Probes for GFAP

Some compulsive disorders have been considered to stem from the loss of control over coping strategies, such as displacement. However, the cellular mechanisms involved in the acquisition of coping behaviours and their subsequent compulsive manifestation in vulnerable individuals have not been elucidated. Considering the role of the locus coeruleus (LC) noradrenaline-dependent system in stress and related excessive behaviours, we hypothesised that neuroplastic changes in the LC may be associated with the acquisition of an adjunctive polydipsic water drinking, a prototypical displacement behaviour, and the ensuing development of compulsion in vulnerable individuals. Thus, male Sprague Dawley rats were characterised for their tendency, or not, to develop compulsive polydipsic drinking in a schedule-induced polydipsia (SIP) procedure before their fresh brains were harvested. A new quantification tool for RNAscope assays revealed that the development of compulsive adjunctive behaviour was associated with a low mRNA copy number of the plasticity marker Arc in the LC which appeared to be driven by specific adaptations in an ensemble of tyrosine hydroxylase (TH)+, zif268- neurons. This ensemble was specifically engaged by the expression of compulsive adjunctive behaviour, not by stress, because its functional recruitment was not observed in individuals that no longer had access to the water bottle before sacrifice, while it consistently correlated with the levels of polydipsic water drinking only when it had become compulsive. Together these findings suggest that downregulation of Arc mRNA levels in a population of a TH+/zif268- LC neurons represents a signature of the tendency to develop compulsive coping behaviours.

Following injury in the central nervous system, a population of astrocytes occupy the lesion site, form glial bridges and facilitate axon regeneration. These astrocytes originate primarily from resident astrocytes or NG2+ oligodendrocyte progenitor cells. However, the extent to which these cell types give rise to the lesion-filling astrocytes, and whether the astrocytes derived from different cell types contribute similarly to optic nerve regeneration remain unclear. Here we examine the distribution of astrocytes and NG2+ cells in an optic nerve crush model. We show that optic nerve astrocytes partially fill the injury site over time after a crush injury. Viral mediated expression of a growth-promoting factor, ciliary neurotrophic factor (CNTF), in retinal ganglion cells (RGCs) promotes axon regeneration without altering the lesion size or the degree of lesion-filling GFAP+ cells. Strikingly, using inducible NG2CreER driver mice, we found that CNTF overexpression in RGCs increases the occupancy of NG2+ cell-derived astrocytes in the optic nerve lesion. An EdU pulse-chase experiment shows that the increase in NG2 cell-derived astrocytes is not due to an increase in cell proliferation. Lastly, we performed RNA-sequencing on the injured optic nerve and reveal that CNTF overexpression in RGCs results in significant changes in the expression of distinct genes, including those that encode chemokines, growth factor receptors, and immune cell modulators. Even though CNTF-induced axon regeneration has long been recognized, this is the first evidence of this procedure affecting glial cell fate at the optic nerve crush site. We discuss possible implication of these results for axon regeneration.

The pro-inflammatory role of interleukin-6 (IL-6) is well-characterized. Blockade of IL-6, by Tocilizumab, is used in patients with rheumatoid arthritis and those diagnosed with cytokine storm. However, brain-produced IL-6 has recently emerged as a critical mediator of gut/adipose communication with the brain. Central nervous system (CNS) IL-6 is engaged by peripheral and central signals regulating energy homeostasis. IL-6 is critical for mediating hypophagia and weight loss effects of a GLP-1 analog, exendin-4, a clinically utilized drug. However, neuroanatomical substrates and behavioral mechanisms of brain IL-6 energy balance control remain poorly understood. We propose that the lateral hypothalamus (LH) is an IL-6-harboring brain region, key to food intake and food reward control. Microinjections of IL-6 into the LH reduced chow and palatable food intake in male rats. In contrast, female rats responded with reduced motivated behavior for sucrose, measured by the progressive ratio operant conditioning test, a behavioral mechanism previously not linked to IL-6. To test whether IL-6, produced in the LH, is necessary for ingestive and motivated behaviors, and body weight homeostasis, virogenetic knockdown by infusion of AAV-siRNA-IL6 into the LH was utilized. Attenuation of LH IL-6 resulted in a potent increase in sucrose-motivated behavior, without any effect on ingestive behavior or body weight in female rats. In contrast, the treatment did not affect any parameters measured (chow intake, sucrose-motivated behavior, locomotion, and body weight) in chow-fed males. However, when challenged with a high-fat/high-sugar diet, the male LH IL-6 knockdown rats displayed rapid weight gain and hyperphagia. Together, our data suggest that LH-produced IL-6 is necessary and sufficient for ingestive behavior and weight homeostasis in male rats. In females, IL-6 in the LH plays a critical role in food-motivated, but not ingestive behavior control or weight regulation. Thus, collectively these data support the idea that brain-produced IL-6 engages the hypothalamus to control feeding behavior.

Neuron navigator 2 (NAV2, RAINB1, POMFIL2, HELAD1, unc53H2) is essential for nervous system development. In the present study the spatial distribution of Nav2 transcript in mouse CNS during embryonic, postnatal and adult life is examined. Because multiple NAV2 proteins are predicted based on alternate promoter usage and RNA splicing, in situ hybridization was performed using probes designed to the 5' and 3' ends of the Nav2 transcript, and PCR products using primer sets spanning the length of the mRNA were also examined by real time PCR (qPCR). These studies support full-length Nav2 transcript as the predominant form in the wild-type mouse CNS. The developing cortex, hippocampus, thalamus, olfactory bulb, and granule cells (GC) within the cerebellum show the highest expression, with a similar staining pattern using either the 5'Nav2 or 3'Nav2 probe. Nav2 is expressed in GC precursors migrating over the cerebellar primordium as well as in the postmitotic premigratory cells of the external granule cell layer (EGL). It is expressed in the cornu ammonis (CA) and dentate gyrus (DG) throughout hippocampal development. In situ hybridization was combined with immunohistochemistry for Ki67, CTIP2 and Nissl staining to follow Nav2 transcript location during cortical development, where it is observed in neuroepithelial cells exiting the germinal compartments, as well as later in the cortical plate (CP) and developing cortical layers. The highest levels of Nav2 in all brain regions studied are observed in late gestation and early postnatal life which coincides with times when neurons are migrating and differentiating. A hypomorphic mouse that lacks the full-length transcript but expresses shorter transcript shows little staining in the CNS with either probe set except at the base of the cerebellum, where a shorter Nav2 transcript is detected. Using dual fluorescent probe in situ hybridization studies, these cells are identified as oligodendrocytes and are detected using both Olig1 and the 3'Nav2 probe. The identification of full-length Nav2 as the primary transcript in numerous brain regions suggests NAV2 could play a role in CNS development beyond that of its well-established role in the cerebellum

Nonarteritic anterior ischemic optic neuropathy (NAION) is a common acute optic neuropathy and cause of irreversible vision loss in those older than 50 years of age. There is currently no effective treatment for NAION and yet the biological mechanisms leading to neuronal loss are not fully understood. Glial cells activation and intercommunication mediated by molecules such as gap junction protein Connexin 43 (Cx43) is thought to modulate neuronal fate in central nervous system disorders. In this study, we investigated retinal glial changes and neuronal loss following a novel NAION animal model using a 577 nm laser. We induced unilateral photochemical thrombosis using rose bengal at the optic nerve head vasculature in adult C57BL/6 mice using a 577 nm laser and performed morphometric analysis of the retinal structure using serial in vivo optical coherence tomography (OCT) and histology for glial and neuronal markers. OCT imaging revealed peripapillary thickening of the retinal ganglion cell complex (GCC, baseline: 79.5 ± 1.0 μm, n = 8; NAION: 93.0 ± 2.5 μm, n = 8, P < 0.01) and total retina (baseline: 202.9 ± 2.4 μm, n = 8; NAION: 228.1 ± 6.8 μm, n = 8, P < 0.01) at day 1 after NAION, and significant GCC thinning (baseline 78.3 ± 2.1 μm, n = 6; NAION: 72.2 ± 1.9 μm, n = 5, P < 0.05) at day 21. NAION induced a significant increase in retinal VEGF levels at day 1 (control: 2319 ± 195, n = 5; NAION: 4549 ± 683 gray mean value, n = 5, P < 0.05), which correlated with retinal thickness (r = 0.89, P < 0.05). NAION led to increased mRNA levels for Cx43 (Gj1a) at day 1 (control: 1.291 ± 0.38; NAION: 3.360 ± 0.58 puncta/mm2, n = 5, P < 0.05), which was not associated with changes in mRNA levels of glial fibrillary acidic protein (Gfap) at the same time (control: 2800 ± 0.59; NAION: 4690 ± 0.90 puncta/mm2 n = 5, P = 0.19). Retinal ganglion cell loss at day 21 was confirmed by a 30% decrease in Brn3a+ cells (control: 2844 ± 235; NAION: 2001 ± 264 cells/mm2, n = 4, P < 0.05). We described a novel protocol of NAION induction by photochemical thrombosis using a 577 nm laser, leading to retinal edema and VEGF increase at day 1 and RGCs loss at day 21 after injury, consistent with the pathophysiology of human NAION. Early changes in glial cells intercommunication revealed by increased Cx43+ gap junctions are consistent with a retinal glial role in mediating cell-to-cell signaling after an ischemic insult. Our study demonstrates an early glial response in a novel NAION animal model and reveals glial intercommunication molecules such as Cx43 as a promising therapeutic target in acute NAION.