Purinergic modulation of respiration via medullary raphe nuclei in rats

Cao Y, Song G. Purinergic modulation of respiration via medullary raphe nuclei in rats.
Respir Physiol Neurobiol. 2006 Apr 28

Link
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=Abstract&list_uids=16750433&query_hl=6&itool=pubmed_docsum

Background

ATP is a neurotransmitter that acts on purinergic receptors that can be divided into two main categories: ionotrophic P2X receptors that are permeable to Na+, K+ and Ca ++ and slower acting metabotropic receptors P2Y. In each class there are several subtypes. In the carotid body ATP acts on P2X receptors affecting transduction of information related to the level of arterial hypoxia. P2X receptors are also found pre- and post-synaptically in respiratory control regions including the nucleus tractus solitarius, dorsal vagal motor nucleus and ventral medullary chemoreceptive regions sensitive to levels of inhaled CO2. Purinergic receptors are located on neurons containing several neurotransmitters including GABA, glutamate, and hypocretin/orexin. Thus, ventilation and arousal state may be modulated directly and indirectly by ATP acting through purinergic receptors. In this study the role of ATP acting on P2X on two different raphe nuclei (raphe magnus (RM) and raphe pallidus (RP)). Both nuclei have direct projections to phrenic motor neurons. Electrical or chemical stimulation of these raphe nuclei affects ventilation in different ways. Stimulation of the RM, which contains Gabanergic and serotoninergic(5-HT) neurons, causes inhibition of breathing, and stimulation of the RP that contains predominantly serotoninergic neurons causes facilitation of breathing.

Hypothesis

The purposes of the present study were to

1. compare the effects of microinjections of ATP into the 2 raphe nuclei on ventilation, and
2. determine if the responses we caused by ATP acting on P2X receptors in these nuclei.

Methods

1. Adult male or female Wistar rats were anesthetized with an IP injection of urethane with given atropine given to decrease tracheal secretions. Some animals were allowed to breathe spontaneously, while others were tracheatomized, paralyzed, and ventilated. End-tidal CO2 was maintained between 5.0 and 5.5%.
2. To access the brain for microinjection of raphe nuclei and C5 rootlets, the rat’s head was placed into a sterotaxic apparatus to expose the brainstem by occipital craniotomy and removal of the posterior portion of the cerebellum.
3. Phrenic nerve recordings were made using a bipolar silver hook electrodes. In some animals ventilatory outflow was determined using diaphragmatic electromyography (EMG).
4. Microinjections of artificial CSF, ATP (0.1M or 0.2M), and/or the broad spectrum P2X receptor antagonist phosphate-6-azophenyl-2’,4’-disulfonci acid (PPAD) occurred following identification of the RP and RM using electrical stimulation.
5. At the end of the studies, immunohistochemical studies were conducted to determine the locations of the microinjections.
6. Parameters that were evaluated included the amplitude of the integrated phrenic nerve recording or diaphragmatic EMG averaged over 10 seconds. The frequency was determined by measuring the number of respiratory cycles over this time period. Statistical tests consisted of Student’s t tests and 95% confidence intervals. Significance was accepted at P<0.05.

Results

1. In spontaneously breathing or paralyzed and ventilated rats, microinjection of 0.2M ATP into the RM caused a marked inhibition of breathing with a decrease of both the amplitude and frequency of the phrenic nerve discharge. The effect was no longer present 30 minutes after microinjection.
2. Injection of 0.2 M ATP into the RP caused facilitation of ventilation with an increase in both the phrenic nerve amplitude and frequency of spontaneously breathing or paralyzed and ventilated rats. By 20 minutes the effect was no longer present in the first group and by 10 minutes the effects were gone in the ventilated groups. Thus, length but not the type of response to ATP was altered by the preparation of the rat.
3. Responses to microinjection or ATP into the RP and RM were dose dependent.
4. The broad spectrum P2X antagonist PPAD, itself did not affect ventilatory responses of the PD or PM. However, injection of PPAD prior to ATP microinjection attenuated the inhibitory effect of ATP in the RM and the facilitative effect of ATP in the RP.
5. Injections of aCSF of the same volume and osmotic pressure as ATP into the RP or RM had no effect on respiration.

Discussion and Clinical significance

This study indicated that ATP acting through P2X receptors affected ventilation in opposite ways when microinjected into the RP and RM of rats. The differences in the effects of ATP on ventilation in these 2 regions are thought to be due to stimulating inhibitory GABAergic neurons in the RM and excitatory 5-HT neurons in the RP. Previous studies have shown that the facilitation could be blocked by systemic or local administration of a 5-HT receptor blocker. Excitation by P2X receptor activation can occur by opening post-synaptic channels that cause depolarization or presynaptically stimulating the release of glutamate. Although PPAD attenuated the effects of ATP in both the RP and RM, it did not totally block it suggesting the ATP may be acting on other purinergic receptors. Studies in many areas affecting patient health including pain, muscular dystrophy, arousal, seizure activity, regulation of sympathetic nerve activity and control of breathing are currently elucidating the importance of ATP (and its metabolite adenosine) and the receptors that ATP acts on in regulation of normal and pathological functions. Investigations are underway to develop specific purinergic receptor antagonists to modulate effects of pain, stroke, and potentially abnormalities in the control of breathing.

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