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.

High CO2/H+ dialysis in the caudal ventrolateral medulla (Loeschcke's area) increases ventilation in wakefulness

“…In the responsive group, six of 8 animals had the probe close to the ventrolateral surface of medulla, while two animals had the probes located more dorsally. Figure 1B also schematically shows (highlighted areas in the left side) an approximate distribution of H + -sensitive neurons described by Ribas-Salgueiro et al (2003). In the responsive group, five of 8 animals had the probe within the H + - sensitive neurons containing-region while the other 3 were just adjacent to this area.…”

“…We decided to group the animals using their response to CO 2 dialysis as criteria rather than the anatomical localization of dialysis probes, because the anatomical boundaries in this caudal region proposed to be involved in central chemoreception are not well established. In the study by Ribas-Salgueiro et al, (2003), in which they described the presence of neurons sensitive to H + stimulus in the caudal region, they found low H + -sensitive neurons loosely distributed in the caudal region studied, while high H + -sensitive neurons were located close to the ventral surface of the medulla. The description of anatomy and the methods that they used to measure rostro-caudal level were imprecise.…”

“…2) Douglas et al (2001) showed the activation of neurons in this caudal region by mapping the distribution of c-fos expression after acid-base stimulation. 3) Ribas-Salgueiro et al (2003) demonstrated, in anaesthetized rats the presence of neurons that fired spontaneously (not coupled to the respiratory cycle) and were responsive to acidic stimulus in the cVLM. This region contains 3 rd order (propriobulbar) neurons identified by retrograde tracing of retrovirus injected into the diaphragm (Dobbins and Feldman, 1994) and respiratory modulated GABAergic neurons involved in blood pressure regulation (Mandel and Schreihofer, 2006).…”

Central Chemoreceptors: Locations and Functions

“…10). Recordings from slice preparations with subsequent anatomical verification, from 5HT cells in culture, and from neurons transgenically labeled as serotonergic all show a specific increase in activity of 5HT neurons to increased CO 2 /H + and, in some neurons, a decrease in activity (31, 214–217). Medullary 5HT neurons exhibit an inherent sensitivity to CO 2 /H + .…”

“…The rostral cluster in the pons projects mainly to more rostral brain structures and is concerned with the functions of these neurons while the caudal cluster projects to spinal cord as well as to other brainstem sites and is more concerned with the functions of these regions including the regulation of breathing and central chemoreception (20, 31, 99–101, 215–217). In 1995, George Richerson (214), in a study using medullary slices in vitro to look for neurons responsive to acidic stimulation in the rostral ventral medulla, described CO 2 /H + responsive neurons at two locations, one possibly within the subsequently described RTN region, the other in the midline raphe. In anesthetized rats in vivo , focal acidification of the midline raphe by microinjection of acetazolamide increased respiratory output (15) indicating the presence of functionally significant chemoreception, a result substantiated shortly thereafter by focal acidification in conscious rats (182) and goats (97, 98) by reverse microdialysis of aCSF equilibrated with increased CO 2 .…”