Al Ghamdi, Kholoud Saad
Populations of spinal cord dorsal horn neurons and their role in nociception.
PhD thesis, University of Glasgow.
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Nociception involves detection of tissue damage by specialized receptors; nociceptors. These convey information to the first synaptic relays in the dorsal horn of the spinal cord. Within the dorsal horn itself are the dorsal horn neurons which can be divided into two broad classes, based on their axonal projections: projection neurons and interneurons.
The neurokinin 1 receptor (NK1r), the main target for substance P, is expressed by most projection neurons and many interneurons in the dorsal horn. These NK1r-expressing neurons show a bimodal size distribution in lamina I. The 1st part of the project tested the hypothesis that large NK1r-immunoreactive cells in this lamina are projection neurons, while the small cells are interneurons. Rats were anaesthetised and received injections of tracers into two supraspinal areas that are likely to label all contralateral lamina I projection neurons. The rats were re-anaesthetized and perfused 3 days later and 1341 NK1r-positive cells were analysed, of which 441 were retrogradely labelled. Cross-sectional soma areas of projection neurons were larger than those of cells that were not retrogradely labelled. This difference was highly significant. Nearly all (99.4%) of the NK1r-expressing cells that were not retrogradely labelled had soma areas <200 microm2, while only 9.8% of the retrogradely labelled NK1r-expressing cells had somata <200 microm2. These results provide a means of distinguishing lamina I NK1r-expressing projection neurons from interneurons based on their soma sizes without the need of retrograde tracing surgeries.
Lamina I contains another population of projection neurons that lack or weakly express the NK1 receptor and consists of very large cells: giant cells, which are coated with the glycine and gamma-aminobutyric acid (GABA) receptor associated protein, gephyrin. There is also a group of large NK1r-expressing projection neurons with cell bodies in laminae III-IV and dendrites that pass dorsally to enter lamina I. Extracellular signal-regulated kinase (ERK) is expressed in dorsal horn neurons and is activated (phosphorylated) by noxious stimuli. In the 2nd part of the project, ERK phosphorylation in NK1r-expressing neurons as well as in lamina I giant cells was investigated following different type of noxious stimuli. Anaesthetised rats received noxious cutaneous, deep or visceral stimuli. They remained anaesthetized for 5 min after the end of the stimulus, and were then fixed by perfusion. Spinal cord sections were immunoreacted to reveal NK1r, gephyrin and phosphorylated ERK (pERK). Among the NK1r-expressing lamina I neurons, pERK was detected in both projection (somata >200 microm2) neurons and interneurons, with a significantly higher proportion in the larger cells, after all types of noxious stimulation. There was no significant difference in the frequency of pERK expression between the three morphological classes (fusiform, pyramidal and multipolar) of lamina I NK1r-expressing projection neurons after these stimuli. Most of the giant cells contained pERK after noxious cutaneous stimuli, but few did so following noxious deep stimulation. Only a few of laminae III-IV NK1r-expressing projection cells contained pERK after noxious deep or visceral stimulation, and the labelling in these was very weak. Results from the present study indicate that different types of neurons have different roles in conveying nociceptive information.
The superficial dorsal horn (SDH) is also a vital area for modulating nociception and contains large number of excitatory and inhibitory interneurons. Glutamate, released by primary afferents and local excitatory neurons, acts on G-protein-coupled metabotropic glutamate receptors (mGlus). Group I mGlus (mGlu1 and mGlu5) are strongly expressed in the SDH. It has been reported that intrathecal administration of the mGlu1/5 agonist 3,5-dihydroxyphenylglycine (DHPG) induces spontaneous nociceptive behaviours, which are ERK-dependent. In the 3rd part of the project, ERK phosphorylation in mGlu5-expressing neurons following the administration of DHPG was investigated. Anaesthetized rats underwent a laminectomy procedure. DHPG or saline was applied to their exposed lumbar cord for 8 minutes after which they were perfused. Sections from the lumbar spinal cord were immunoreacted to reveal mGlu5, pERK and one of various markers for excitatory or inhibitory interneurons. Following DHPG (but not saline), numerous pERK-positive cells were seen in the SDH, particularly lamina II, and the great majority of these were mGlu5-positive. ERK phosphorylation was detected in both inhibitory and excitatory mGlu5-expressing cells, suggesting that type I mGlus have a complex role in nociceptive processing.
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