The properties and synaptic inputs of cholinergic interneurones.

The central autonomic area (CAA) contains interneurones which directly influence sympathetic preganglionic neurones (Deuchars et al., 2005). Sensory afferent terminals are located around lamina X and form close appositions with interneurones (Hofstetter et al., 2005). CAA neurones have been identified as being premotor to somatic motor neurones (Stepien et al., 2010). This suggests that these interneurones may coordinate autonomic and motor outflow from the spinal cord and therefore, may receive inputs from primary afferents. This study aims to address how these interneurones fit into circuits involved in autonomic control. To investigate whether interneurones in CAA received direct inputs from myelinated primary afferents, immunohistochemistry for vesicular glutamate transporter 1 (VGLUT1) was conducted in GAD67-GFP mice anaesthetised with pentobarbital (60mg/kg; i.p) and transcardially perfused with 4% paraformaldehyde. Close appositions were observed between VGLUT1 primary afferent terminals and interneurones immunoreactive for GFP (GABAergic) or Choline Acetyltransferase. To determine how interneurones responded to stimulation at the dorsal root entry zone, 7-12 day old mice and 12-21 day old rats, of either sex, were anaesthetised by urethane (2g/kg; i.p). Transcardiac perfusion was followed by decapitation and 300µm transverse slices of lumbar and thoracic spinal cord were cut. Viable interneurones in laminae VII and X were identified and whole cell patch clamp recordings performed. A stimulating electrode was placed at the dorsal root entry zone to deliver stimuli (5-100 Hz) of 1 ms duration. The evoked excitatory postsynaptic potential (EPSP) amplitude was recorded and the latency to onset of the EPSP measured. The standard deviation (SD) of latency gave an indication of variability in latency (jitter). Values are means ± S.E.M, compared by 2 sample t-test. Mouse thoracic interneurones had an action potential threshold of -35.06 ± 1.99 mV, action potential amplitude of 49.52 ± 2.64 mV and afterhyperpolarisation amplitude of -19.45 ± 1.08 mv (n= 15). EPSP jitter was 2.31± 0.01 µs (n= 7). EPSPs followed high frequency tetanic stimulation, without failures indicating that they are likely elicited by monosynaptic primary afferents, and were significantly antagonised by 20 µM NBQX (an excitatory amino acid receptor antagonist) (7.68 ± 0.64 mV in control (n= 7) to 3.28 ± 0.45 mV (n= 7) in NBQX; p< 0.001). In rats, EPSP jitter was 4.23 ± 0.51 µs (n= 16) and EPSP amplitude was significantly reduced with NBQX (6.43 ± 0.04 mV (n= 16) in control to 0.42 ± 0.24 mV (n= 9) in 20 µM NBQX; p< 0.001). Post hoc analysis revealed extensive axon ramifications in both the dorsal and ventral horn. The direct action of primary afferents onto interneurones in the CAA may provide a avenue for the co-ordination of autonomic and motor activity. [ABSTRACT FROM AUTHOR]