Spinal cord neuronal circuitry involving dorsal horn projection cells

Baseer, Najma (2014) Spinal cord neuronal circuitry involving dorsal horn projection cells. PhD thesis, University of Glasgow.

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The spinal cord dorsal horn is involved in the processing and transmission of sensory information to the brain. There are several distinct populations of dorsal horn projection cells that constitute the major output of the spinal cord. These cells are mostly found in lamina I and are scattered throughout the deep dorsal horn. There is a population of large lamina III projection cells that expresses the neurokinin 1 receptor (NK1r), which is the main target for substance P released by nociceptive primary afferents. These cells are densely innervated by peptidergic nociceptive afferents and more sparsely by low-threshold myelinated afferents. In addition, they also receive selective innervation from neuropeptide Y-containing inhibitory interneurons. However, not much is known about their input from glutamatergic spinal neurons. It has already been reported that the great majority of large lamina III NK1r expressing cells project to caudal ventrolateral medulla (CVLM) therefore in this study these cells were easily identified without retrograde tracer injection. Preliminary observations showed that these cells received contacts from preprodynorphin (PPD)-containing excitatory axons. The first part of the study tested the hypothesis that lamina III projection cells are selectively targeted by PPD-containing excitatory spinal neurons. Spinal cord sections from lumbar segments of the rat underwent immunocytochemical processing including combined confocal and electron microscopy to look for the presence of synapses at the sites of contact. The results showed that lamina III NK1r cells received numerous contacts from non-primary boutons that expressed vesicular glutamate transporter 2 (VGLUT2), and formed asymmetrical synapses on their dendrites and cell bodies. These synapses were significantly smaller than those formed by peptidergic afferents but provided a substantial proportion of the glutamatergic input to lamina III NK1r projection cells. Furthermore, it was observed that PPD was found to be present in ~58% of the VGLUT2 boutons that contacted these cells while a considerably smaller proportion of (5-7%) VGLUT2 boutons in laminae I-IV expressed PPD. These results indicate a highly selective targeting of the lamina III projection neurons by glutamatergic neurons that express PPD. Fine myelinated (Aδ) nociceptors are responsible for the perception of fast, well-localised pain. Very little is known about their postsynaptic targets in the spinal cord, and therefore about their roles in the neuronal circuits that process nociceptive information. In the second part of the study, Fluorogold injections were made into the lateral parabrachial region (LPb) of the rat brain on one side and cholera toxin B subunit (CTb) was injected into the sciatic nerve on the contralateral side to assess whether Aδ nociceptors provide input to lamina I projection cells. The vast majority of lamina I projection neurons belong to the spinoparabrachial tract, and these can be divided into two major groups: those that express NK1r, and those that do not. The results suggested that CTb labelled a distinct set of Aδ nociceptors, most of which lack neuropeptides. CTb-labelled Aδ afferents formed contacts on 43% of the spinoparabrachial lamina I neurons that lacked the NK1r, but on a significantly smaller proportion (26%) of NK1r projection cells. Combined confocal and electron microscopy established that the contacts were associated with synapses. Furthermore, the contact density of CTb labelled boutons was considerably higher on the NK1r- cells than on those with the NK1r. These results provide further evidence that primary afferents input to projection cells is organized in a specialized way and that both NK1r+ and NK1r- lamina I projection neurons are directly innervated by Aδ nociceptors, thus may have an important role in the perception of fast pain. Lamina I of the rat spinal cord dorsal horn contains a population of large spinoparabrachial projection neurons (giant cells) that receive numerous synapses from both excitatory (VGLUT2) and inhibitory (VGAT) interneurons. The giant cells are selectively innervated by GABAergic axons that express neuronal-nitric oxide synthase (nNOS) and are thought to originate from local inhibitory interneurons. In the rat, the nNOS inhibitory cells belong to a distinct functional population that differs from other inhibitory interneurons in terms of somatostatin receptor (sst2A) expression and also in responsiveness to painful stimuli. There is a population of inhibitory interneurons that express green fluorescent protein (GFP) in lamina II of mice in which GFP is under control of the prion promoter (PrP) and the great majority of these cells also express nNOS. In this part of the study, the inhibitory synaptic input from nNOS-containing GFP boutons to giant lamina I cells was investigated. The great majority of lamina I projection neurons express NK1 receptor; therefore, the possibility that lamina I NK1r-expressing projection neurons received innervation from GFP+/nNOS+ axons was also tested. Since retrograde tracing technique was not used in this part of the study, lamina I projection cells were identified based on the observations made in the previous studies in the rat. Lamina I giant cells were recognized with antibodies against glycine receptor associated protein gephyrin as well as VGLUT2 and VGAT boutons, all of which provide dense innervation to these cells while only those lamina I NK1cells were included in the sample that were large and strongly immunoreactive for NK1r. The results indicated that although GFP axons accounted for only 7-9% of the GABAergic boutons in superficial dorsal horn, they provided over 70% of the inhibitory synapses on most of the giant cells in the PrP-GFP mouse and the great majority of these boutons also contained nNOS. Moreover, a subset of large lamina I NK1r-expressing cells (18/60) received a substantial inhibitory input (> 30%) from GFP+ boutons while the majority of these neurons showed sparse (< 15%) synaptic input. Recently, it has been reported that loss of some inhibitory interneurons in mice lacking the transcription factor Bhlhb5 results in exaggerated itch, and the cells that are lost include many of those that would normally express nNOS. Therefore, in the final set of experiments was designed to test whether there is a reduction in the inhibitory synaptic input to the giant cells in Bhlhb5-/- mouse. Spinal cord sections from Bhlhb5-/- mice and the wild type littermates were processed and analysed to determine any difference in the inhibitory nNOS input to lamina I giant cells belonging to either group. The giant cells from the knockout mice showed a substantial reduction (~80%) in their inhibitory nNOS input; with a moderate reduction in their overall GABAergic input (~35%). There was a considerable increase in nNOS-/VGAT+ boutons in the Bhlhb5-/- mouse (18 ± 4.6 and 37.7 ± 8.2/100 µm of the dendrite in WT and KO, respectively), suggesting some compensation from other nNOS-negative inhibitory interneurons. These results suggest that the loss of nNOS-containing inhibitory synaptic input to lamina I projection cells may contribute to the abnormal scratching behaviour seen in the Bhlhb5-/- mouse. This raises the possibility that the giant cells and a subset of large lamina I NK1r-expressing cells are involved in perception of itch.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Spinal Cord, neurokinin 1 receptor, itch, pain, dynorphin
Subjects: R Medicine > RC Internal medicine > RC0321 Neuroscience. Biological psychiatry. Neuropsychiatry
Colleges/Schools: College of Medical Veterinary and Life Sciences
Funder's Name: UNSPECIFIED
Supervisor's Name: Todd, Professor Andrew
Date of Award: 2014
Depositing User: Dr Najma Baseer
Unique ID: glathesis:2014-5596
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Oct 2014 14:11
Last Modified: 19 Mar 2015 11:20
URI: http://theses.gla.ac.uk/id/eprint/5596

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