Joseph S. Erlichman

Central chemoreceptor stimulus in the terrestrial, pulmonate snail, Helix aspersa

We studied the effect of hypercapnic and fixed acid central chemoreceptor stimulation on the pneumostome in the pulmonate snail, Helix aspersa. We found that focal stimulation of the central chemoreceptor area of the pulmonate snail brain with hypercapnic solutions more effectively increased the pneumostomal area than did fixed acid stimulation at the same extracellular pH. Disrupting intracellular pH regulation by inhibiting Cl- transport, either pharmacologically (DIDS) or by ion substitution (Cl(-)-free perfusate), enhanced pneumostomal responses to CO2. While maintaining a constant perfusate pH, addition of NH4Cl to the perfusate resulted in pneumostomal closure; whereas removal of NH4Cl from the bath resulted in pneumostomal opening. In conclusion, the ventilatory response to CO2 in H. aspersa does not require Cl- transport or conductance. Furthermore, changing pHi alone is an adequate stimulus for the central chemoreceptors in the snail.

CO2 chemoreception in the pulmonate snail, Helix aspersa

We have studied the response of the pneumostome to CO2, O2 and combined CO2 and O2 in intact snails. We found that pneumostomal opening increases in response to both hypercapnia and mild hypoxia. We determined which neural structures were essential for the pneumostomal response to CO2 by eliminating parts of the nervous system: the subesophageal ganglia and an intact anal nerve were necessary and sufficient elements for the CO2 response. Within the subesophageal ganglia, we identified a discrete region on the medial margin of the visceral ganglion that was capable of increasing pneumostomal area when focally stimulated with 6% CO2. Ion substitution experiments indicated that pneumostomal responses to hypercapnia were not mediated by the pneumostomal motor neurons themselves, but rather by interneurons connected polysynaptically to the motor neurons controlling pneumostomal function. In conclusion, intact H. aspersa have a ventilatory response to CO2, and this response is mediated by CO2 sensitive cells located in a small area of the central nervous system.

Comparative aspects of central CO2 chemoreception

We compare and contrast the putative mechanisms underlying CO2 chemoreceptor function in air breathing vertebrates and terrestrial pulmonate snails. We discuss the role of intracellular pH (pHi) in central respiratory responses to CO2 and describe a variety of patterns of pHi regulation in chemosensory areas. One pattern, in which pHi retains a fixed relationship to the CO2 stimulus over time, seems well suited to chemoreceptor cells. Alphastat regulation of ventilation is apparent in both air breathing vertebrates and terrestrial pulmonate snails. Diethyl pyrocarbonate inhibits respiratory responses to hypercapnia in both groups of animals. The neuronal basis of chemosensitivity is similar, in that putative chemoreceptor cells depolarize during hypercapnic stimulation, but the ionic basis of excitability appears to be a potassium conductance in the vertebrates studied to date and a calcium conductance in the snails. Despite divergent evolutionary histories, chemosensory responses and mechanisms are remarkably similar in air breathing vertebrates and terrestrial pulmonate snails.

Carbonic anhydrase and CO2 chemoreception in the pulmonate snail Helix aspersa

We have studied the effects of carbonic anhydrase inhibition on the hypercapnic ventilatory response of the pulmonate snail, Helix aspersa, in an isolated brain-pneumostome preparation. We found that the cell permeant carbonic anhydrase inhibitor, acetazolamide (ACTZ), increased pneumostomal opening and ventilation during normocapnia (2-3% CO2) and decreased the rate of pneumostomal response to step changes in CO2 (4.5%), but did not change the steady-state ventilatory response to elevated CO2 (4.5%) compared to the inactive ACTZ analogue, N2-substituted 2-acetylamino-1,3,4-thiadiazole (Cl 13850). In contrast, the cell impermeant carbonic anhydrase inhibitor, quartenary ammonium sulfonilamide (QAS), had no effect on the pneumostomal response to CO2 compared to Cl 13850. Using Hanssons histochemical technique to stain for carbonic anhydrase activity, we identified a small number of neurons in the subesophageal ganglia that exhibited carbonic anhydrase activity. Some of these cells were in the region of CO2-sensitivity. In conclusion, carbonic anhydrase inhibition slows the ventilatory response to rapid changes in CO2, but does not affect the intrinsic ability of H. aspersa to respond to CO2. The ventilatory effects of carbonic anhydrase inhibition may be attributed to the intracellular actions of the carbonic anhydrase enzyme.

Diethyl pyrocarbonate (DEPC) inhibits CO2 chemosensitivity in Helix aspersa.

Central CO2 chemoreceptors in poikilothermic vertebrates may not regulate ventilation at a particular pH setpoint; central chemoreceptor responses may more accurately reflect the relative charge state (alpha) of the imidazole of histidine. We have tested the alphastat hypothesis in the terrestrial, air breathing, pulmonate snail, Helix aspersa, by chemically modifying histidine residues in the central CO2 chemoreceptor area of this animal using diethyl pyrocarbonate (DEPC). After focal application of 20 mM DEPC to the central CO2 chemoreceptor region, the pneumostome, a respiratory, CO2 responsive organ in the snail, no longer responded to hypercapnic, acidotic stimulation of the central chemoreceptor area. However, pneumostomal responses to hypoxic stimulation of the pneumostome and to focal stimulation of the central chemoreceptor area with sodium nitroprusside, a respiratory stimulant in H. aspersa, remained intact after DEPC treatment. Furthermore, DEPC treatment of the central chemoreceptor area blocked pneumostomal responses to ammonia pre-pulse treatment, which changes intracellular pH, while extracellular pH is held constant. These results resemble mammalian responses to DEPC treatment and indicate that central chemoreceptor responses in H. aspersa may originate from changes in the alpha of intracellular histidine residues.