated translation of choline acetyltransferase in the CPEB2 KO dorsal motor nucleus of vagus resulted in hyperactivation of parasympathetic signaling-induced bronchoconstriction, as evidenced by increased pulmonary acetylcholine and phosphorylated myosin light chain 2 in bronchial smooth muscles. Specific deletion of CPEB2 in cholinergic neurons sufficiently caused increased apnea in neonatal pups and airway hyper-reactivity in adult mice. Moreover, inhalation of an anticholinergic bronchodilator reduced apnea episodes in global and cholinergic CPEB2-KO mice. Together, the elevated airway constriction induced by cholinergic transmission in KO neonates may account for the respiratory defect and mortality.

To understand the origination of sympathetic nerve discharge (SND), I developed an in vitro brain stem-spinal cord preparation from neonatal rats. Ascorbic acid (3 mM) was added into the bath solution to increase the viability of preparations. At 24 degrees C, rhythmic SND (recorded from the splanchnic nerve) was consistently observed, but it became quiescent at <16 degrees C. Respiratory-related SND (rSND) was discernible and was well correlated with C(4) root activity. Power spectral analysis of SND revealed a dominant 2-Hz oscillation. In most preparations (86%), such oscillation was persistent, whereas it only slightly reduced its magnitude after isolation from the brain stem. The removal of neural structures rostral to the superior cerebellar artery (equivalent to the level of facial nuclei) reduced rSND, increased tonic SND, but did not affect the temporal coupling between SND and C(4) root activity. Our data suggest a prominent contribution of SND from the neural mechanisms confined within the neonatal rat spinal cord. This ascorbic acid-enhanced in vitro preparation is a very useful model to study neural mechanisms underlying sympathorespiratory integration.

Sympathetic nerves conveying central commands to regulate visceral functions often display activities in synchronous bursts. To understand how individual fibers fire synchronously, we establish "oligofiber recording techniques" to record "several" nerve fiber activities simultaneously, using in vitro splanchnic sympathetic nerve-thoracic spinal cord preparations of neonatal rats as experimental models. While distinct spike potentials were easily recorded from collagenase-dissociated sympathetic fibers, a problem arising from synchronous nerve discharges is a higher incidence of complex waveforms resulted from spike overlapping. Because commercial softwares do not provide an explicit solution for spike overlapping, a series of custom-made LabVIEW programs incorporated with MATLAB scripts was therefore written for spike sorting. Spikes were represented as data points after waveform feature extraction and automatically grouped by k-means clustering followed by principal component analysis (PCA) to verify their waveform homogeneity. For dissimilar waveforms with exceeding Hotelling's T 2 distances from the cluster centroids, a unique data-based subtraction algorithm (SA) was used to determine if they were the complex waveforms resulted from superimposing a spike pattern close to the cluster centroid with the other signals that could be observed in original recordings. In comparisons with commercial software, higher accuracy was achieved by analyses using our algorithms for the synthetic data that contained synchronous spiking and complex waveforms. Moreover, both T 2 -selected and SA-retrieved spikes were combined as unit activities. Quantitative analyses were performed to evaluate if unit activities truly originated from single fibers. We conclude that applications of our programs can help to resolve synchronous sympathetic nerve discharges (SND).

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Nitric oxide (NO) is a diffusible gas and has multifarious effects on both pre- and postsynaptic events. As a consequence of complex excitatory and inhibitory integrations, NO effects on neuronal activities are heterogeneous. Using in vitro preparations of neonatal rats that retain the splanchnic sympathetic nerves and the thoracic spinal cord as an experimental model, we report here that either enhancement or attenuation of NO production in the neonatal rat spinal cords could increase, decrease, or not change the spontaneous firing behaviors recorded from splanchnic sympathetic single fibers. To elucidate the mathematical features of NO-mediated heterogeneous responses, the ratios of changes in firing were plotted against their original firing rates. In log-log plots, a linear data distribution demonstrated that NO-mediated heterogeneity in sympathetic firing responses was well described by a power function. Selective antagonists were applied to test if glycinergic, GABAergic, glutamatergic, and cholinergic neurotransmission in the spinal cord are involved in NO-mediated power-law firing modulations (plFM). NO-mediated plFM diminished in the presence of mecamylamine (an open-channel blocker of nicotinic cholinergic receptors), indicating that endogenous nicotinic receptor activities were essential for plFM. Applications of strychnine (a glycine receptor blocker), gabazine (a GABAA receptor blocker), or kynurenate (a broad-spectrum ionotropic glutamate receptor blocker) also caused plFM. However, strychnine- or kynurenate-induced plFM was diminished by L-NAME (an NO synthase inhibitor) pretreatments, indicating that the involvements of glycine or ionotropic glutamate receptor activities in plFM were secondary to NO signaling. To recapitulate the arithmetic natures of the plFM, the plFM were simulated by firing changes in two components: a step increment and a fractional reduction of their basal firing activities. Ionotropic glutamate receptor activities were found to participate in plFM by both components. In contrast, GABAA receptor activities are involved in the component of fractional reduction only. These findings suggest that NO orchestrates a repertoire of excitatory and inhibitory neurotransmissions, incurs a shunting effect on postsynaptic membrane properties, and thus, alters sympathetic firing in a manner of plFM. We propose that the plFM mediated by NO forms a basic scheme of differential controls for heterogeneous sympathetic regulation of visceral functions.