Proceedings of The Physiological Society
Trinity College Dublin (2003) J Physiol 551P, C51
Evidence for the existence of GABAergic and glutamatergic neurones within the nucleus intermedius with synaptic connections to the nucleus of the solitary tract in rat
Mark L. Dallas, Susan A. Deuchars, Carol J. Milligan, Dave I. Lewis and Jim Deuchars
School of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, UK
In an unpublished study we noted that injections of retrograde tracers into the nucleus of the solitary tract (NTS) in the medulla oblongata resulted in labelled neurones in the nucleus intermedius (InM; T.F.C. Batten, personal communication). Here we utilise in situ hybridisation and electrophysiological studies to investigate the connections from the InM to the NTS.
For in situ hybridisation, rats (n = 5, 150 g) were humanely killed by anaesthetising with sodium pentobarbitone (60 mg kg-1, I.P.) followed by transcardial perfusion with 4 % paraformaldehyde in 0.1 M phosphate buffer. Brainstem slices (30 µm) were hybridised with digoxigenin-UTP-labelled GAD65, GAD67 and VGlut2 sense and antisense RNA probes using a modified version of the manufacturer's protocol (www.biochem.roche.com). mRNA for GAD65, GAD67 or VGlut2 was visualised in a number of regions in the medulla oblongata but neurones within the InM were notably mRNA positive for both inhibitory and excitatory markers. For electrophysiology, male Wistar rats (15-21 days) were terminally anaesthetized with sodium pentobarbitone (120 mg kg-1, I.P.) and 300 µm coronal slices of medulla oblongata prepared. Whole cell patch clamp recordings were made from 15 neurones located within dorsal and dorsomedial (n = 12) or medial (n = 3) regions of the NTS, at room temperature. These NTS neurones had an action potential amplitude of 61.3 ± 1.66 mV (mean ± S.E.M.), an action potential duration of 4.8 ± 0.35 ms and an AHP amplitude of 15.7 ± 1.7 mV.
Electrical stimulation of the InM, using bipolar stimulating electrodes (voltage 9-18 V, duration 100 µs), elicited excitatory and/or inhibitory synaptic potentials in 12/15 NTS neurones. EPSPs had an amplitude (AMP) of 6.01 ± 0.5 mV, width at half-amplitude (HW) of 52.1 ± 5.12 ms and 10-90 % rise time (RT) of 0.73 ± 0.09 mV ms-1 (n = 10), and were blocked by 1 mM kynurenic acid indicating that they were mediated by excitatory amino acids. IPSPs (AMP = 6.01 ± 0.41 mV, HW = 51.3 ± 1.88 ms, RT = 0.53 ± 0.04 mV ms-1(n = 9)) in seven NTS neurones were GABAergic as they were blocked by bicuculline (10 µM) while the remaining two were mediated by both GABA and glycine as they were abolished by co-application of bicuculline (10 µM) and strychnine (2 µM). Further to this, recordings were also made from InM neurones where stimulation of the NTS resulted in antidromic action potentials in these neurones (n = 2/2). The function and the pharmacology of these excitatory and inhibitory synaptic pathways are currently being investigated.
The University of Leeds and The Wellcome Trust supported this work
Where applicable, experiments conform with Society ethical requirements