Chemosensitivity of rat medullary raphe neurones in primary tissue culture

Abstract

1. The medullary raphe, within the ventromedial medulla (VMM), contains putative central respiratory chemoreceptors. To study the mechanisms of chemosensitivity in the raphe, rat VMM neurones were maintained in primary dissociated tissue culture, and studied using perforated patch-clamp recordings. Baseline electrophysiological properties were similar to raphe neurones in brain slices and in vivo. 2. Neurones were exposed to changes in CO2 from 5% to 3 or 9% while maintaining a constant [NaHCO3]. Fifty-one per cent of neurones (n = 210) did not change their firing rate by more than 20% in response to hypercapnic acidosis. However, 22% of neurones responded to 9% CO2 with an increase in firing rate ('stimulated'), and 27% of neurones responded with a decrease in firing rate ('inhibited'). 3. Chemosensitivity has often been considered an all-or-none property. Instead, a method was developed to quantify the degree of chemosensitivity. Stimulated neurones had a mean increase in firing rate to 298 +/- 215% of control when pH decreased from 7.40 to 7.19. Inhibited neurones had a mean increase in firing rate to 232 +/- 265% of control when pH increased from 7. 38 to 7.57. 4. Neurones were also exposed to isocapnic acidosis. All CO2-stimulated neurones tested (n = 15) were also stimulated by isocapnic acidosis, and all CO2-inhibited neurones tested (n = 19) were inhibited by isocapnic acidosis. Neurones with no response to hypercapnic acidosis also had no response to isocapnic acidosis (n = 12). Thus, the effects of CO2 on these neurones were mediated in part via changes in pH. 5. In stimulated neurones, acidosis induced a small increase in the after-hyperpolarization level of 1.38 +/- 1. 15 mV per -0.2 pH units, which was dependent on the level of tonic depolarizing current injection. In voltage clamp mode at a holding potential near resting potential, there were small and inconsistent changes in whole-cell conductance and holding current in both stimulated and inhibited neurones. These results suggest that pH modulates a conductance in stimulated neurones that is activated during repetitive firing, with a reversal potential close to resting potential. 6. The two subtypes of chemosensitive VMM neurones could be distinguished by characteristics other than their response to acidosis. Stimulated neurones had a large multipolar soma, whereas inhibited neurones had a small fusiform soma. Stimulated neurones were more likely than inhibited neurones to fire with the highly regular pattern typical of serotonergic raphe neurones in vivo. 7. Within the medullary raphe, chemosensitivity is a specialization of two distinct neuronal phenotypes. The response of these neurones to physiologically relevant changes in pH is of the magnitude that suggests that this chemosensitivity plays a functional role. Elucidating their mechanisms in vitro may help to define the cellular mechanisms of central chemoreception in vivo.

Figure 1. Microdissection of the medullary raphe and surrounding ventromedial medulla (VMM) for preparation of cell culture

A, ventral view of the cat medulla showing borders of cuts made on the VMM. The locations of the major landmarks of the neonatal rat are similar. V-XII, cranial nerves. C1, 1st cervical root. Adapted with permission from Feldman (1986). B, transverse section of the rat medulla showing wedge of tissue removed for cell culture. NA, nucleus ambiguus. NTS, nucleus tractus solitarius. LPG, nucleus lateralis paragigantocellularis. VII, 7th nerve nucleus.

Figure 3. Hypercapnic acidosis stimulated some neurones cultured from the VMM

A, example of a ‘stimulated’ chemosensitive neurone. Shown is firing rate vs. time as hypocapnic alkalosis and hypercapnic acidosis were induced by changing CO₂ from 5% to 3% and from 5% to 9%. B, example of a second stimulated neurone. Shown are firing rate (top) and pH (bottom). Acid/base changes were induced as in A. The times of transition between CO₂ levels are shown (vertical dashed lines). C, example of a third stimulated neurone. Shown are firing rate (top) and pH (bottom). The numbers above the firing rate plot correspond to the intervals for calculations of mean firing rate and pH in D and E. D, steady state firing rate vs. pH for the neurone shown in C. Numbers correspond to the intervals at different CO₂ levels. Error bars are ±s.d.E, firing rate (percentage of control) vs. pH for each transition between CO₂ levels (same neurone as in C and D). The firing rates at the test CO₂ levels were normalized to the firing rate for the adjacent intervals at 5% CO₂. For this neurone, a change in pH from a mean of 7.39 to a mean of 7.23 resulted in a mean increase in firing rate to 285% of control. Note the logarithmic vertical scale, discontinuous at 85%.

Figure 4. Summary of chemosensitivity of neurones stimulated by hypercapnic acidosis

A, mean and median firing rate (percentage of control) are plotted against pH for 25 stimulated neurones exposed to at least six transitions in CO₂. B, histogram of responses to hypercapnic acidosis of the same stimulated neurones as in A. Responses were normalized to a change in pH of −0.2 units. Most neurones increased their firing rate to more than 200% of control when CO₂ was increased from 5% to 9%. C, histogram of responses to hypocapnic alkalosis of the same stimulated neurones as in A. Responses were normalized to a change in pH of +0.2 units. Most neurones decreased their firing rate to below 50% of control when CO₂ was decreased from 5% to 3%.

Figure 6. Summary of chemosensitivity of neurones inhibited by hypercapnic acidosis

A, mean and median firing rate (percentage of control) are plotted against pH for 19 inhibited neurones exposed to at least six transitions in CO₂. B, histogram of responses to hypercapnic acidosis of the same inhibited neurones as in A. Responses were normalized to a change in pH of −0.2 units. Most neurones decreased their firing rate to less than 60% of control when CO₂ was increased from 5% to 9%. C, histogram of responses to hypocapnic alkalosis of the same inhibited neurones as in A. Responses were normalized to a change in pH of +0.2 units. Most neurones had a smaller response to alkalosis than to acidosis. In most cases firing rate did not increase above 200% of control when CO₂ was decreased from 5% to 3%.

Figure 2. Baseline electrophysiological properties of cultured VMM neurones

A, membrane potential of a neurone that fired spontaneously with a highly regular firing pattern. For this neurone, the s.d. of the relative ISI was 0.16, the CV of ISI was 0.14, and the mean firing rate was 1.65 Hz. B, membrane potential of a different neurone that fired more irregularly, but with a prominent AHP. For short periods of time, this neurone could fire more regularly, but without the same high degree of regularity as the neurone in A. For this neurone, the s.d. of the relative ISI was 1.90, the CV of ISI was 1.89, and the mean firing rate was 0.24 Hz. C, firing rate vs. time for a cultured VMM neurone. Blockade of ionotropic glutamate and GABA receptors led to an increase in firing rate.

Figure 5. Hypercapnic acidosis inhibited some neurones cultured from the VMM

A, example of an ‘inhibited’ neurone. Shown is firing rate vs. time as acid/base changes were induced by changing CO₂ from 5% to 9% and from 5% to 3%. B, example of a second inhibited neurone. Note that this neurone did not fire until CO₂ dropped below the baseline of 5%. This was not the case for most neurones, since tonic current injection was used in most neurones that were not spontaneously active to induce firing near 1 Hz at 5% CO₂.

Figure 7. Summary of chemosensitivity in VMM neurones

Plotted is a histogram of the chemosensitivity index (CI) for 134 cultured VMM neurones defined as stimulated, inhibited, or unresponsive to respiratory acidosis. The CI is a calculated value that normalizes the response of a neurone to hypercapnic acidosis of −0.2 pH units. A large percentage of these neurones increased or decreased their firing rate by ±50% of control in response to hypercapnic acidosis. Note log scale.

Figure 8. Chemosensitive neurones also responded to isocapnic acid/base changes

A, stimulated neurone. Changes in firing rate were induced by a decrease in CO₂ from 5% to 3% and by an increase in CO₂ from 5% to 9%. When this neurone was subsequently exposed to isocapnic acidosis, there was also an increase in firing rate. Letter C on the abscissa is the control period at 5% CO₂ and pH 7.4. B, inhibited neurone. This neurone was inhibited by both hypercapnic acidosis and by isocapnic acidosis.

Figure 9. Effect of acid/base changes on membrane potential of stimulated and inhibited neurones

A, membrane potential of a stimulated neurone during exposure to 5% CO₂. Note the regular firing pattern (s.d. of relative ISI was 0.33 for entire recording) and the prominent AHP. B, membrane potential of the same neurone as in A during exposure to 9% CO₂. The firing rate increased and the firing pattern became even more regular. C, membrane potential of an inhibited neurone at 3% CO₂. Note that the firing pattern is more irregular (s.d. of relative ISI was 1.38) than the neurone in A and B. D, membrane potential of the same neurone as in C after CO₂ was increased to 9%. Note that firing rate decreased.

Figure 10. Stimulated neurones were more likely to fire with a high degree of regularity than inhibited neurones

A, histogram of relative ISI for a stimulated neurone. The distribution of relative ISI was narrow, with a peak at 1.0, indicating a high degree of regularity. The CV of ISI for this neurone was 0.09, and the s.d. of relative ISI was 0.33. B, histogram of relative ISI for an inhibited neurone. The distribution of relative ISI was broad, indicating a large degree of irregularity. The CV of ISI for this neurone was 0.78, and the s.d. of the relative ISI was 1.50. C, s.d. of relative ISI vs. firing rate for 79 stimulated (▪) and inhibited (○) neurones. Stimulated neurones were more likely to have a low s.d. of relative ISI than inhibited neurones (P < 0.0004). Note double log scale. When the CV of ISI was plotted against firing rate, the distribution was similar, and the difference between the two groups was also statistically significant (P < 0.0004).

Figure 11. Acid/base disturbances induced small and inconsistent changes in resting whole-cell conductance

A, stimulated neurone with a CI of 212%. Acidosis induced a decrease in the outward holding current. Recording was made in voltage clamp at a holding potential of −50 mV (resting potential, −57 mV). There was a consistent decrease in outward holding current with hypercapnic acidosis, although the maximum change was only 10 pA. B, whole-cell conductance of the same neurone as in A, measured from a holding potential of −50 mV, with voltage steps to −60 mV. Recording was simultaneous with that in A. Hypercapnic acidosis induced a small decrease in whole-cell conductance to an average of 92% of control. C, inhibited neurone. Alkalosis had no effect on holding current in this inhibited neurone (same neurone as in Fig. 5B). Holding potential, −60 mV (resting potential, −50 mV). D, resting whole-cell conductance of the same neurone as in C, measured from −60 to −70 mV. Recording was simultaneous with that in C. There was no change in whole-cell conductance with hypocapnic alkalosis.

Figure 12. Morphology of stimulated neurones was different from that of inhibited neurones

A, ‘video lucida’ drawings of the first 10 well-visualized stimulated neurones. Note that all stimulated neurones had medium to large sized somata. Most had irregular cell bodies, often with three major poles. In all cases, there were at least three major processes coming off the soma. Subsequent recordings have shown these features to be highly consistent. B, ‘video lucida’ drawings of the first 13 well-visualized inhibited neurones. Note that all inhibited neurones had small to medium sized somata, and all but one had fusiform cell bodies. In most cases there were two major processes, one coming off each pole of the soma. Subsequent recordings have shown these features to also be highly consistent.