Intracellular analysis in vivo of different barosensitive bulbospinal neurons in the rat rostral ventrolateral medulla

Abstract

Neurons located in the rostral ventrolateral medulla (RVLM) with projections to the intermediolateral column (IML) in the spinal cord were electrophysiologically characterized and anatomically identified using an intracellular recording technique in vivo. A group of spontaneously active neurons was antidromically activated by electrical stimulation of the IML in the thoracic spinal cord (T2-T3 level). The axonal conduction velocities ranged from 1.5 m/sec to 11.0 m/sec; mean value, 5.5 ± 2.6 m/sec (±SD). The firing pattern and changes in membrane potential in relation to the cardiac cycle were investigated in these bulbospinal neurons. A first group discharged action potentials with higher frequency at the end of the diastolic/beginning of the systolic period. The average of the neuronal membrane potentials demonstrated depolarizing potentials at the end of the diastolic/beginning of the systolic period. These depolarizing potentials increased in magnitude when the neurons were hyperpolarized. Therefore, they were characterized as EPSPs. The baroreceptor reflex activation produced by the increase in systemic arterial pressure following intravenous injection of phenylephrine elicited hyperpolarization, a decrease in the rate of discharge, and an increase in the membrane input resistance, suggesting that a disfacilitatory effect was produced by the activation of baroreceptor inputs on these bulbospinal neurons. Conversely, the inactivation of the baroreceptor reflex by intravenous injection of sodium nitroprusside produced depolarization and an increase in the firing rate. These neurons were characterized as baroreceptor-sensitive type I neurons. A second group of bulbospinal neuron in the RVLM was differentiated from the first group because it demonstrated a decrease in the frequency of discharge at the end of the diastolic/beginning of the systolic period. The average of the membrane potentials showed hyperpolarizing potentials that decreased in magnitude when the neuron was hyperpolarized. These hyperpolarizing potentials occurred at the end of the diastolic/beginning of the systolic period and were reversed in polarity after intracellular injections of chloride ions for several minutes. Therefore, these potentials were characterized as chloride-dependent IPSPs locked to the cardiac cycle. In some of these neurons, the electrical stimulation of the IML produced, in addition to the antidromic action potential, a monosynaptic EPSP with a shorter latency. Based on these unique characteristics, these neurons were defined as barosensitive type II neurons. During constant baroreceptor inactivation achieved by the hypotension produced by intravenous infusions of sodium nitroprusside, the pattern of discharge of barosensitive type II neurons became very regular, and the IPSPs locked to the cardiac cycle were absent. The recorded neurons were intracellularly injected with biocytin, incubated with the avidin-Texas red complex, and visualized under fluorescent microscopy. The histological sections were then processed for phenylethanolamine-N-methyltransferase (PNMT) immunoreactivity, using a double labeling immunohistofluorescence procedure. Although all neurons were found in the RVLM and 22 out of 25 of them were surrounded by catecholaminergic neurons, none of the investigated barosensitive neurons demonstrated PNMT immunoreactivity. These data provide direct evidence for two different types of sympathoexcitatory bulbospinal neurons in the RVLM, each receiving functionally opposing baroreceptor inputs. Even though they were found close to the PNMT-containing neurons, they did not appear to be catecholaminergic. The results support the idea that noncatecholaminergic bulbospinal neurons in the RVLM are important in cardiovascular control.