Inhibition of protein kinase A activity depresses phrenic drive and glycinergic signalling, but not rhythmogenesis in anaesthetized rat.

The cAMP-protein kinase A (PKA) pathway plays a critical role in regulating neuronal activity. Yet, how PKA signalling shapes the population activity of neurons that regulate respiratory rhythm and motor patterns in vivo is poorly defined. We determined the respiratory effects of focally inhibiting endogenous PKA activity in defined classes of respiratory neurons in the ventrolateral medulla and spinal cord by microinjection of the membrane-permeable PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) in urethane-anaesthetized adult Sprague Dawley rats. Phrenic nerve activity, end-tidal CO2 and arterial pressure were recorded. Rp-cAMPS in the preBötzinger complex (preBötC) caused powerful, dose-dependent depression of phrenic burst amplitude and inspiratory period. Rp-cAMPS powerfully depressed burst amplitude in the phrenic premotor nucleus, but had no effect at the phrenic motor nucleus, suggesting a lack of persistent PKA activity here. Surprisingly, inhibition of PKA activity in the preBötC increased phrenic burst frequency, whereas in the Bötzinger complex phrenic frequency decreased. Pretreating the preBötC with strychnine, but not bicuculline, blocked the Rp-cAMPS-evoked increase in frequency, but not the depression of phrenic burst amplitude. We conclude that endogenous PKA activity in excitatory inspiratory preBötzinger neurons and phrenic premotor neurons, but not motor neurons, regulates network inspiratory drive currents that underpin the intensity of phrenic nerve discharge. We show that inhibition of PKA activity reduces tonic glycinergic transmission that normally restrains the frequency of rhythmic respiratory activity. Finally, we suggest that the maintenance of the respiratory rhythm in vivo is not dependent on endogenous cAMP-PKA signalling.

Discharge rate profiles of paratrigeminal nucleus neurons throughout a pressor event in non-anaesthetized rats.

Located in the lower brainstem, the paratrigeminal nucleus (Pa5) is related to cardiorespiratory autonomic reflex functions. To characterize the structures' role in blood pressure regulation and the cardiovascular reflex responses Pa5 unit activity was evaluated during a phenylephrine-produced pressor response in non-anaesthetized rats by means of simultaneous many-unit recording. Ninety five percent of the identified Pa5 responded to baroreceptor stimulation, 77% increasing and 23% decreasing firing rates. Cross-correlation analysis of neuron electrical behavior referenced to the heart beat event revealed that 65% of the featured cardiac cycle-locked rhythmic activity. The identification of neurons that change firing rates in response to increases of arterial pressure with cardiac cycle-locked rhythmic activity, further supports for a role for the nucleus in moment to moment control of blood pressure. The largest changes in firing rate occurred in the units with low resting firing rates in response to the ascending phase of the pressor event. Thus, the group displaying both cardiac cycle-locked and other rhythmic activities within the ranges of cardiac and respiratory rates or arterial pressure low frequencies, is probably the most influential regarding homoeostatic reflex responses. The findings advance the notion that the dynamic control of blood pressure involves lower brainstem integration of cardiac and respiratory reflexes.

Cardiovascular and baroreceptor functions of the paratrigeminal nucleus for pressor effects in non-anaesthetized rats.

Located in the lower brainstem, the paratrigeminal nucleus (Pa5) is related to cardiorespiratory autonomic reflex functions. To characterize the structures' role in blood pressure regulation and baroreflex response, both resting cardiovascular parameters and reflex responses were evaluated during phenylephrine-produced pressor responses in non-anaesthetized rats with or without bilateral chemical Pa5 ablation. The Pa5-ablated animals, in contrast to the Pa5-intact control animals, presented increased resting arterial pressure (115+/-4 vs. 100+/-3 mm Hg), decreased heart (293+/-10 vs. 315+/-7 bpm) and increase of the respiratory (104+/-3 vs. 94+/-5 rpm) rates, larger pressor responses and reduced baroreflex index (1.6+/-0.2 vs. 2.8+/-0.2, p<0.05). The cardiovascular changes, compatible to those produced by nucleus of the solitary tract (NTS) lesions in non-anaesthetized rats, indicate a reduction of both the sympathetic and cardiac components of the baroreflex response. Further analyses showed the Pa5 mediates reflex responses to smaller blood pressure increases, while the NTS would be predominantly active in surges over 40 mm Hg. Thus, the integrity of the Pa5 is important for resting blood pressure maintenance as for a full baroreceptor response.

Expression and characterization of LTx2, a neurotoxin from Lasiodora sp. effecting on calcium channels.

Here, we described the expression and characterization of the recombinant toxin LTx2, which was previously isolated from the venomous cDNA library of a Brazilian spider, Lasiodora sp. (Mygalomorphae, Theraphosidae). The recombinant toxin found in the soluble and insoluble fractions was purified by reverse phase high-performance liquid chromatography (HPLC). Ca2+ imaging analysis revealed that the recombinant LTx2 acts on calcium channels of BC3H1 cells, blocking L-type calcium channels.