The working heart brainstem preparation (WHBP) is a well-established model for investigating integrative and synaptic mechanisms of cardiorespiratory control in the ponto-medullary brainstem of mature and neonatal rats. (Dutschmann & Paton, 2002a). Whilst intracellular recording of ventral medullary respiratory neurones was possible using sharp microelectrodes (Dutschmann & Paton 2002a, b), pharmacological analysis was restricted due to the limited time of stable recordings (~15 min). An alternative approach is whole-cell patch-clamp (w-c-p) which has been performed in the WHBP but from superficial brainstem nuclei (see Paton et al. 1999). However, the ventrolateral medullary neurones are out of reach of patch pipettes due to their depth from the dorsal medullary surface. Therefore, in the present study we have developed a new approach to allow the use of w-c-p recording from the ventral medullary respiratory group in the WHBP of neonatal rats.
Neonatal rats of between 3 and 6 days were anaesthetised deeply in a saturated atmosphere of halothane and a WHBP employed (Dutschmann et al. 2000). To achieve access to the ventral respiratory group, we exposed the ponto-medullary brainstem laterally by removing the temporal bones of the skull including the bulla. Following this exposure, the ponto-medullary brainstem could be visualised under a dissection microscope. The recording sites were identified by orientating patch pipette tips slightly dorsal (200-400 µm) to the rootlets of the hypoglossal rootlets. Recording success was assisted by stripping off overlying superficial layers of the spinal trigeminal fibre tract/nuclei (300-400 µm), thereby reducing the distance from the medullary surface to the neurones. Patch piptettes (2.5-3 MV, tip size 1-2 µm) were filled with the following (mM): 130 potassium gluconate, 10 Hepes, 11 EGTA, 4 NaCl, 2 MgCl2, 1 CaCl2, 2 ATP, 0.5 GTP and 5 glucose.
To screen for appropriate co-ordinates, extracellular recordings were performed initially and revealed that ventral respiratory group neurones were located at a depth of 500-800 µm below the lateral surface of the intact brainstem spanning the obex (n = 20). Subsequently, w-c-p recordings were made from all major groups of respiratory neurones: inspiratory (n = 5), post-inspiratory (n = 2), augmenting expiratory (n = 3). We achieved 1-2 successful w-c-p recordings per WHBP, which were stable for 20-65 min without obvious changes in resting membrane potential or input resistance.
Thus w-c-p recordings from ventral medullary respiratory neurones can be obtained using a lateral approach in the WHBP. This approach will now permit pharmacological studies to determine cellular mechanisms of respiratory rhythm and pattern generation as well as its modulation by various sensory inputs.
British Heart Foundation and Deutsche Forschungsgemeinschaft (SFB 430/C9) funded research.