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Rostral ventral medulla (RVM) contains significant numbers of local GABAergic neurons which may subserve respiratory chemosensory and baroreceptor reflexes. Nicotinic mechanisms stimulate release of GABA in certain brainstem neurons. Whether the GABAergic neurons at RVM express nicotinic cholinergic receptors (nAChRs) is not known. We used glutamic acid decarboxylase 67-kDa isoform (GAD67) and parvalbumin (PV) as anatomical markers to identify the GABAergic neurons of the RVM and caudal pons and performed double labeling to evaluate the expression of alpha-7 and alpha-4 nAChRs by GAD67 and PV-imnunoreactive (ir) cells at these sites. GAD67-ir cells were found at the ventrolateral pontomedullary border in areas adjacent to the A5 noradrenergic cell group and overlapping the facial nucleus lateral subnuclei and para-facial zones. Of 205 GAD67-ir cells labeled at these sites, 74% exhibited immunoreactivity for alpha-7 nAChRs. Alpha-4 immunoreactivity was also present in 35% of GAD67-ir cells at these sites. The PV-ir cells of RVM and caudal pons were found medial to the facial nucleus and lateral to the pyramid in a column distinct from the GAD67-ir cells. Virtually all the PV-ir cells demonstrated immunoreactivity for alpha-4 nAChR (95%) and alpha-7 (93%) subunits of nAChRs. Differential expression of GAD67 and PV by neurons at the pontomedullary border implies that PV may not be a valid marker for GABAergic neurons. The expression of alpha-4 and alpha-7 nAChRs by GAD67-ir cells suggests nicotinic cholinergic modulation of GABAergic signaling at these ventrolateral pontomedullary sites.

Previous studies have shown that molecules of the taste transduction pathway may serve as biochemical markers for chemoreceptive cells in respiratory and gastrointestinal tracts. In this study, we tested the hypothesis that brainstem neurons contain signaling molecules similar to those in taste buds which may sense the chemical composition of brain extracellular fluids. We used the reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunohistochemical techniques to evaluate presence of different bitter-responsive type 2 taste receptors (T2Rs), their associated G-protein α-gustducin, the downstream signaling molecules phospholipase C isoform β2 (PLC-β2) and transient receptor potential melastatin 5 (TRPM5) in the brainstem of rats. RT-PCR confirmed the mRNA coding for α-gustducin, PLC-β2, TRPM5 and rT2R1 but not that of rT2R16, rT2R26 and rT2R38 in the medulla oblongata. Western blotting confirmed the presence of α-gustducin at the protein level in rat brainstem. Immunohistochemistry identified cells expressing α-gustducin and PLC-β2 at multiple cardiorespiratory and CO 2 /H + chemosensory sites, including rostral ventral medulla, facial, parapyramidal, solitary tract, hypoglossal and raphe nuclei. In the medullary raphe, α-gustducin and PLC-β2 were colocalized with a subpopulation of tryptophan hydroxylase (TPH)-immunoreactive serotonergic neurons, a subset of which has respiratory CO 2 /H + chemosensitivity. Presence of the T2R1 gene and other genes and proteins of the bitter taste transduction pathway in the brainstem implies additional functions for taste receptors and their effector molecules apart from their gustatory function.

In cats anesthetized with chloralose-urethane (40 mg/kg chloralose; 200 mg/kg urethane) pH sensitivity of neurons in the caudal chemosensitive area on the ventrolateral surface of the medulla oblongata was examined while monitoring phrenic nerve activity simultaneously. pH was varied by superfusion of the ventral medullary surface with mock cerebrospinal fluid (CSF) of different pH (pH 7.4 control, 7.0 and 7.8). A total of 130 units from 21 cats changed their firing rate in response to CSF-pH changes. These were subdivided into 3 groups. InGroup I, 31 respiratory pH sensitivie units increased their firing rate in response to decreased mock CSF-pH, noxious pinch, joint movement in contralateral forelimb, and increased inspired CO2. These responses may have originated from respiratory center neurons.Group II consisted of 59 non-respiratory pH sensitive units whose firing rate changed in an inverse manner with CSF-pH changes. Of these, 30 responded to contralateral distal forelimb movement, 15 to hair manipulation, 9 to heavy pressure and 5 to noxious pinch. Increased inspired CO2 (rebreathing) did not modify activity. The response to pH is believed to be from non-specific neurons.Group III consisted of 40 non-respiratory pH sensitive units responding to CSF-pH changes and to increased inspired CO2. The firing rate was irregular, the interval distribution approaching an exponential function. It may be postulated that the impulse frequency of chemosensitive impulses may be irregular at the site of impulse generation, the irregularity decreasing by convergence during transmission to the respiratory centers. The time course of Group III chemosensitive units was similar to phrenic nerve responses.

Physiological investigations have indicated that the ventrolateral surface of the medulla oblongata is involved in the chemical drive to respiration. In this investigation, light and electron microscopic investigations of the 3 chemosensitive regions reveal the following. (1) Evaginations of the ventral surface abut the overlying pia mater thereby delimiting discrete compartments; invaginations of the surface delimit wide cisternae lined with basement membrane. Neuronal elements with numerous synapses. were found scattered among astrocytic processes of the marginal glia in intermediate and caudal chemosensitive areas Microvasculature are conspicuously absent from the marginal glia. Intramedullary vessels are surrounded by perivascular spaces and the endothelium shows zonulae occludentes at cell junctions. (2) Horseradish peroxidase (HRP) applied to the ventral surface diffused throughout the interstitial and perivascular compartments, into synaptic clefts and neuronal soma. Diffusion of HRP into blood vessels was blocked at zonulae occludentes. Following intravenous of HRP, no reaction product was found outside cerebral vasculature in chemosensitive areas. (3) In spontaneously breathing cats, 2% procaine applied to the caudal chemosensitive area resulted in respiratory depression which began with the second breath. It is proposed, that substances which stimulate or depress respiration, when applied to the ventral medullary surface, produce their effects on superficial neurons located in the intermediate and caudal chemosensitive areas after diffusion through interstitial spaces.