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We investigated the CO2 responsiveness of GABAergic neurons in the ventral medullary surface (VMS), a putative chemoreceptive area using a 67-kDa isoform of GABA-synthesizing enzyme (GAD67)-green fluorescence protein (GFP) knock-in neonatal mouse, in which GFP is specifically expressed in GABAergic neurons. The slice was prepared by transversely sectioning at the level of the rostral rootlet of the XII nerve and the rostral end of the inferior olive in mock cerebrospinal fluid (CSF). Each medullary slice was continuously superfused with hypocapnic CSF. GFP-positive neurons in the VMS were selected by using fluorescent optics and their membrane potentials and firing activities were analyzed with a perforated patch recording technique. Thereafter, superfusion was changed from hypocapnic to hypercapnic CSF. In 4 out of 8 GABAergic neurons in the VMS, perfusion with hypercapnic CSF induced more than a 20% decrease in the discharge frequency and hyperpolarized the neurons. The remaining 4 GFP-positive neurons were CO2-insensitive. GABAergic neurons in the VMS have chemosensitivity. Inhibition of chemosensitive GABAergic neural activity in the VMS may induce increases in respiratory output in response to hypercapnia.

The characteristics of GABAergic neurons involved in respiratory control have not been fully understood because identification of GABAergic neurons has so far been difficult in living tissues. In the present in vitro study, we succeeded in analysing the electrophysiological as well as morphological characteristics of GABAergic neurons in the pre-Bötzinger complex. We used 67-kDa isoform of glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) (Delta neo) knock-in (GAD67(GFP/+)) mice, which enabled us to identify GABAergic neurons in living tissues. We prepared medullary transverse slices that contained the pre-Bötzinger complex from these neonatal mice. The fluorescence intensity of the pre-Bötzinger complex region was relatively high among areas of the ventral medulla. Activities of GFP-positive neurons in the pre-Bötzinger complex were recorded in a perforated whole-cell patch-clamp mode. Six of 32 GFP-positive neurons were respiratory and the remaining 26 neurons were non-respiratory; the respiratory neurons were exclusively inspiratory, receiving excitatory post-synaptic potentials during the inspiratory phase. In addition, six inspiratory and one expiratory neuron of 30 GFP-negative neurons were recorded in the pre-Bötzinger complex. GFP-positive inspiratory neurons showed high membrane resistance and mild adaptation of spike frequency in response to depolarizing current pulses. GFP-positive inspiratory neurons had bipolar, triangular or crescent-shaped somata and GFP-negative inspiratory neurons had multipolar-shaped somata. The somata of GFP-positive inspiratory neurons were smaller than those of GFP-negative inspiratory neurons. We suggest that GABAergic inhibition not by expiratory neurons but by inspiratory neurons that have particular electrophysiological and morphological properties is involved in the respiratory neuronal network of the pre-Bötzinger complex.

Nicotine exposure is a risk factor in several breathing disorders Nicotinic acetylcholine receptors (nAChRs) exist in the ventrolateral medulla, an important site for respiratory control. We examined the effects of nicotinic acetylcholine neurotransmission on central respiratory control by addition of a nAChR agonist or one of various antagonists into superfusion medium in the isolated brainstem-spinal cord from neonatal rats. Ventral C4 neuronal activity was monitored as central respiratory output, and activities of respiratory neurons in the ventrolateral medulla were recorded in whole-cell configuration. RJR-2403 (0.1-10 mM), alpha4beta2 nAChR agonist induced dose-dependent increases in respiratory frequency. Non-selective nAChR antagonist mecamylamine (0.1-100 mM), alpha4beta2 antagonist dihydro-beta-erythroidine (0.1-100 mM), alpha7 antagonist methyllycaconitine (0.1-100 mM), and a-bungarotoxin (0.01-10 mM) all induced dose-dependent reductions in C4 respiratory rate. We next examined effects of 20 mM dihydro-beta-erythroidine and 20mM methyllycaconitine on respiratory neurons. Dihydro-beta-erythroidine induces hyperpolarization and decreases intraburst firing frequency of inspiratory and preinspiratory neurons. In contrast, methyllycaconitine has no effect on the membrane potential of inspiratory neurons, but does decrease their intraburst firing frequency while inducing hyperpolarization and decreasing intraburst firing frequency in preinspiratory neurons. These findings indicate that alpha4beta2 nAChR is involved in both inspiratory and preinspiratory neurons, whereas alpha7 nAChR functions only in preinspiratory neurons to modulate C4 respiratory rate.