Highly H+-sensitive neurons in the caudal ventrolateral medulla of the rat

Abstract

The ventral surface of the caudal ventrolateral medulla (cVLM) has been shown to generate intense respiratory responses after surface acid-base stimulation. With respect to their chemosensitive characteristics, cVLM neurons have been less studied than other rostral-most regions of the brainstem. The purpose of these experiments was to determine the bioelectric responses of cVLM neurons to acidic stimuli and to determine their chemosensitive properties. Using extracellular and microiontophoretic techniques, we recorded electrical activities from 117 neurons in an area close to the ventral surface of the cVLM in anaesthetised rats. All neurons were tested for their sensitivity to H+. The fluorescent probe BCECF was used to measure extracellular pH changes produced by the microiontophoretic injection of H+ in brainstem slices. This procedure provided an estimation of the local changes in pH produced by microiontophoretic H+ application in the anaesthetised rat. Neurons coupled to the respiratory cycle, R (n = 51), were not responsive to direct stimulation with H+. Sixty-six neurons that did respond to H+ stimulation were uncoupled from respiration, and identified as NR neurons. These neurons presented distinct ranges of H+ sensitivity. The neuronal sensitivity to H+ was mainly assessed by the slope of the stimulus-response curve, where the steeper the slope, the higher the H+ sensitivity. On this basis, NR neurons were classed as being either weakly or highly sensitive to H+. NR neurons with a high H+ sensitivity (n = 12) showed an average value of 34.17 +/- 7.44 spikes s-1 (100 nC)-1 (mean +/- S.D.) for maximal slope and an EC50 of 126.76 +/- 33 nC. Suprathreshold H+ stimulation of highly sensitive NR neurons elicited bursting pattern responses coupled to the respiratory cycle. The bursting responses, which were synchronised with the inspiratory phase and the early expiratory phase of the respiratory cycle, lasted for several seconds before returning to the steady state firing pattern characteristic of the pre-stimulus condition. These NR neurons, which possess the capacity to detect distinct H+ concentrations in the extracellular microenvironment, are excellent candidates to serve in a chemoreceptor capacity in the caudal medulla.

Figure 1. Calibration of the acidification procedure in the extracellular space of nervous tissue

A, calibration curve for BCECF under in vitro conditions. Ratios of the fluorescence emitted by BCECF at 490 nm and 450 nm excitation wavelengths are plotted against distinct pH buffered solutions. K_d: dissociation constant. B, BCECF calibration in brainstem slices. The relationship between the charge of the stimuli (top x axis) and the change in the tissue pH is represented by filled squares and its regression by the continuous line. The relationship between time (bottom x axis) and pH is represented by filled circles and its regression by the dashed line. Inset shows three images of a brainstem slice and the size effects of three different H⁺ stimulations. White arrows show the areas where pH decreased. Calibration bars for images: 100 μm. Vertical calibration bar: grey scale for changes in fluorescence induced by H⁺ stimulation.

Figure 2. Respiratory-coupled neuron (R) unresponsive to H⁺ stimulation

R neuron activity and respiratory cycles with increasing H⁺ stimulations. A, upper panel, extracellular recording of a cVLM neuron with respiratory-related activity; middle panel, average firing frequency; and lower panel, respiratory cycle recording from an anaesthetised rat. B and C, upper panels, average firing frequency; lower panels, respiratory cycle recording from an anaesthetised rat. Application time of stimulus (for 100, 350, and 600 nC total charge) is depicted by the bar. There was no increase in firing rate after stimulation. Calibration bar shows time base. The arrow indicates expiration phase.

Figure 3. Response of a low H⁺-sensitive non-respiratory (NR) cVLM neuron to H⁺ stimulation

A, NR neuron activity and respiratory cycles with increasing H⁺ stimulations. Upper panel, respiratory cycle recording from an anaesthetised rat, middle panel, extracellular recording of a cVLM neuron, lower panel, average firing frequency. The arrow indicates expiration phase. Application time of stimulus (108, 150 and 224 nC) is depicted by the bar. Calibration bar shows time base. These neurons showed low-level responsiveness to proton stimulation.

Figure 5. Relationship between focal pH change and maximal firing rate responses of NR neurons in cVLM

A, estimated pH and proton charge during stimulation are plotted against the frequency of action potentials displayed by NR neurons. Continuous traces, dose-response curves of low H⁺-sensitive neurons. Dashed traces, dose-response curves of highly H⁺-sensitive neuron. B and C, statistical differences in slopes and EC₅₀ of both groups, NR neurons of low and high H⁺sensitivity (**P < 0.001).

Figure 4. Response of a highly H⁺-sensitive cVLM neuron to acid stimulation

A, lack of response of a highly H⁺-sensitive NR neuron to non-specific cationic (Na⁺) stimulation. Upper panel, extracellular recording of a cVLM neuron; lower panel, average firing frequency. B, selective response to H⁺ stimulation of a NR neuron. Upper panel, extracellular recording of a cVLM neuron; lower panel, average firing frequency. C and D, average firing frequency with increasing H⁺ stimulations. E, respiratory cycle recording from the anaesthetised rat. The arrow indicates expiration phase. Application time of stimulus (for 56, 60 and 84 nC total charge) is depicted by the bar. Note that the duration of the response was proportional to the intensity and duration of the stimulation. Calibration bar shows time base.

Figure 6. Bursting response of a highly H⁺-sensitive NR neuron of the cVLM to H⁺ stimulation

A, suprathreshold response to a local injection of 290 nC of H⁺ (bar). Note that the neuron started to increase its firing rate during the second half of the stimulation period and rapidly passed to bursting activity which was synchronous with the respiratory cycle. Upper panel, respiratory recording; middle panel, extracellular recording of the cVLM neuron; lower panel, frequency histograms of action potential firing (bin: 100 ms). The bursts of neuronal activity were synchronised with the respiratory cycles and lasted more than 12 s. The frequency histogram in the lower panel shows the intraburst frequency. B, the pre-stimulus condition; C, a parcel of the bursting response. The data in C correspond to the square marked as C in A. The arrow indicates expiration phase. Note that bursts were synchronised with the inspiratory phase and the early expiratory phase of the respiratory cycle. Calibration bar shows time base.

Figure 7. Disciminant analysis of resting activity of NR neurons in cVLM

Comparison of the rate and regularity of the discharge pattern from low H⁺ sensitivity neurons (open circles) and the high H⁺ sensitivity neurons (filled symbols: circles indicate bursting neurons and squares indicate non-bursting neurons) in cVLM. Logarithmic plot of the mean vs.s.d. of NR neurons. The continuous line represents the discriminant function separating the low and the high H⁺ sensitivity groups with an error < 16 %. Notice the lower rate and the higher regularity of the high H⁺ sensitivity neuron discharges. The dashed line represents the discriminant function proposed by Mason (1997) for serotonergic neurons.

Figure 8. Illustrative diagrams of coronal sections of the caudal ventrolateral medulla of the rat showing the location of the tested neurons

High H⁺-sensitive neurons (black stars) were located near the surface of the medulla. Low H⁺-sensitive neurons (black circles) were widely distributed in the caudal ventrolateral medulla. Respiratory-related neurons (open circles) were located within the ventral respiratory group of the caudal medulla. Abbreviations: 12, hypoglossal nucleus; ECu, external cuneate nucleus; py, pyramidal tract; Sol, nucleus of the solitary tract; Sp5C, spinal trigeminal nucleus, caudal part; Sp5I, spinal trigeminal nucleus, interpolar part. Coordinates: millimetres caudal to the interaural line.