In Situ Analysis of the Mu-Opioid Receptor Splice Variants in Adult Rat Brain

<p>The original intention of this study was to exploit the specificity of circularisable ligation probes (CLiPs) in a unique approach of in situ genotyping the mu-opioid receptor (MOR) splice variants. CLiPs were designed to target a PCR generated MOR-1 template in vivo. The ligation results were consistent with circularised CLiPs, however due to the inherent limitations of this method the more conventional technique of fluorescent in situ hybridisation (FISH) was substituted for CLiPs to analyse to distribution of MOR splice variants in rat brain. Utilising FISH, the aim was to produce RNA probes (riboprobes) approximately the same size as the target specific region of CLiPs (~60-70 nt) to analyse the distribution patterns of MOR splice variants in rat brain. Five short (70-222 nt) riboprobes were generated to exons 1, 3, 4 and 9, and the 5' UTR + exon 1 of the Rattus norvegicus MOR gene (Oprm) to be utilised in FISH. The exon 1, 4 and 5' UTR + exon 1 riboprobes were shown to localise to MOR mRNA in brain structures previously reported to express MORs. These riboprobes also localised to mRNA within the Purkinje cells of the adult rat cerebellum, where it is generally accepted that only DOR is expressed in the rat cerebellum. MOR mRNA was visualised in many structures in the rat brain, including the dendate gyrus, inferior olive and spinal trigeminal nucleus. Riboprobes generated to the 5' UTR + exon 1 and exon 4 showed differential distribution patterns, the functional significance of this discovery is unknown, however these results implicate a role for FISH in tracking the distribution patterns of untranslated and translated mRNA. The use of novel new short riboprobes represents a technically difficult yet feasible technique for mapping MOR mRNA distribution in adult rat brain.</p>

In Situ Analysis of the Mu-Opioid Receptor Splice Variants in Adult Rat Brain

<p>The original intention of this study was to exploit the specificity of circularisable ligation probes (CLiPs) in a unique approach of in situ genotyping the mu-opioid receptor (MOR) splice variants. CLiPs were designed to target a PCR generated MOR-1 template in vivo. The ligation results were consistent with circularised CLiPs, however due to the inherent limitations of this method the more conventional technique of fluorescent in situ hybridisation (FISH) was substituted for CLiPs to analyse to distribution of MOR splice variants in rat brain. Utilising FISH, the aim was to produce RNA probes (riboprobes) approximately the same size as the target specific region of CLiPs (~60-70 nt) to analyse the distribution patterns of MOR splice variants in rat brain. Five short (70-222 nt) riboprobes were generated to exons 1, 3, 4 and 9, and the 5' UTR + exon 1 of the Rattus norvegicus MOR gene (Oprm) to be utilised in FISH. The exon 1, 4 and 5' UTR + exon 1 riboprobes were shown to localise to MOR mRNA in brain structures previously reported to express MORs. These riboprobes also localised to mRNA within the Purkinje cells of the adult rat cerebellum, where it is generally accepted that only DOR is expressed in the rat cerebellum. MOR mRNA was visualised in many structures in the rat brain, including the dendate gyrus, inferior olive and spinal trigeminal nucleus. Riboprobes generated to the 5' UTR + exon 1 and exon 4 showed differential distribution patterns, the functional significance of this discovery is unknown, however these results implicate a role for FISH in tracking the distribution patterns of untranslated and translated mRNA. The use of novel new short riboprobes represents a technically difficult yet feasible technique for mapping MOR mRNA distribution in adult rat brain.</p>

Essential roles of plexin-B3+ oligodendrocyte precursor cells in the pathogenesis of Alzheimer’s disease

The role of oligodendrocyte lineage cells, the largest glial population in the adult central nervous system (CNS), in the pathogenesis of Alzheimer’s disease (AD) remains elusive. Here, we developed a culture method for adult oligodendrocyte progenitor cells (aOPCs). Fibroblast growth factor 2 (FGF2) promotes survival and proliferation of NG2+ aOPCs in a serum-free defined medium; a subpopulation (~5%) of plexin-B3+ aOPCs was also found. FGF2 withdrawal decreased NG2+, but increased plexin-B3+ aOPCs and Aβ1-42 secretion. Plexin-B3+ aOPCs were distributed throughout the adult rat brain, although less densely than NG2+ aOPCs. Spreading depolarization induced delayed cortical plexin-B3+ aOPC gliosis in the ipsilateral remote cortex. Furthermore, extracellular Aβ1-42 accumulation was occasionally found around plexin-B3+ aOPCs near the lesions. In AD brains, virtually all cortical SPs were immunostained for plexin-B3, and plexin-B3 levels increased significantly in the Sarkosyl-soluble fractions. These findings suggest that plexin-B3+ aOPCs may play essential roles in AD pathogenesis, as natural Aβ-secreting cells.

Complete spatial characterisation of N-glycosylation upon striatal neuroinflammation in the rodent brain

Background Neuroinflammation is an underlying pathology of all neurological conditions, the understanding of which is still being comprehended. A specific molecular pathway that has been overlooked in neuroinflammation is glycosylation (i.e., post-translational addition of glycans to the protein structure). N-glycosylation is a specific type of glycosylation with a cardinal role in the central nervous system (CNS), which is highlighted by congenital glycosylation diseases that result in neuropathological symptoms such as epilepsy and mental retardation. Changes in N-glycosylation can ultimately affect glycoproteins’ functions, which will have an impact on cell machinery. Therefore, characterisation of N-glycosylation alterations in a neuroinflammatory scenario can provide a potential target for future therapies. Methods With that aim, the unilateral intrastriatal injection of lipopolysaccharide (LPS) in the adult rat brain was used as a model of neuroinflammation. In vivo and post-mortem, quantitative and spatial characterisation of both neuroinflammation and N-glycome was performed at 1-week post-injection of LPS. These aspects were investigated through a multifaceted approach based on positron emission tomography (PET), quantitative histology, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), liquid chromatography and matrix-assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI). Results In the brain region showing LPS-induced neuroinflammation, a significant decrease in the abundance of sialylated and core fucosylated structures was seen (approximately 7.5% and 8.5%, respectively), whereas oligomannose N-glycans were significantly increased (13.5%). This was confirmed by MALDI-MSI, which provided a high-resolution spatial distribution of N-glycans, allowing precise comparison between normal and diseased brain hemispheres. Conclusions Together, our data show for the first time the complete profiling of N-glycomic changes in a well-characterised animal model of neuroinflammation. These data represent a pioneering step to identify critical targets that may modulate neuroinflammation in neurodegenerative diseases.

Complete Spatial Characterisation of N-glycosylation upon Striatal Neuroinflammation in the Rodent Brain

Background: Neuroinflammation is an underlying pathology of all neurological conditions, the understanding of which is still being comprehended. A specific molecular pathway that has been overlooked in neuroinflammation is glycosylation (i.e. post-translational addition of glycans to the protein structure). N- glycosylation is a specific type of glycosylation with a cardinal role in the central nervous system (CNS), which is highlighted by congenital glycosylation diseases that result in neuropathological symptoms such as epilepsy and mental retardation. Changes in N- glycosylation can ultimately affect glycoproteins' functions, which will have an impact on cell machinery. Therefore characterisation of N- glycosylation alterations in a neuroinflammatory scenario can provide a potential target for future therapies. Methods: With that aim, the unilateral intrastriatal injection of Lipopolysaccharide (LPS) in the adult rat brain was used as a model of neuroinflammation. In vivo and post-mortem, quantitative and spatial characterisation of both neuroinflammation and N- glycome was performed at one-week post-injection of LPS. These aspects were investigated through a multifaceted approach based on positron emission tomography (PET), quantitative histology, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), liquid chromatography and matrix-assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI). Results: In the brain region showing LPS-induced neuroinflammation, a significant decrease in the abundance of sialylated and core fucosylated structures was seen (approximately 7.5% and 8.5%, respectively), whereas oligomannose N- glycans were significantly increased (13.5%). This was confirmed by MALDI-MSI, which provided a high-resolution spatial distribution of N- glycans allowing precise comparison between normal and diseased brain hemispheres. Conclusions: Together, our data show for the first time the complete profiling of N- glycomic changes in a well-characterised animal model of neuroinflammation. These data represent a pioneering step to identify critical targets that may modulate neuroinflammation in neurodegenerative diseases.

Photoperiod-Induced Neurotransmitter Switching in the Circadian Pacemaker Regulates Hypothalamic Dopamine Expression

Light, circadian clocks, and rhythmic behaviors interact closely to produce a temporal order that is essential for the survival of most living organisms. In mammals, the principal circadian pacemaker in the brain is the suprachiasmatic nucleus (SCN), which receives direct retinal input and synchronizes itself and other brain regions to the external light-dark cycle. Altered day length (photoperiod) and disrupted circadian rhythms are associated with impaired memory and mood in both humans and animal models. Prior work demonstrated that altering photoperiod can change neurotransmitter (NT) expression in the periventricular nucleus (PeVN) of the hypothalamus in adult rat brain. Here we show that neuromedin S-(NMS-) and vasoactive intestinal polypeptide-(VIP-) expressing neurons in the SCN also display photoperiod-induced neurotransmitter switching. Such photoperiod-dependent NT plasticity is retained in Bmal1-KO mice, indicating that NT plasticity in the SCN does not require a functional circadian clock. Utilizing a conditional viral DO-DIO vector as an historical marker of NT expression in the SCN, we further reveal that short-day photoperiod induces a cluster of non-NMS-expressing neurons to undergo NT switching and acquire the NMS phenotype. Selective chemogenetic activation of NMS neurons, but not VIP neurons, during the dark phase induces a significant delay in the timing of locomotor activity onset and is sufficient to increase the number of dopaminergic neurons in the PeVN. Our findings provide novel insights into molecular adaptations of the SCN neuronal network in response to altered photoperiod that affect neuronal circuit function in the hypothalamus and lead to changes in circadian behavior.

The distribution of oxytocin and the oxytocin receptor in rat brain: relation to regions active in migraine

Background Recent work, both clinical and experimental, suggests that the hypothalamic hormone oxytocin (OT) and its receptor (OTR) may be involved in migraine pathophysiology. In order to better understand possible central actions of OT in migraine/headache pathogenesis, we mapped the distribution of OT and OTR in nerve cells and fibers in rat brain with a focus on areas related to migraine attacks and/or shown previously to contain calcitonin gene related peptide (CGRP), another neuropeptide involved in migraine. Methods Distribution of OT and OTR in the adult, rat brain was qualitatively examined with immunohistochemistry using a series of well characterized specific antibodies. Results As expected, OT was extensively localized in the cell somas of two hypothalamic nuclei, the supraoptic (SO or SON) and paraventricular nuclei (Pa or PVN). OT also was found in many other regions of the brain where it was localized mainly in nerve fibers. In contrast, OTR staining in the brain was mainly observed in cell somas with very little expression in fibers. The most distinct OTR expression was found in the hippocampus, the pons and the substantia nigra. In some regions of the brain (e.g. the amygdala and the hypothalamus), both OT and OTR were expressed (match). Mismatch between the peptide and its receptor was primarily observed in the cerebral and cerebellar cortex (OT expression) and hippocampus (OTR expression). Conclusions We compared OT/OTR distribution in the CNS with that of CGRP and identified regions related to migraine. In particular, regions suggested as “migraine generators”, showed correspondence among the three mappings. These findings suggest central OT pathways may contribute to the role of the hypothalamus in migraine attacks.