Axon terminals possessing alpha2C-adrenergic receptors densely innervate neurons in the rat lateral spinal nucleus which respond to noxious stimulation.

The lateral spinal nucleus (LSN) in the rat spinal cord contains projection neurons that are densely innervated by peptidergic varicosities which probably originate from spinal interneurons. The alpha2C-adrenoceptor (alpha2C-AR) is present on axon terminals in this nucleus and therefore norepinephrine is likely to modulate input to LSN neurons. We investigated the involvement of LSN neurons in nociceptive transmission and their relationship with axons that possess alpha2C-ARs. Double-labeling immunostaining experiments showed that alpha2C-ARs are present on axon terminals of excitatory and inhibitory interneurons that frequently contain colocalised peptides. Electron microscopy revealed that alpha2C-AR terminals are presynaptic to dendrites and somata of LSN neurons and predominantly form asymmetric synapses. We retrogradely labeled LSN neurons that project to the caudal ventrolateral medulla and combined this with induction of c-Fos expression by peripheral noxious thermal stimulation along with immunolabelling for the alpha2C-AR and the substance P (neurokinin-1) receptor. This enabled us to identify neuronkinin-1 projection neurons in the LSN that express c-Fos and to determine if such cells receive contacts from alpha2C-AR terminals. The results show that some LSN neurons are activated by noxious stimulation and that this input is likely to be modulated by norepinephrine acting on alpha2C-ARs which are present on axon terminals that are presynaptic to LSN neurons.

Axon terminals possessing the alpha 2c-adrenergic receptor in the rat dorsal horn are predominantly excitatory.

In this study we used confocal microscopy to show that most (83.67%) alpha(2C)-adrenergic receptor (alpha(2C)-AR)-immunoreactive terminals in the superficial dorsal horn contain the vesicular glutamate transporter 2 and hence are glutamatergic. Few (11.33%) terminals contain glutamic acid decarboxylase (a marker for GABAergic axons) and none were associated with the B subunit of cholera toxin (a marker for myelinated primary afferents) or the vesicular glutamate transporter 1. These data indicate that most dorsal horn axons possessing the alpha(2C)-AR are excitatory and add further support to the suggestion that they originate principally from spinal interneurons.

An investigation of neurones that possess the alpha 2C-adrenergic receptor in the rat dorsal horn.

The function of the alpha(2C) subclass of adrenergic receptor in the spinal cord is unclear at present. Immunoreactivity for this receptor is found predominantly on axon terminals of the superficial dorsal horn but limited information is available about the properties and origin of these axons. The aim of this study was to determine which classes of neurone give rise to axons that possess this receptor and to investigate the synaptic organisation of these terminals. A series of double-labelling experiments was performed to investigate the relationship between the alpha(2C) receptor and each one of 14 chemical markers that label various types of axon terminal in the dorsal horn. Tissue was examined with two-colour confocal laser scanning microscopy. Quantitative analysis revealed that alpha(2C)-adrenergic receptors are not present on terminals of unmyelinated or peptidergic primary afferents and descending noradrenergic or serotoninergic axons. They were found on a proportion of terminals belonging to a mixed population of excitatory and inhibitory spinal interneurones, including those that contain neurotensin, somatostatin, enkephalin, GABA and neuropeptide Y. However, a greater proportion of terminals originating from excitatory interneurones were found to possess the receptor. Electron microscopic analysis revealed that alpha(2C)-adrenergic receptor immunoreactivity is predominantly associated with axon terminals that are presynaptic to dendrites but a small proportion of immunoreactive terminals formed axo-axonic synaptic arrangements. These studies indicate that noradrenaline can modulate transmission in the dorsal horn by acting through alpha(2C)-adrenergic receptors on terminals of spinal interneurones.

Myelinated and unmyelinated primary afferent axons form contacts with cholinergic interneurons in the spinal dorsal horn.

Cholinergic interneurons in laminae III/IV of the dorsal horn contain co-localised gamma-aminobutyric acid (GABA) and frequently form axoaxonic synapses with terminals of primary afferents. They are therefore probably last-order interneurons involved in presynaptic inhibition. The purpose of the present investigation was to determine if these cells receive direct input from primary afferents. Relationships between primary afferents and interneurons were investigated in adult rats. Myelinated primary afferents were labelled with the B-subunit of cholera toxin (CTb). Unmyelinated afferents were labelled with IB4 lectin and an antibody to identify calcitonin-gene-related peptide (CGRP). Cholinergic neurons were labelled with an antibody raised against choline acetyltransferase and examined with a confocal microscope. Cells were reconstructed with NeuroLucida for Confocal and afferent contacts plotted. Interneurons (N=30) received an average of 20.2+/-11.9 (SD) contacts from CTb-labelled primary afferents, which were preferentially distributed on proximal and intermediate dendrites. Interneurons with dendrites which extended into lamina II (N=20) received an average of 27.4+/-19.0 IB4 contacts (on intermediate and distal dendrites) and 9.2+/-6.8 CGRP contacts. It is concluded that cholinergic interneurons receive contacts from both myelinated and unmyelinated primary afferents and different classes of afferent target particular dendritic domains. Cholinergic interneurons are likely to be components of an inhibitory feedback pathway that is monosynaptically activated by primary afferents.