Neuronal circuits using neurotransmitters are essential for generating an eupnoeic breathing pattern in mammals. The different respiratory neurons scattered in the ponto-medullary brainstem are coupled via various synaptic connections which allow generation and modulation of the three neuronal phases of respiration namely inspiration, postinspiration and expiration. Apart from gamma-aminobutyric acid (GABA), glycine and glutamate which are known to be involved in respiratory rhythm generation, acetylcholine (ACh) exerts significant respiratory effects. Organophosphorus compounds (OP), used as pesticides or chemical warfare agents, act via inhibition of the enzyme acetylcholineesterase (AChE) leading to accumulation of ACh followed by central und peripheral respiratory arrest. Concerning the implication of ACh in the central control of breathing the existing results in newer literature are not consistent among different types of experiments and in different species studied. Furthermore, the knowledge on differentiated expression pattern of all ACh-receptor (AChR) subtypes in respiratory releated areas within the brainstem is incomplete.

To interpret the central effects of OP and ACh agonists on breathing the presence and accurate distribution pattern of muscarinic and nicotinic acetylcholine receptors (AChR) in the adult rat brainstem was investigated using immunhistochemistry. After intracardial perfusion with paraformaldehyd (PFA) following PBS (phosphate buffered saline) the brains were postfixed by immersion in PFA and cryoprotected. Fourteen micrometer cryostat sections of the hindbrain were stained using antibody specific protocols. All used antibodies were commercially available. M35, a monoclonal antibody which was shown to detect all five muscarinic acetylcholine receptor subunits, as well as subunit specific ones for m1, m2 and m3 AChRs were used. Two neuronal nicotinic AChRs were detected using antibodies specific for the 7 and the 2 subunit. The latter ones were found in areas of rat brainstem and medulla by bright field and fluorescence microscopy. Apart from these nicotinic receptors muscarinic AChR subunits were also found to have specific distribution patterns in the respiratory related areas. It awaits further experiments to define the percentage of cells which express both receptor subtypes.

These findings provide for the first time immunhistochemical evidence for the expressing of different cholinergic receptors in the respiratory related areas of the rat brainstem and allow targeted application of OP and other compounds effecting ACh to give information about the role of acetylcholine in the central modulation of breathing. (Supported by contract research project for the Bundeswehr Medical Service)