Alfredo J. Garcia

Development of in vivo ventilatory and single chemosensitive neuron responses to hypercapnia in rats.

We used pressure plethysmography to study breathing patterns of neonatal and adult rats acutely exposed to elevated levels of CO2. Ventilation (VE) increased progressively with increasing inspired CO2. The rise in VE was associated with an increase in tidal volume, but not respiratory rate. In all animals studied, the CO2 sensitivity (determined from the slope of the VE vs. inspired % CO2 curve) was variable on a day to day basis. Chemosensitivity was high in neonates 1 day after birth (P1) and fell throughout the first week to a minimum at about P8. Chemosensitivity rose again to somewhat higher values in P10 through adult rats. The developmental pattern of these in vivo ventilatory responses was different than individual locus coeruleus (LC) neuron responses to increased CO2. The membrane potential (V(m)) of LC neurons was measured using perforated patch (amphotericin B) techniques in brain slices. At all ages studied, LC neurons increased their firing rate by approximately 44% in response to hypercapnic acidosis (10% CO2, pH 7.0). Thus the in vivo ventilatory response to hypercapnia was not correlated with the V(m) response of individual LC neurons to hypercapnic acidosis in neonatal rats. These data suggest that CO2 sensitivity of ventilation in rats may exist in two forms, a high-sensitivity neonatal (or fetal) form and a lower-sensitivity adult form, with a critical window of very low sensitivity during the period of transition between the two (approximately P8).

Developmental changes of the response of in vivo ventilation to acute and chronic hypercapnia in rats

As part of our studies of the role of changes of intracellular pH (pHi) in central chemosensitive neurons, we began studying the in vivo ventilatory response to increased inspired CO2. Our hope was to identify distinct sub-populations of rats with widely different CO2 sensitivity of in vivo ventilation so that we could study the cellular responses to hypercapnia (pHi changes and increased chemosensitive neuron firing rate) and look for a correlation with the in vivo ventilatory patterns. Ventilation in neonatal Sprague-Dawley rats (P1-P21) was studied using a dual chamber Respiromax Plethysmograph. Weight specific minute ventilation (VE) was measured as a function of inspired CO2 over the range of 0-5%. VE increased linearly with increasing CO2 due entirely to increased tidal volume (no change in respiratory frequency) [1]. The VE response to increased CO2 varied among individuals and from day to day in a given individual. The CO2 sensitivity of ventilation (determined as the slope of the VE vs.inspired % CO2 curve) showed a biphasic relationship with age. CO2 sensitivity was high in newborn rats (P1) and decreased to a minimum value at P7-P8. It increased again to reach a level at P14 that was very similar to the CO2 sensitivity in adults [1]. The basis for this developmental pattern is unknown but it is not due to changes in the CO2 responsiveness of chemosensitive neurons from the locus coeruleus, since these neurons show a constant increased firing rate of about 44% in response to hypercapnia (10% CO2). In an attempt to vary CO2 sensitivity, we reared neonatal rats in a chronic hypercapnic (CH) environment [2] and studied the effect of this treatment on VE and on CO2 sensitivity of VE. We reared time pregnant mothers in an environment of constant CH (7% CO2) for 1week prior to the birth of the pups and maintained the mother and pups in the CH environment until the neonates were tested (at least 6additional days). These neonates exhibited retarded growth (smaller by 1-2g) for the first 2weeks of life but attained the same weight as control rats (reared in room air) by P16. These CH rats exhibited higher CO2 sensitivity than control rats at days P6-P9 and then showed lower values that were indistinguishable from control rats from P10->P19. Other litters were exposed to severe CH (10%) using the same protocol as for the 7% CH rats. Litters were culled in these severe CH rats (1/3 of the litter sacrificed at birth), but even so these rats showed marked growth retardation that got worse with increasing age. These rats exhibited an even higher CO2 sensitivity than control or 7% CH rats at P6-P9 and then showed values that were similar to control rats at P10->P19. VE increased with increasing exposure to CH from (mean ± SE; n = 14) 892 ± 100 (control) to 960 ± 109 (7% CH) to 1127 ± 108 (10% CH)ml-min-1-kg-1. We suggest that CH results in a slowing of development so that CO2 sensitivity of VE remains elevated longer after birth. The biphasic developmental pattern suggests that after birth rats display a neonatal pattern of chemosensitivity that decreases during the first week of life and is replaced after the second week by a more adult form of chemosensitivity. A critical period of low in seen between these two periods. The CO2 sensitivity of VE mechanistic basis for this biphasic pattern is unknown as is the effect of CH on the properties of central chemosensitive neurons and both of these questions should be fruitful areas of future work.