R. Kerr

A quantitative study of spinothalamic neurons in laminae I, III, and IV in lumbar and cervical segments of the rat spinal cord

The major ascending outputs from superficial spinal dorsal horn consist of projection neurons in lamina I, together with neurons in laminae III–IV that express the neurokinin 1 receptor (NK1r) and have dendrites that enter the superficial laminae. Some neurons in each of these populations belong to the spinothalamic tract, which conveys nociceptive information via the thalamus to cortical areas involved in pain. A projection from the cervical superficial dorsal horn to the posterior triangular nucleus (PoT) has recently been identified. PoT is at the caudal end of the thalamus and was not included in injection sites in many previous retrograde tracing studies. We have injected various tracers (cholera toxin B subunit, Fluoro‐Gold, and fluorescent latex microspheres) into the thalamus to estimate the number of spinothalamic neurons in each of these two populations, and to investigate their projection targets. Most lamina I and lamina III/IV NK1r‐immunoreactive spinothalamic neurons in cervical and lumbar segments could be labeled from injections centered on PoT. Our results suggest that there are 90 lamina I spinothalamic neurons per side in C7 and 15 in L4 and that some of those in C7 only project to PoT. We found that 85% of the lamina III/IV NK1r‐immunoreactive neurons in C6 and 17% of those in L5 belong to the spinothalamic tract, and these apparently project exclusively to the caudal thalamus, including PoT. Because PoT projects to second somatosensory and insular cortices, our results suggest that these are major targets for information conveyed by both these populations of spinothalamic neurons. J. Comp. Neurol. 511:1–18, 2008.

Characterisation of axon terminals in the rat dorsal horn that are immunoreactive for serotonin 5-HT3A receptor subunits

Serotonin 5-HT3 receptors are abundant in the superficial dorsal horn and are likely to have an involvement in processing of nociceptive information. It has been shown previously that 5-HT3 receptors are present on primary afferent terminals and some dorsal horn cells. The primary aim of the present study was to determine what classes of primary afferent possess 5-HT3A receptor subunits. We performed a series of double- and triple-labelling immunofluorescence experiments. Subunits were labelled with an anti-peptide antibody and primary afferent axons were identified by the presence of calcitonin gene-related peptide (CGRP) and binding of the lectin IB4. Quantitative confocal microscopic analysis revealed that approximately 10% of axons displaying 5-HT3A immunoreactivity were also labelled for CGRP but that only 3% of these fibres bind IB4. We also investigated the relationship between immunoreactivity for the subunit and descending serotoninergic systems, axons originating from inhibitory neurons that contain glutamic acid decarboxylase, and axons of a subpopulation of excitatory neurons that contain neurotensin. None of these types of axon was associated with immunoreactivity for receptor subunits. Ultrastructural studies confirmed that punctate immunoreactive structures observed with the light microscope were axon terminals. These terminals invariably formed asymmetric synaptic junctions with dendritic profiles and often contained a mixture of granular and agranular vesicles. Some terminals formed glomerular-like arrangements. Immunoreactive cells were also examined and were found to contain intense patches of reaction product within the cytoplasm. We conclude that the majority (about 87%) of dorsal horn axons that are immunoreactive for 5-HT3A receptor subunits do not originate from the subtypes of primary afferent fibres that bind IB4 or contain CGRP. It is likely that most of these axons have an excitatory action and they may originate from dorsal horn interneurons and/or fine myelinated primary afferent fibres.

Colocalization of glycine and GABA in synapses on spinomedullary neurons

Spinomedullary neurons of the postsynaptic dorsal column pathway in adult cats were retrogradely labelled with horseradish peroxidase. Postembedding immunogold reactions were performed with antisera which recognise GABA or glycine to determine if synaptic boutons in contact with these neurons contain both transmitters. Analysis of series of ultrathin sections revealed that synaptic profiles with strong immunogold reactions for GABA usually also displayed strong immunogold reactions for glycine. Pre-embedding immunocytochemistry was performed on sections containing labelled cells with a monoclonal antibody which recognises the glycine receptor-associated protein, gephyrin. Many synapses onto postsynaptic dorsal column neurons were associated with gephyrin-like immunoreactivity and these typically contained irregularly shaped vesicles. Immunogold reactions showed that synaptic profiles apposed to gephyrin-immunoreactive junctions contained GABA and glycine. The evidence suggests that glycine is a neurotransmitter at synapses on spinomedullary neurons and that it is colocalized with GABA.

SYNAPTIC CONNECTIONS OF DORSAL HORN GROUP II SPINAL INTERNEURONS : SYNAPSES FORMED WITH THE INTERNEURONS AND BY THEIR AXON COLLATERALS

Five dorsal horn interneurons with monosynaptic input from group II primary afferent fibres were physiologically characterized and intracellularly labelled with horseradish peroxidase. The cells were prepared for combined light and electron microscopy, and synaptic arrangements formed by axon collaterals of interneurons and synapses formed with their dendrites and somata were examined with the electron microscope. Immunogold reactions for γ‐aminobutyric acid, glycine and glutamate were performed to determine if these synapses were excitatory or inhibitory. Axon collaterals in lamina VI formed synapses with somata and dendrites of other neurons, and collaterals of one cell also formed axoaxonic synapses. It was concluded that one cell from the sample was inhibitory, whereas the remainder were probably excitatory. Dendrites and cell bodies of interneurons were contacted by several types of synaptic bouton. The first type of bouton displayed immunoreactivity for glutamate, the second type contained both γ‐aminobutyric acid and glycine, the third type contained glycine alone, and the fourth type contained γ‐aminobutyric acid alone. Some large glutamatergic boutons were postsynaptic to other boutons. Presynaptic boutons at these axoaxonic synapses always contained γ‐aminobutyric acid but a minority also contained glycine.

Inhibitory amino acid transmitters associated with axons in presynaptic apposition to cutaneous primary afferent axons in the cat spinal cord

The purpose of the present study was to characterize the transmitter content of structures in presynaptic apposition to the central terminals of cutaneous afferent fibers in the dorsal horn of the spinal cord. Axons in the Aαβ conduction velocity range were identified in adult cats, stained intra‐axonally with horseradish peroxidase, and prepared for combined light and electron microscopy. In total, we labeled two slowly adapting (Type 1) axons, two hair‐follicle afferents, and one rapidly adapting (Krause) afferent. Ninety‐nine labeled boutons were examined through complete series of serial sections. Approximately 80% of boutons originating from rapidly adapting and hair‐follicle afferents were postsynaptic to other axons, but only 50% of boutons from slowly adapting axons were associated with this type of arrangement. Postembedding immunogold reactions revealed that between 80% (for slowly adapting axons) and 100% (for rapidly adapting axons) of boutons presynaptic to primary afferents were immunoreactive for γ‐aminobutyric acid (GABA). The vast majority of these terminals (in excess of 80%) were also enriched with glycine. Therefore, presynaptic inhibition of these three functional classes of Aαβ cutaneous primary afferents is mediated principally by the subgroup of GABAergic interneuron that also contains glycine. J. Comp. Neurol. 452:154–162, 2002.

Differential expression of the muscarinic m2 acetylcholine receptor by small and large motoneurons of the rat spinal cord

The purpose of this study was to determine if motoneurons in the spinal ventral horn express the muscarinic m2 acetylcholine receptor. Motoneurons were retrogradely labelled in adult rats by intramuscular injection of Fluorogold and examined with confocal microscopy for evidence of immunoreactivity for the receptor. The cells were also double-labelled for choline acetyltransferase to determine if they were contacted by cholinergic axons. Almost all large motoneurons (diameters greater than 35 microm) displayed intense immunoreactivity for the receptor which was evenly distributed along the plasma membrane. Small cells were immunonegative for the receptor or weakly labelled. As large cells probably correspond to alpha-motoneurons and receive many cholinergic contacts, it is concluded that acetylcholine can influence this type of motoneuron by acting through the m2 receptor.

Absence of co-localized glutamic acid decarboxylase and neuropeptides in noradrenergic axons of the rat spinal cord

Patterns of co-localization of immunoreactivity for dopamine beta-hydroxylase (the synthetic enzyme for noradrenaline) and glutamic acid decarboxylase (the synthetic enzyme for GABA) or each one of six neuropeptides (neuropeptide Y, substance P, met-enkephalin, galanin, dynorphin A and somatostatin) were investigated with dual-colour confocal laser scanning microscopy in axons of cervical, thoracic and lumbar spinal segments of six adult rats. Four regions of the grey matter were studied (laminae I-II, V, IX and X) and, in thoracic segments, the intermediolateral cell column was also examined. The extent of co-localization was estimated by direct assessment of merged pairs of optical sections and by automated image analysis. Significant co-localization was found for neuropeptide Y in axons of the intermediolateral cell column of thoracic segments and in lamina X of cervical and thoracic segments. None of the other peptides or glutamic acid decarboxylase were found to coexist at significant levels with dopamine beta-hydroxylase and hence it is likely that this group of neuropeptides and GABA are not co-transmitters of bulbospinal noradrenergic axons in the rat.

An immunocytochemical investigation of the relationship between substance P and the neurokinin-1 receptor in the lateral horn of the rat thoracic spinal cord

The relationship between substance P-containing axons and sympathetic preganglionic neurons possessing the neurokinin-1 receptor was investigated in the lateral horn of the rat thoracic spinal cord. Sympathetic preganglionic neurons were labelled retrogradely with Fluorogold. Sections containing labelled cells were reacted with antibodies against choline acetyltransferase, substance P and the neurokinin-1 receptor and examined with three-colour confocal laser scanning microscopy. In all, 95 sympathetic preganglionic neurons were examined and 79% of these were immunoreactive for the neurokinin-1 receptor. Substance P-immunoreactive axons not only made contacts with preganglionic neurons which were immunoreactive for the receptor but also made contacts with cells which did not express the receptor. Dendrites, labelled with immunoreactivity for choline actyltransferase, also received contacts from substance P-immunoreactive varicosities but this was not related to the presence or the absence of receptor. An electron microscopic analysis was performed to investigate the relationship between substance P-containing boutons and dendrites possessing the neurokinin-1 receptor. Immunoreactivity for substance P was detected with peroxidase immunocytochemistry and immunoreactivity for the receptor was detected with the silver-intensified gold method. Substance P-containing boutons made synapses with dendrites which were positively and negatively labelled for the receptor. Receptor immunoreactivity was not usually present at synapses formed by substance P boutons with neurokinin-1-immunoreactive dendrites. It is concluded that substance P may modulate much of the activity of sympathetic preganglionic neurons through an indirect non-synaptic mechanism.

Evidence for the presence of neurokinin-1 receptors on dorsal horn spinocerebellar tract cells in the rat

Dorsal horn spinocerebellar tract cells of adult rats were labelled by retrograde axonal transport with the B subunit of cholera toxin. Sections were prepared from lumbar and thoracic spinal segments and incubated with antisera which specifically recognise neurokinin-1 receptor protein and substance P. Labelled cells and immunoreactivity for the receptor and substance P were identified by using three different fluorophores and the relationships between them were assessed in single optical sections with three-colour confocal laser scanning microscopy. Forty-eight cells were examined and 23 of them displayed immunoreactivity for the receptor. Many substance P-immunoreactive profiles were present in lamina V and some formed contacts with spinocerebellar tract cells possessing neurokinin-1 receptor immunoreactivity. The evidence suggests that substance P may influence the activity of a subpopulation of dorsal horn spinocerebellar tract cells by acting through neurokinin-1 receptors.

Glutamate receptor subunits associated with rat sympathetic preganglionic neurons

The purpose of this study was to determine what subunits of the glutamate (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)) receptor are expressed by sympathetic preganglionic neurons in the spinal cord of adult rats. Preganglionic neurons were retrogradely labelled with Fluorogold, double-labelled with choline acetyltransferase immunofluorescence, and examined with confocal microscopy for evidence of immunoreactivity for GluR1, GluR2, GluR2/3 and GluR4 subunits. Quantitative analysis revealed that 92, 63 and 85% of preganglionic cells in the T8 segment express GluR1, GluR2 and GluR2/3 subunits, respectively. Cells were not immunoreactive for the GluR4 subunit. This evidence is consistent with the idea that most sympathetic preganglionic neurons form heteromeric AMPA receptors. Cells with GluR2 subunits will assemble receptors which are impermeable to calcium ions and may be resistant to excitotoxic cell death.