Calretinin-expressing interneurons in the mouse spinal cord

Mustapa, Marami binti (2018) Calretinin-expressing interneurons in the mouse spinal cord. PhD thesis, University of Glasgow.

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Skin and organs in the body are innervated by primary afferents that respond to both noxious and non-noxious stimuli. The sensory information from the surrounding structures is transmitted into the dorsal horn by these afferents following their sensory modality and the region of the body that they innervate. The information is interpreted by neuronal circuits involving excitatory and inhibitory interneurons that lie in the dorsal horn of the spinal cord.
This current study involves characterisation of a specific population of inhibitory neurons that are presumably involved in presynaptic inhibition towards central terminal of non-peptidergic C fibres that terminate in a complex known as synaptic glomeruli (type I) in the middle of lamina II of the dorsal horn. The inhibition is formed through axoaxonic synapses on the central axons of these primary afferent fibres. A preliminary work carried out by Dr David I Hughes using CR::eGFP mouse line found that calcium binding protein, calretinin (CR) is expressed mostly by excitatory interneurons and a small percentage by inhibitory interneurons. Electron microscopic study was performed in these animals and he found that axonal boutons originated from dorsal horn calretinin cells were presynaptic to the central axon of type I glomeruli, which belong to CMrgD afferents. He also found that these cells have islet morphology which suggested that they are inhibitory cells.
The main objective of the project was to characterize a population of inhibitory CR interneurons in the superficial dorsal horn (SDH) and their synaptic connection with CMrgD primary afferent fibres. To achieve this several transgenic mouse lines that labelled inhibitory interneurons in the dorsal horn were used including lines in which Nociceptin and RorB transgenes were either expressed under the control of the regulatory elements of the Nociceptin gene or knocked into the RorB locus, respectively. Using Nociceptin::eGFP mice it was found that 93% of the cells that expressed eGFP were positive for Pax2 thus showed that these animals labelled inhibitory interneurons in the dorsal horn. In this mouse lines it was found that inhibitory CR cells in lamina II formed a band of dendrites and axons that perfectly overlapped with non-peptidergic C fibres plexus that were labelled with Isolectin B4 (IB4). The great majority of inhibitory interneurons (78%) in the SDH of Nociceptin::eGFP mice were immunoreactive for eGFP. Based on this result, Nociceptin::eGFP mice were then crossed with CRCre and Ai9 mice to allow reconstructions of individual neurons. It was found that inhibitory CR interneurons resemble islet cells and had dendrites projecting extensively in the rostrocaudal axis with a limited dorsoventral extent that arborized within lamina II. Due to a few limitations with Nociceptin::eGFP mice, this project was then continued with a knock-in mouse line, RorBeGFP mouse that also labelled inhibitory interneurons in the dorsal horn. It was found that more of lamina II inhibitory CR-immunoreactive cells were labelled in RorβeGFP mouse line than the Nociceptin::eGFP mouse, thus showed that RorBeGFP mice appeared to be a better choice for investigating the inhibitory CR cells than Nociceptin::eGFP mice.
In this study it was found that inhibitory CR cells do not co-expressed other neurochemical markers of the inhibitory interneurons such as galanin, nNOS, NPY and PV. This showed that CR represent another neurochemically distinct group of inhibitory interneurons in the dorsal horn. To investigate the synaptic connection between CR islet cells with CMrgD afferents, the third part of the study was carried out using the RorBCreERT2 mouse line with tamoxifen induction. In this study, it was found that intraperitoneal injection of tamoxifen in early stage of postnatal life resulted in more labelling of cells, meanwhile less cells were labelled if tamoxifen were given at the later stage of postnatal life. Surprisingly, under optimal conditions, only a quarter of the inhibitory CR cells in lamina II were immunoreactive for tdTomato. However, the majority of tdTomato cells in the SDH are inhibitory interneurons and immunoreactive for CR, thus showed that this mouse line was highly selective for inhibitory CR cells. Using this mouse line, it was found that most of CMrgD afferents central terminals were in contact with axonal boutons of inhibitory CR cells and the excitatory synapses on the dendritic trees of inhibitory CR cells were derived from CMrgD afferents. About 40% of the inhibitory CR axonal boutons are in contact with non-peptidergic C-nociceptors and electron microscopic revealed that the axons belonged to the inhibitory CR cells formed axoaxonic synapses with CMrgD afferents. A few studies claimed there is a loss of these cells after peripheral nerve injury. However, in this study there were no loss of cells seen following sciatic nerve transection with no alteration on the rostrocaudal and dorsoventral extent of inhibitory CR cells in the affected area on RorBCreERT2; Ai9 mice. The results from this study suggest that a subset of lamina II inhibitory interneurons that express CR form axoaxonic synapses on the central boutons of type I synaptic glomeruli which belong to CMrgD afferents and they also receive excitatory inputs from these primary afferents. As a conclusion, results from this study are in parallel with the recommendation that different neurochemical populations of inhibitory neurons have distinct functional roles and also emphasize the important of presynaptic inhibition on central terminals of CMrgD primary afferents in maintaining normal somatosensation.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Calretinin, inhibitory interneuron, spinal cord, superficial dorsal horn, pre-synaptic inhibition.
Subjects: R Medicine > RC Internal medicine > RC0321 Neuroscience. Biological psychiatry. Neuropsychiatry
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Psychology & Neuroscience > Centre for Neuroscience
Supervisor's Name: Todd, Professor Andrew
Date of Award: 2018
Depositing User: Marami Mustapa
Unique ID: glathesis:2018-41204
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 25 Apr 2019 12:37
Last Modified: 12 Jun 2019 12:44
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