We hypothesized that the direct stimulus of the central chemoreceptor
neurons is the CO2/H+-induced change in intracellular pH (pHi). If it
is true, pi-Ii responses during hypercapnic stimulation should be exhi
bited in the central chemoreceptor neurons in the ventral medullary su
rface (VMS) and some neurons in the CO2/H+ sensitive regions such as t
he nucleus tractus solitarii of the medial dorsal medulla (MDM). To te
st this hypothesis, the cultured VMS and MDM neurons (control) derived
from one day-old neonate rats were labeled with H+-sensitive fluoresc
ent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF),
and were exposed to perfusate of various pHs. The H+-sensitive neuron
s were determined by a rapid decrease in the intracellular BCECF fluor
escence intensity. In almost all the MDM neurons (99.6%) and 94% of th
e VMS neurons, the intracellular BCECF fluorescence intensity remained
unchanged when the extracellular pH (pHo) was decreased. In contrast,
in 0.4% of the MDM neurons (8/1800) and in 6% of the VMS neurons (111
/1800), the intracellular BCECF fluorescence intensity decreased when
the pHo was decreased from 7.4 to 7.2. This subpopulation of MDM and V
MS neurons were considered to be H+-sensitive neurons. The H+-sensitiv
e neurons in the VMS showed positive immunoreactivity to glutamate (57
%, 17/30) and glutamic acid decarboxylase (23%, 7/30), but no immunore
activity to choline acetyltransferase, tyrosine hydroxylase, phenyleth
anolamine N-methyltransferase, somatostatin, serotonin and substance P
. These results indicate that the H+-sensitive neurons are present spe
cifically in the VMS, and are mainly glutamatergic and GABAergic. (C)
1997 Elsevier Science B.V.