Noradrenergic tuning, not simple rate effects, produces temperature‐sensitivity of the respiratory network in bullfrogs

Abstract

Noradrenergic tuning, not simple rate effects, produces temperature-sensitivity of the respiratory network in bullfrogs Animals match ventilation to metabolic and acid-base regulatory demands during changes in body temperature. Across vertebrates ventilation increases at warmer temperatures and decreases at cooler temperatures. At the organismal level this is attributed to metabolic feedback and/or alpha-stat pH regulation; however, the cellular mechanisms that produce ventilatory output in response to brain temperature changes remain unclear. To identify mechanisms underlying temperature-sensitivity of the respiratory control network, I used brainstem-spinal cord preparations producing spontaneously active, rhythmic motor output similar to breathing in vivo of adult bullfrogs Lithobates catesbeianus. In vitro brainstem-spinal cords were superfused with artificial cerebrospinal fluid (aCSF) equilibrated at 90% O 2 , 1.3% CO 2 , and balance N 2 .Whole nerve recordings from the trigeminal (V) and vagus (X) nerves were used for measuring respiratory-related activity. I applied temperature ramps from 20ºC to 15ºC and then to 25ºC; each step lasted 15 minutes. Bursting frequency was analyzed for the last 5 minutes of each step and then normalized to percent of baseline (20ºC). Consistent with in vivo and in vitro data (Bícego-Nahas and Branco, 1999;Morales and Hedrick, 2002), I demonstrate that the frequency of respiratory-related nerve activity is stable iv across high temperatures, but not lower temperatures (One-way ANOVA p<0.0002; burst frequency different at 15ºC compared to 20ºC and compared to 25ºC, but no difference between 20ºC and 25ºC; Tukey's Multiple Comparison post hoc test). The locus coeruleus (LC) is a nucleus of the respiratory network and is the main supplier of norepinephrine in the brain. LC neurons from bullfrogs are paradoxically activated by decreases in temperature suggesting that firing frequencies inversely proportional to temperature may play a role in setting the respiratory frequency across temperatures. To identify the role of norepinephrine in generating the respiratory frequency, I applied the temperature protocol while blocking the main adrenergic receptors (AR). For blocking α 1 AR, preparations were superfused with aCSF containing prazosin and for blocking α 2 AR I used RX821002. I found that bursting stability at high temperatures is disrupted when α 1 AR are blocked (One-way ANOVA p<0.0001; burst frequency significantly lower at 15ºC compared to 20ºC and 25ºC, and significantly lower at 20ºC compared to 25ºC; Tukey's Multiple Comparison post hoc test). In contrast, stable pattern across warm temperatures was maintained while the inhibition of bursting at 15°C was lost when α 2 AR were blocked (One-way ANOVA p=0.025; Tukey's Multiple Comparison post hoc test showed no difference in burst frequency between 15ºC, 20ºC and 25ºC). These results imply that norepinephrine tuning through different receptors, rather than simple Q 10 effects, plays a major role in generating the breathing ...