title: The organisation of the monoaminergic and cholinergic systems in the spinal cord
Creators: name: Stewart, William
Publisher: name: University of Glasgow
Year: 2001
Subjects: subject: Neurosciences.

description: Within the central nervous system, both the monoaminergic and cholinergic systems are recognised as having a significant role in the regulation of nociceptive processing at the level of the spinal dorsal horn producing a depression of the nociceptive signal. Despite this, relatively little is known of the mechanisms and neuronal circuits which are responsible for the action of these systems. In producing their action the descending monoaminergic pathways from the brainstem, which utilise serotonin and noradrenaline may potentially operate via pre- and/or post-synaptic pathways at either direct or indirect sites. For both the monoaminergic and cholinergic systems multiple receptor subtypes exist and the role of each individual subtype is ill understood. In this series of experiments, the relationship of the monoamines to identified neurons of the dorsal horn is first examined. Then, in a separate series of studies the distribution of the muscarinic m2 receptor is examined in the spinal cord with particular reference to the distribution of this receptor in the dorsal horn and the cells which are targeted. Adult Albino Swiss rats were used throughout. These were anaesthetised with pentobarbatone (1ml of 60mg/ml, i.p.) and perfused prior to removal of mid-lumbar or thoracic spinal cord blocks. These blocks were then sectioned in either the transverse or parasagittal plane then reacted with the primary antibody cocktail required for each protocol. Following this the sections were transferred to solutions of fluorophore-coupled secondary antibodies or, for ultrastructural studies, antibodies coupled to biotin or immunogold. The sections were then processed for analysis using three-colour confocal laser scanning microscopy or electron microscopy. In sections reacted with serotonin a dense plexus of axons and terminals was identified in the dorsal horn corresponding to the region of lamina I/IIo from which axons emerged which appeared to encircle the somata and proximal dendrites of neurons within lamina III/IV forming 'basket-like' associations. Numerous noradrenergic profiles (revealed with dopamine-beta-hydroxylase immunoreactivity) were also noted corresponding to lamina I/II however these did not appear to cluster around the proximal regions of dorsal horn neurons. Examining the relationship of these monoaminergic axons and terminals to specific dorsal horn neuronal subtypes demonstrated few contacts with no evidence of clustering on either excitatory or inhibitory dorsal horn interneurons revealed with antibodies to GABA, glycine, somatostatin, neurotensin, choline acetyltransferase, nitric oxide synthase (NOS) or the mu-opioid receptor. In contrast, cells immunoreactive for the substance P receptor neurokinin-1 (NK-1), located in lamina III/IV and lamina I, received relatively high contact frequencies form serotonin when compared to the dorsal horn interneurons. These contacts were clustered around the proximal dendrites and somata. In addition in approximately 50% of the NK-1-immunoreactive neurons in lamina III/IV the 'basket-like' arrangement of serotonin axons was observed. This correlated with a higher frequency of contacts on these cells. Ultrastructural observations on the relationship of serotonin terminals to GABA-immunoreactive somata showed several close associations however synaptic specialisations were not identified. In material reacted with the muscarinic m2 acetylcholine receptor immunolabelled profiles were identified in the dorsal horn, ventral horn, intermediate grey and around the central canal in lamina X. In the dorsal horn approximately 23% of neurons were immunoreactive for the m2 receptor. Examination of the relationship of this receptor to specific dorsal horn neuronal subtypes revealed that some cells immunoreactive for either GABA, NOS, NK-1, parvalbumin or the somatostatin2A receptor were m2 positive with the ratio in part dependant on laminar location. No neurotensin-immunoreactive neurons were m2 receptor-immunoreactive. Throughout the dorsal horn and lamina X 41% of cholinergic neurons were m2 receptor-immunoreactive. In the ventral horn larger rather than smaller motoneurons were found to be receptor positive and these neurons were also noted to receive greater numbers of cholinergic terminals. These investigations have demonstrated that the cells of the dorsal horn which are NK-1-immunoreactive are unique amongst the neurons studied in receiving significantly greater frequencies of serotonin contacts than the dorsal horn interneurons examined.