Characterization of the chemosensitive response of individual solitary complex neurons from adult rats

Abstract

We studied the CO(2)/H(+)-chemosensitive responses of individual solitary complex (SC) neurons from adult rats by simultaneously measuring the intracellular pH (pH(i)) and electrical responses to hypercapnic acidosis (HA). SC neurons were recorded using the blind whole cell patch-clamp technique and loading the soma with the pH-sensitive dye pyranine through the patch pipette. We found that SC neurons from adult rats have a lower steady-state pH(i) than SC neurons from neonatal rats. In the presence of chemical and electrical synaptic blockade, adult SC neurons have firing rate responses to HA (percentage of neurons activated or inhibited and the magnitude of response as determined by the chemosensitivity index) that are similar to SC neurons from neonatal rats. They also have a typical response to isohydric hypercapnia, including decreased DeltapH(i), followed by pH(i) recovery, and increased firing rate. Thus, the chemosensitive response of SC neurons from adults is similar to the chemosensitive response of SC neurons from neonatal rats. Because our findings for adults are similar to previously reported values for neurons from neonatal rats, we conclude that intrinsic chemosensitivity is established early in development for SC neurons and is maintained throughout adulthood.

Fig. 1.

A: calibration curve for pyranine in individual solitary complex (SC) neurons from adult rats. Each point represents an N_fl value at a given pH for a single SC neuron. R_fl values measured during the experiment were divided by the R_fl value at pH 7.4 measured at the end of an experiment, which then gave N_fl. A sigmoidal curve was fit to the data over a range of pH from 6.2 to 8.6. The equation for this curve (Methods) was used to convert N_fl values into pH_i. B: pH_i response of SC neurons from adult rats to an acidification induced by an NH₄Cl prepulse followed by either artificial cerebrospinal fluid (aCSF) (□) or 0 Na⁺ (▪) solution. The control experiment (NH₄Cl prepulse followed by aCSF) exhibited recovery from NH₄Cl-induced acidification back to the initial pH_i. In 0 Na⁺ solution, the neuron had a maintained acidification with pH_i recovery inhibited. Upon replacing 0 Na⁺ solution with aCSF, pH_i recovered rapidly back to initial pH_i.

Fig. 2.

The pH_i and firing rate response of an individual SC neuron from an adult rat that was activated by hypercapnic acidosis (HA). A: experimental protocol used. B: sample fluorescence images of the SC neuronal cell body in response to each solution. C: pH_i response of the SC neuron to HA (15% CO₂) over time, which was a maintained acidification with a lack of pH_i recovery. Notice that once the HA solution was removed, pH_i returned back toward initial pH_i. D: firing rate response to HA of the SC neuron over time, which increased in response to HA and returned to initial firing rate once HA was removed. E: 10-s samples of action potentials taken at the time points indicated in D. Notice that action potential frequency increased with HA and then decreased back to initial frequency once HA was removed.

Fig. 3.

A: sample basal firing rate response of an SC neuron to synaptic blockade (SNB). Notice that basal firing rate increases in response to SNB. Inset: presence of SNB (bottom) suppresses the postsynaptic potentials (greater than 5-mV deflections) that can be seen in the absence of SNB (top). B: sample truncated action potential in the absence (left) and presence of SNB (right). Notice that the excitatory postsynaptic potentials seen on the left (signified by arrows) are eliminated by SNB shown on the right. C: average basal firing rate of adult SC neurons in the presence of aCSF and SNB. *Significantly greater firing rate in SNB than the firing rate in aCSF (P < 0.05). The height of a bar represents the mean firing rate with error bar of 1 SE.

Fig. 4.

The pH_i and firing rate response of an individual SC neuron from an adult rat that was activated by hypercapnic acidosis (HA) in the presence of SNB. A: experimental protocol used. B: pH_i response of the SC neuron to HA over time, which was not changed by SNB (i.e., a maintained acidification with a lack of pH_i recovery). Notice that once the HA solution was removed, pH_i returned back toward the initial pH_i. C: firing rate response to HA over time of the SC neuron, which increased in response to HA and returned back toward initial firing rate once the HA was removed. We hyperpolarized the V_m (indicated by hyp) by injecting negative DC current to bring the firing rate back toward initial firing rate before exposing the neuron to HA. D: 10-s samples of action potentials taken at the time points indicated in C. Notice that action potential frequency increased with HA and then decreased back to initial frequency once HA was removed.

Fig. 5.

The pH_i and firing rate response of an individual SC neuron from an adult rat that was activated by hypercapnic acidosis (HA) in the presence of SNB + CARB. A: experimental protocol used. B: pH_i response to HA acidosis over time of the SC neuron. Notice the similar pattern of pH_i response to HA as in SNB (Fig. 4), but the much larger HA-induced acidification in SNB + CARB. C: firing rate response of the SC neuron to HA over time, which increased reversibly in response to HA. D: 10-s samples of action potentials taken at the time points indicated in C. notice that action potential frequency increased with HA and then decreased back to initial frequency once HA was removed.

Fig. 6.

A: summary of the percentage of SC neurons from adult rats that respond to hypercapnic acidosis (HA). We found that the % activated in the presence of only aCSF (open bar) was not changed by SNB (gray bar) or SNB + CARB (solid bar). The percentage of inhibited neurons (9% in aCSF) was similarly unaffected by SNB (7%) and by SNB + CARB (19%). N values are denoted on each bar and height of bars represent the mean values. B: summary of the chemosensitivity index (CI) of SC neurons from adult rats that are activated in response to HA. We found that the CI of activated SC neurons from adult rats in the presence of aCSF only (white bar) was not affected by SNB (gray bar) or SNB + CARB (black bar). Height of bar represents mean CI, and error bar represents 1 SE.

Fig. 7.

The pH_i and firing rate response of an individual SC neuron from an adult rat that was activated by isohydric hypercapnia (IH) in the presence of SNB. A: experimental protocol used. B: pH_i response to IH over time of the SC neuron. Notice that there is pH_i recovery during IH and that once the IH solution was removed, there was an alkaline overshoot in pH_i that returned back to a value above initial pH_i. C: firing rate response to IH over time of the SC neuron, which increased reversibly in response to IH. D: 10-s samples of action potentials taken at the time points indicated in C. Notice that action potential frequency increased with IH and then decreased back to initial frequency once the IH solution was removed.

Fig. 8.

A: average magnitude of acidification (reported as a negative change in pH_i) that is induced by either HA or IH in CO₂-activated SC neurons from adult rats. Note that IH induced a statistically smaller acidification than HA (*P < 0.001). B: average rate of pH_i recovery from hypercapnia-induced acidification for SC neurons from adult rats in response to HA and IH. Very few (4/19) SC neurons exhibited at most a small pH_i recovery from HA-induced acidification, whereas a majority (13/19) of SC neurons showed clear pH_i recovery from IH-induced acidification. Of the 13/19 SC neurons that exhibited pH_i recovery in response to the IH-induced acidification, 10/13 SC neurons exhibited an alkaline overshoot in response to IH removal. In all cases, the height of a bar represents either the mean ΔpH_i or the mean pH_i recovery rate with the error bar being 1 SE. C: average firing rate change induced by either HA or IH. Both hypercapnic solutions caused a significant increase in firing rate (P <0.05 for HA and P < 0.001 for IH), but there was no significant difference in the change of firing rate induced by IH compared with HA. The height of a bar represents the mean firing rate, and the error bar is 1 SE.