Melvin D. Burton

RET proto-oncogene is important for the development of respiratory CO2 sensitivity

Brain stem muscarinic cholinergic pathways are important in respiratory carbon dioxide (CO2) chemosensitivity. Defects in the muscarinic system have been reported in children with congenital/developmental disorders of respiratory control such as sudden infant death syndrome (SIDS) and congenital central hypoventilation syndrome (CCHS). This early onset of disease suggests a possible genetic basis. The muscarinic system is part of the autonomic nervous system which develops from the neural crest. Ret proto-oncogene is important for this development. Thus, a potential role for ret in the development of respiratory CO2 chemosensitivity was considered. Using plethysmography, we assessed the ventilatory response to inhaled CO2 in the unanesthetized offsprings of ret +/- mice. Fractional increases in minute ventilation during hypercapnia relative to isocapnia were 5.1 +/- 3.2, 3.0 +/- 1.6 and 1.4 +/- 0.8 for the ret +/+, ret +/- and ret +/- mice, respectively. The ret knockout mice have a depressed ventilatory response to inhaled CO2. Therefore, the ret gene is an important factor in the pathway of neuronal development which allow respiratory CO2 chemosensitivity.

Neurotransmitters in central respiratory control

A diverse group of processes are involved in central control of ventilation. Both fast acting neurotransmitters and slower acting neuromodulators are involved in the central respiratory drive. This review deals with fast acting neurotransmitters that are essential centrally in the ventilatory response to H(+)/CO(2) and to acute hypoxia. Data are reviewed to show that the central response to H(+)/CO(2) is primarily at sites in the medulla, the most prominent being the ventral medullary surface (VMS), and that acetylcholine is the key neurotransmitter in this process. Genetic abnormalities in the cholinergic system lead to states of hypoventilation in man and that knock out mice for genes responsible for neural crest development have none or diminished CO(2) ventilatory response. In the acute ventilatory response to hypoxia the afferent impulses from the carotid body reach the nucleus tractus solitarius (NTS) releasing glutamate which stimulates ventilation. Glutamate release also occurs in the VMS. Hypoxia is also associated with release of GABA in the mid-brain and a biphasic change in concentration of another inhibitory amino acid, taurine. Collectively changes in these amino acids can account for the ventilatory output in response to acute hypoxia. Future studies should provide more data on molecular and genetic basis of central respiratory drive and the role of neurotransmitter in this essential function.

Acetylcholine and central respiratory control: perturbations of acetylcholine synthesis in the isolated brainstem of the neonatal rat

The brainstem neurochemical processes which support spontaneous ventilation are not known. Cholinergic transmission may play an important role. If this is true, perturbations in acetylcholine (ACh) turnover should alter ventilatory output in a predictable manner. Using the isolated superfused brainstem-spinal axis from the neonatal rat, the effects of modifiers of ACh synthesis on spontaneous C-4 (phrenic) output were determined. 3-Bromopyruvate and hydroxycitrate, inhibitors of acetyl-CoA (substrate for ACh synthesis) formation, caused depression of the C-4 output in a dose-dependent manner when added to the superfusate. Triethylcholine, a false-transmitter generating choline analog, caused a similar depression. Citrate, a cytosolic precursor to acetyl-CoA formation, caused stimulation of C-4 (phrenic) output. The stimulatory effects of citrate were blocked by the muscarinic cholinergic blocker, atropine. These findings are consistent with the view that the ACh synthetic pathway provides a continuous and important input to the normal brainstem elements that support ventilation.

The central respiratory effects of acetylcholine vary with CSF pH.

Hydrogen ion concentration [H+] centrally is a major determinant of ventilation. Its action involves central cholinergic mechanisms. The point(s) where increased [H+] induces its changes in the cholinergic system is unclear. If H+ acts presynaptically by increasing endogenous ACh synthesis and release, its effect should be absent when ACh is supplied exogenously. If H+ acts postsynaptically by changing ACh degradation or ACh receptor sensitivity, its effect should persist in the presence of exogenous ACh. We perfused the brain ventricular system in spontaneously breathing anesthetized dogs with progressively higher concentrations of ACh (0-52.8 mM) in cerebrospinal fluid (CSF) at pH 7.4 and CSF pH 7.1. Increasing concentrations of ACh increased ventilation > 4-fold in a linear manner in the presence of non-acidic and acidic CSF. With acidic CSF the ACh ventilatory response line was shifted to a higher y-intercept, resulting in a higher ventilation at any [ACh]. These findings are consistent with the hypothesis that central acidosis augments ventilation by postsynaptic cholinergic events.

Correlation in stimulated respiratory neural noise

Noise in spontaneous respiratory neural activity of the neonatal rat isolated brainstem-spinal cord preparation stimulated with acetylcholine (ACh) exhibits positive correlation. Neural activity from the C4 (phrenic) ventral spinal rootlet, integrated and corrected for slowly changing trend, is interpreted as a fractal record in time by rescaled range, relative dispersional, and power spectral analyses. The Hurst exponent H measured from time series of 64 consecutive signal levels recorded at 2 s intervals during perfusion of the preparation with artificial cerebrospinal fluid containing ACh at concentrations 62.5 to 1000 &mgr;M increases to a maximum of 0.875+/-0.087 (SD) at 250 &mgr;M ACh and decreases with higher ACh concentration. Corrections for bias in measurement of H were made using two different kinds of simulated fractional Gaussian noise. Within limits of experimental procedure and short data series, we conclude that in the presence of added ACh of concentration 250 to 500 &mgr;M, noise which occurs in spontaneous respiratory-related neural activity in the isolated brainstem-spinal cord preparation observed at uniform time intervals exhibits positive correlation. (c) 1995 American Institute of Physics.

Potential role for imidazole in the rhythmic respiratory activity of the in vitro neonatal rat brainstem

We used the imidazole-binding agent, diethylpyrocarbonate (DEPC), to test the hypothesis that rhythmic respiratory activity of the in vitro neonatal rat brainstem-spinal cord preparation was functionally dependent on imidazole. Neural activity was recorded from spinal nerves (C1-C4) during superfusion with 95%O2/5%CO2 buffer at pH 7.3 and T = 26 degrees C. Superfusate containing DEPC (40 mM) caused cessation of rhythmic activity within minutes. In eight of 33 preparations, microinjection of DEPC (32 nmol) onto the ventral medullary surface (VMS) reduced burst amplitude by at least 50% within 10 min, and in 12 of 33 preparations, microinjection of DEPC produced neural apnea. Therefore, we conclude that proteins containing imidazole near the VMS are critically important for the maintenance of rhythmic respiratory activity in vitro. Furthermore, alphastat regulation of respiration may be an essential trait of this preparation.

Fractal Noise in Breathing

Our understanding of respiration derives from applications of a variety of physical and life science disciplines, methods, and models to a critical physiological process: exchange and balance of oxygen and carbon dioxide. We know that breathing at rest arises from a diversity of interrelated and interactive physical and chemical mechanisms involving molecular and cellular processes in the brainstem which include-among other phenomena common to the central nervous system-metabolism, synaptic transmission of neurochemicals, neurochemical-mediated alteration of neural cell membrane potential, transmembrane ion conductance, neural electrical signal propagation, and neuromodulation by afferent chemoreceptive and mechanoreceptive inputs.

Viability of the neonatal rat isolated brainstem preparation by 31P MRS

The isolated brainstem-spinal axis from the neonatal rat is an established model for studying neuronal responses of the ventilatory control system, however, its viability has not been clearly established. We studied the brainstem-spinal axis from newborn rats at 8.5 T with 31P NMR spectroscopy. The relative pattern of high energy phosphates (HEPs) was similar to that reported for the in vivo neonatal brain. The average pHi was 0.2 to 0.4 units less than the pHi for the in vivo neonatal brain. The HEPs and pHi were stable for 6 h, suggesting extended in vitro viability.

Role of Acetylcholine as an Essential Neurotransmitter in Central Respiratory Drive

John Scott Haldane in the 1930’s suggested that the profound effect of CO2 on the level of ventilation was related to its ability to change brain hydrogen ion concentration, and that the increase in ventilation seen with a rise in PCO2 was because of a fall in brain pH. Pappenheimer and associates (12) in the 1960’s showed that in the unanesthetized goat brain interstitial fluid pH determined the level of ventilation in chronic acid-base disorders. Site of action of H+ in the brain has been shown to be in at least three superficial areas on the ventral surface of the medulla. How hydrogen ions act at these “chemosensitive” sites is not clear(10). The possibilities are: 1- hydrogen ion gradient across the neuronal membrane; 2- H+ changes affect ions or ionic channels, and 3- H+ causes the release or activation of neurotransmsitters. Our studies have concentrated on neurotransmitter release, particularly acetylcholine.