The midbrain periaqueductal grey has no role in the generation of the respiratory motor pattern, but provides command function for the modulation of respiratory activity

https://doi.org/10.1016/j.resp.2014.07.011Get rights and content

Highlights

  • Strong respiratory modulation can be triggered from in situ PAG.

  • Inhibition of the PAG has no effect on the eupnoeic respiratory motor pattern.

  • The perfused brainstem preparation is model to study PAG mediated cardio-respiratory commands.

Abstract

It has previously been shown that stimulation of cell-columns in the periaqueductal grey (PAG) triggers site-specific cardiorespiratory effects. These are believed to facilitate changes in behaviour through coordinated changes in autonomic outflow. Here, we investigated whether PAG-evoked respiratory commands can be studied in situ using the decerebrate perfused brainstem preparation. Phrenic, vagus and abdominal iliohypogastric nerves were recorded before and after microinjection of l-glutamate (30–50 nl, 10 mM) or isoguvacine (GABA-receptor agonist, 30–50 nl, 10 mM) into the PAG. l-glutamate microinjection triggered a range of site-specific respiratory modulations (n = 17 preparations). Subsequent microinjection of isoguvacine into the same PAG sites had no effect on the baseline respiratory motor pattern or rhythm. We conclude that while the PAG has no function in respiratory pattern generation, PAG-evoked respiratory modulations can be evoked in situ in the absence of higher brain centres and while homeostatic parameters that may affect respiratory drive are held static.

Introduction

The midbrain periaqueductal grey (PAG), in conjunction with superior and inferior collicular structures, is implicated in initiating coordinated alterations in respiratory, cardiovascular and thermoregulatory parameters. These alterations presumably facilitate behaviours that are simultaneously elicited from the PAG in response to stressful stimuli (Bandler et al., 2000, Dampney et al., 2013).

The PAG is organised into anatomically and functionally distinct columns (Carrive, 1993), that upon stimulation produce specific patterns of autonomic and somatic motor activity. Some of these are implicated in the modulation of respiratory motor patterns corresponding to vocalisation (Carrive et al., 1989a, Carrive et al., 1989b, Carrive et al., 1987, Hayward et al., 2004, Subramanian et al., 2008) or the alteration of ventilation in anticipation of exercise (Green et al., 2007, Paterson, 2014). There is also limited evidence that the PAG may modulate airway defence reflexes such as cough (Jakus et al., 2008, Sessle et al., 1981).

Much attention has been focussed on the anatomical projections of various PAG regions in an effort to grasp the means by which these neurons modulate autonomic and somatic activity. Many studies focus on the modulation of cardiovascular parameters and/or consider respiration purely in terms of vocalisation (Bandler and Carrive, 1988, Carrive et al., 1989a, Carrive et al., 1989b). However, both the magnitude and nature of changes in the respiratory pattern evoked following activation of the dorsomedial (dmPAG), lateral (lPAG) and dorsolateral (dlPAG) PAG show marked variation while changes in sympathetic nerve discharge remain very similar throughout (Iigaya et al., 2010). Similarly, stimulation of the caudal portion of the dmPAG and dlPAG produces changes in respiratory parameters which are greater than those evoked more rostrally, while cardiovascular responses remained consistent (Iigaya et al., 2010, Zhang et al., 2007).

A wide variety of respiratory and vocalisation-related modulations of the respiratory motor pattern have been reported in cats (Subramanian et al., 2008, Subramanian et al., 2007, Zhang et al., 1994). Stimulation or disinhibition of the dmPAG in anaesthetised rats produces increased respiratory frequency and increased tonic drive to the diaphragm (Hayward et al., 2003). Similarly, tachypnoeic responses have been observed on stimulation of the dorsolateral PAG in rats (Huang et al., 2000). Importantly, differentiation in the character of the respiratory response has also been observed in different PAG columns, with stimulation of the dlPAG producing a larger effect upon the respiratory rate and stimulation of the lPAG producing a larger effect upon the amplitude of phrenic nerve discharge (Iigaya et al., 2010).

In the current study, we report that activation of the PAG produces region-specific respiratory patterns in the decerebrate in situ perfused brainstem preparation of the rat. In contrast to previous studies, responses were successfully evoked in the absence of the hypothalamus, indicating that these responses were produced via direct projections to the pontomedullary respiratory rhythm and pattern generator. We propose that this preparation, which is unanaesthetised, provides a novel setting for the study of PAG circuits and the means by which these circuits modulate changes in the respiratory motor pattern. Additionally, we investigated whether the PAG provides a contribution to the generation or shaping of the respiratory pattern.

Section snippets

Materials and methods

All experimental procedures were performed in accordance with the Australian code of practice for the care and use of animals for scientific purposes and conform to the principles of international regulations. This study was approved by and carried out in accordance with guidelines put in place by the ethics committee of the Florey Institute of Neuroscience and Mental Health (AEC 12-004).

Results

Microinjections of 30–60 nl l-glutamate into the PAG (n = 29) in 12 perfused brainstem preparations of rat reliably evoked a variety of site-specific respiratory responses. In contrast, local inhibition of the same PAG sites by injection of the GABA receptor agonist isoguvacine (n = 24 in 9 preparations) never produced any consistent change in ongoing respiratory activity. As previously reported for the perfused brainstem preparation (Abdala et al., 2009), AbNA was either absent or showed weak tonic

Discussion

The primary finding of the current study is that distinct, site-specific patterns of activity in respiratory motor outputs can be evoked by stimulation of the PAG in the decerebrate perfused brainstem preparation of the rat. While some of these responses correspond with patterns evoked in previous studies in vivo (Huang et al., 2000, Iigaya et al., 2010), activation or inhibition of the same sites in the perfused brainstem preparation also produced responses that differed significantly. Thus,

Summary

In summary, we conclude that marked changes in the respiratory motor pattern are reproducibly evoked from the midbrain PAG in the unanaesthetised and decerebrate perfused brainstem preparation of the rat. In contrast to previous studies, these responses were evoked in the absence of the dorsomedial hypothalamus and are most likely mediated via direct projections to the brainstem, the pontine parabrachial complex and possibly the RTN/pFRG. Inhibition of responsive sites within the PAG did not

Author contribution

DF, TB, MD performed perfused brainstem experiments and analysed data. SJ provided histology and anatomical verification of the injection sites. DF TB SJ DS and MD interpreted the results and wrote the manuscript.

Acknowledgements

The study was supported by a future fellowship of the Australian Research Council to MD (FT120100953) and a start-up fund of the Florey Institute of Neuroscience and Mental Health. We acknowledge the support of the Victorian Government through the Operational Infrastructure Scheme.

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    This paper is part of a special issue entitled “Non-homeostatic control of respiration”, guest-edited by Dr. Eugene Nalivaiko and Dr. Paul Davenport.

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