MAB377 Sigma-AldrichAnti-NeuN Antibody, clone A60

The adult dentate gyrus produces new neurons that morphologically and functionally integrate into the hippocampal network. In the adult brain, most excitatory synapses are ensheathed by astrocytic perisynaptic processes that regulate synaptic structure and function. However, these processes are formed during embryonic or early postnatal development and it is unknown whether astrocytes can also ensheathe synapses of neurons born during adulthood and, if so, whether they play a role in their synaptic transmission. Here, we used a combination of serial-section immuno-electron microscopy, confocal microscopy, and electrophysiology to examine the formation of perisynaptic processes on adult-born neurons. We found that the afferent and efferent synapses of newborn neurons are ensheathed by astrocytic processes, irrespective of the age of the neurons or the size of their synapses. The quantification of gliogenesis and the distribution of astrocytic processes on synapses formed by adult-born neurons suggest that the majority of these processes are recruited from pre-existing astrocytes. Furthermore, the inhibition of astrocytic glutamate re-uptake significantly reduced postsynaptic currents and increased paired-pulse facilitation in adult-born neurons, suggesting that perisynaptic processes modulate synaptic transmission on these cells. Finally, some processes were found intercalated between newly formed dendritic spines and potential presynaptic partners, suggesting that they may also play a structural role in the connectivity of new spines. Together, these results indicate that pre-existing astrocytes remodel their processes to ensheathe synapses of adult-born neurons and participate to the functional and structural integration of these cells into the hippocampal network.

To investigate the effects of nicotine on retinal alterations in early-stage diabetes in an established rodent model.Sprague-Dawley rats were examined using a combination of confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography to determine changes in retinal structure in response to nicotine exposure, diabetes and the combined effects of nicotine and diabetes. Diabetes was induced by a single injection of 65 mg/kg streptozotocin and nicotine injections were administered subcutaneously daily. Retinal thickness in the superior, inferior, nasal and temporal quadrants were determined based on the spectral domain optical coherence tomography (SD-OCT) volume scans (20° × 20°) centered on the optic disc. Segmentation of discrete retinal layers was performed on a subset of SD-OCT cross-sections to further examine changes in each treatment group. Survival of neurons within the ganglion cell layer (GCL) was assessed by confocal morphometric imaging.The control group did not experience any significant change throughout the study. The nicotine treatment group experienced an average decrease in total retinal thickness (TRT) of 9.4 µm with the majority of the loss localized within the outer nuclear layer (ONL) as determined by segmentation analysis (p less than  0.05). The diabetic group exhibited a trend toward decreased TRT while segmentation analysis of the diabetic retinopathy (DR) group revealed significant thinning within the ONL (p less than  0.05). The combination of nicotine and diabetes revealed a significant increase of 8.9 µm in the TRT (p less than  0.05) accompanied by a decrease in the number of GCL neurons.We demonstrated significant temporal changes in retinal morphology in response to nicotine exposure, diabetes and with the combined effects of nicotine and diabetes. These findings may have implications in determining treatment strategies for diabetic patients using products containing nicotine, such as cigarettes, smokeless tobacco, electronic cigarettes or smoking cessation products.

Glycoprotein-deleted rabies virus (RABV ∆G) is a powerful tool for the analysis of neural circuits. Here, we demonstrate the utility of an anterograde RABV ∆G variant for novel neuroanatomical approaches involving either bulk or sparse neuronal populations. This technology exploits the unique features of RABV ∆G vectors, namely autonomous, rapid high-level expression of transgenes, and limited cytotoxicity. Our vector permits the unambiguous long-range and fine-scale tracing of the entire axonal arbor of individual neurons throughout the brain. Notably, this level of labeling can be achieved following infection with a single viral particle. The vector is effective over a range of ages (greater than 14 months) aiding the studies of neurodegenerative disorders or aging, and infects numerous cell types in all brain regions tested. Lastly, it can also be readily combined with retrograde RABV ∆G variants. Together with other modern technologies, this tool provides new possibilities for the investigation of the anatomy and physiology of neural circuits.

Adult animals continue to modify their behavior throughout life, a process that is highly influenced by past experiences. To shape behavior, specific mechanisms of neural plasticity to learn, remember, and recall information are required. One of the most robust examples of adult plasticity in the brain occurs in the dentate gyrus (DG) of the hippocampus, through the process of adult neurogenesis. Adult neurogenesis is strongly upregulated by external factors such as voluntary wheel running (RUN) and environmental enrichment (EE); however, the functional differences between these two factors remain unclear. Although both manipulations result in increased neurogenesis, RUN dramatically increases the proliferation of newborn cells and EE promotes their survival. We hypothesize that the method by which these newborn neurons are induced influences their functional role. Furthermore, we examine how EE-induced neurons may be primed to encode and recognize features of novel environments due to their previous enrichment experience. Here, we gave mice a challenging contextual fear-conditioning (FC) procedure to tease out the behavioral differences between RUN-induced neurogenesis and EE-induced neurogenesis. Despite the robust increases in neurogenesis seen in the RUN mice, we found that only EE mice were able to discriminate between similar contexts in this task, indicating that EE mice might use a different cognitive strategy when processing contextual information. Furthermore, we showed that this improvement was dependent on EE-induced neurogenesis, suggesting a fundamental functional difference between RUN-induced neurogenesis and EE-induced neurogenesis.

The local renin-angiotensin system has been held to be expressed in many organs, including the eye. It has an important role in the regulation of local fluid homeostasis, cell proliferation, fibrosis, and vascular tone. Mas-receptor (Mas-R) is a potential receptor acting mainly opposite to the well-known angiotensin II receptor type 1. The aim of this study was to determine if Mas-R is expressed in the human eye.Seven enucleated human eyes were used in immunohistochemical detection of Mas-R and its endogenous ligand angiotensin (1-7) [Ang(1-7)]. Both light microscopy and immunofluorescent detection methods were used. A human kidney preparation sample was used as control.The Mas-R was found to have nuclear localization, and localized in the retinal nuclear layers and in the structures of the anterior segment of the eye. A cytoplasmic immunostaining pattern of Ang(1-7) was found in the inner and outer nuclear and plexiform layers of the retina and in the ciliary body.To the best of our knowledge, this is the first report showing Mas-R expression in the human eye. Its localization suggests that it may have a role in physiological and pathological processes in the anterior part of the eye and in the retina.

Alternative splicing (AS) generates vast transcriptomic complexity in the vertebrate nervous system. However, the extent to which trans-acting splicing regulators and their target AS regulatory networks contribute to nervous system development is not well understood. To address these questions, we generated mice lacking the vertebrate- and neural-specific Ser/Arg repeat-related protein of 100 kDa (nSR100/SRRM4). Loss of nSR100 impairs development of the central and peripheral nervous systems in part by disrupting neurite outgrowth, cortical layering in the forebrain, and axon guidance in the corpus callosum. Accompanying these developmental defects are widespread changes in AS that primarily result in shifts to nonneural patterns for different classes of splicing events. The main component of the altered AS program comprises 3- to 27-nucleotide (nt) neural microexons, an emerging class of highly conserved AS events associated with the regulation of protein interaction networks in developing neurons and neurological disorders. Remarkably, inclusion of a 6-nt, nSR100-activated microexon in Unc13b transcripts is sufficient to rescue a neuritogenesis defect in nSR100 mutant primary neurons. These results thus reveal critical in vivo neurodevelopmental functions of nSR100 and further link these functions to a conserved program of neuronal microexon splicing.

Microglia are the primary immune cell in the brain and are postulated to play important roles outside of immunity. Administration of the dual colony-stimulating factor 1 receptor (CSF1R)/c-Kit kinase inhibitor, PLX3397, to adult mice results in the elimination of ~99% of microglia, which remain eliminated for as long as treatment continues. Upon removal of the inhibitor, microglia rapidly repopulate the entire adult brain, stemming from a central nervous system (CNS) resident progenitor cell. Using this method of microglial elimination and repopulation, the role of microglia in both healthy and diseased states can be explored. Here, we examine the responsiveness of newly repopulated microglia to an inflammatory stimulus, as well as determine the impact of these cells on behavior, cognition, and neuroinflammation. Two month-old wild-type mice were placed on either control or PLX3397 diet for 21 d to eliminate microglia. PLX3397 diet was then removed in a subset of animals to allow microglia to repopulate and behavioral testing conducted beginning at 14 d repopulation. Finally, inflammatory profiling of the microglia-repopulated brain in response to lipopolysaccharide (LPS; 0.25 mg/kg) or phosphate buffered saline (PBS) was determined 21 d after inhibitor removal using quantitative real time polymerase chain reaction (RT-PCR), as well as detailed analyses of microglial morphologies. We find mice with repopulated microglia to perform similarly to controls by measures of behavior, cognition, and motor function. Compared to control/resident microglia, repopulated microglia had larger cell bodies and less complex branching in their processes, which resolved over time after inhibitor removal. Inflammatory profiling revealed that the mRNA gene expression of repopulated microglia was similar to normal resident microglia and that these new cells appear functional and responsive to LPS. Overall, these data demonstrate that newly repopulated microglia function similarly to the original resident microglia without any apparent adverse effects in healthy adult mice.

Genetic lineage tracing with electroporation is one of the most powerful techniques to target neural progenitor cells and their progeny. However, the spatiotemporal relationship between neural progenitors and their final phenotype remain poorly understood. One critical factor to analyze the cell fate of progeny is reporter integration into the genome of transfected cells. To address this issue, we performed postnatal and in utero co-electroporations of different fluorescent reporters to label, in both cerebral cortex and olfactory bulb, the progeny of subventricular zone neural progenitors. By comparing fluorescent reporter expression in the adult cell progeny, we show a differential expression pattern within the same cell lineage, depending on electroporation stage and cell identity. Further, while neuronal lineages arise from many progenitors in proliferative zones after few divisions, glial lineages come from fewer progenitors that accomplish many cell divisions. Together, these data provide a useful guide to select a strategy to track the cell fate of a specific cell population and to address whether a different proliferative origin might be correlated with functional heterogeneity.

Selective neuronal loss (SNL) in the reperfused penumbra may impact clinical recovery and is thus important to investigate. Brief proximal middle cerebral artery occlusion (MCAo) results in predominantly striatal SNL, yet cortical damage is more relevant given its behavioral implications and that thrombolytic therapy mainly rescues the cortex. Distal temporary MCAo (tMCAo) does target the cortex, but the optimal occlusion duration that results in isolated SNL has not been determined. In the present study, we assessed different distal tMCAo durations looking for consistently pure SNL.Microclip distal tMCAo (md-tMCAo) was performed in ~6-month old male spontaneously hypertensive rats (SHRs). We previously reported that 45 min md-tMCAo in SHRs results in pan-necrosis in the majority of subjects. Accordingly, three shorter MCAo durations were investigated here in decremental succession, namely 30, 22, and 15 min (n = 3, 3, and 7 subjects, respectively). Recanalization was confirmed by MR angiography just prior to brain collection at 28 days and T2-weighted MRI was obtained for characterization of ischemic lesions. NeuN, OX42, and GFAP immunohistochemistry appraised changes in neurons, microglia, and astrocytes, respectively. Ischemic lesions were categorized into three main types: (1) pan-necrosis; (2) partial infarction; and (3) SNL.Pan-necrosis or partial infarction was present in all 30 min and 22 min subjects, but not in the 15 min group (p less than  0.001), in which isolated cortical SNL was consistently present. MRI revealed characteristic hyperintense abnormalities in all rats with pan-necrosis or partial infarction, but no change in any 15 min subject.We found that 15 min distal MCAo consistently resulted in pure cortical SNL, whereas durations equal or longer than 22 min consistently resulted in infarcts. This model may be of use to study the pathophysiology of cortical SNL and its prevention by appropriate interventions.

Endocannabinoids participate in the control of neurogenesis, neural cell death and gliosis. The pharmacological effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which limits the endocannabinoid degradation, was investigated in the present study. Cell proliferation (phospho-H3(+) or BrdU(+) cells) of the main adult neurogenic zones as well as apoptosis (cleaved caspase-3(+)), astroglia (GFAP(+)), and microglia (Iba1(+) cells) were analyzed in the hippocampus, hypothalamus and striatum of rats intraperitoneally treated with URB597 (0.3 mg/kg/day) at one dose/4-days resting or 5 doses (1 dose/day). Repeated URB597 treatment increased the plasma levels of the N-acylethanolamines oleoylethanolamide, palmitoylethanolamide and arachidonoylethanolamine, reduced the plasma levels of glucose, triglycerides and cholesterol, and induced a transitory body weight decrease. The hippocampi of repeated URB597-treated rats showed a reduced number of phospho-H3(+) and BrdU(+) subgranular cells as well as GFAP(+), Iba1(+) and cleaved caspase-3(+) cells, which was accompanied with decreased hippocampal expression of the cannabinoid CB1 receptor gene Cnr1 and Faah. In the hypothalami of these rats, the number of phospho-H3(+), GFAP(+) and 3-weeks-old BrdU(+) cells was specifically decreased. The reduced striatal expression of CB1 receptor in repeated URB597-treated rats was only associated with a reduced apoptosis. In contrast, the striatum of acute URB597-treated rats showed an increased number of subventricular proliferative, astroglial and apoptotic cells, which was accompanied with increased Faah expression. Main results indicated that FAAH inhibitor URB597 decreased neural proliferation, glia and apoptosis in a brain region-dependent manner, which were coupled to local changes in Faah and/or Cnr1 expression and a negative energy context.