MAB377 Sigma-AldrichAnti-NeuN Antibody, clone A60

Active sensing involves the fusion of internally generated motor events with external sensation. For rodents, active somatosensation includes scanning the immediate environment with the mystacial vibrissae. In doing so, the vibrissae may touch an object at any angle in the whisk cycle. The representation of touch and vibrissa self-motion may in principle be encoded along separate pathways, or share a single pathway, from the periphery to cortex. Past studies established that the spike rates in neurons along the lemniscal pathway from receptors to cortex, which includes the principal trigeminal and ventral-posterior-medial thalamic nuclei, are substantially modulated by touch. In contrast, spike rates along the paralemniscal pathway, which includes the rostral spinal trigeminal interpolaris, posteromedial thalamic, and ventral zona incerta nuclei, are only weakly modulated by touch. Here we find that neurons along the lemniscal pathway robustly encode rhythmic whisking on a cycle-by-cycle basis, while encoding along the paralemniscal pathway is relatively poor. Thus, the representations of both touch and self-motion share one pathway. In fact, some individual neurons carry both signals, so that upstream neurons with a supralinear gain function could, in principle, demodulate these signals to recover the known decoding of touch as a function of vibrissa position in the whisk cycle.

The role of microglia in amyloid-β (Aβ) deposition is controversial. In the present study, an organotypic hippocampal slice culture (OHSC) system with an in vivo-like microglial-neuronal environment was used to investigate the potential contribution of microglia to Aβ plaque formation. We found that microglia ingested Aβ, thereby preventing plaque formation in OHSCs. Conversely, Aβ deposits formed rapidly in microglia-free wild-type slices. The capacity to prevent Aβ plaque formation was absent in forebrain microglia from young adult but not juvenile 5xFamilial Alzheimer's disease (FAD) mice. Since no loss of Aβ clearance capacity was observed in both wild-type and cerebellar microglia from 5xFAD animals, the high Aβ1-42 burden in the forebrain of 5xFAD animals likely underlies the exhaustion of microglial Aβ clearance capacity. These data may therefore explain why Aβ plaque formation has never been described in wild-type mice, and point to a beneficial role of microglia in AD pathology. We also describe a new method to study Aβ plaque formation in a cell culture setting.

We recently demonstrated that human C-reactive protein (CRP), expressed hepatically in transgenic mice (CRPtg), improved the outcome of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). The liver is the primary site of CRP synthesis in humans and in CRPtg mice but is also expressed by both at low levels in the CNS. To determine if CNS expression of human CRP is sufficient to impact EAE, we generated neuronal CRP transgenic mice (nCRPtg) wherein human CRP expression is driven by the neuron-specific Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) gene promoter. We found that hepatically expressed/blood-borne CRP, but not CNS expressed CRP, lessened EAE severity. These outcomes indicate that the protective actions of human CRP in EAE are manifested in the periphery and not in the CNS and reveal a previously unappreciated site specificity for the beneficial actions of CRP in CNS disease.

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.

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.

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.

Status epilepticus (SE) can cause neuronal cell death and impaired behavioral function. Acute exposure to potent acetylcholinesterase inhibitors such as soman (GD) can cause prolonged SE activity, micro-hemorrhage and cell death in the hippocampus, thalamus and piriform cortex. Neuroinflammation is a prominent feature of brain injury with upregulation of multiple pro-inflammatory cytokines including those of the IL-1 family. The highly pleiotropic pro-inflammatory cytokine interleukin-18 (IL-18) belongs to the IL-1 family of cytokines and can propagate neuroinflammation by promoting immune cell infiltration, leukocyte and lymphocyte activation, and angiogenesis and helps facilitate the transition from the innate to the adaptive immune response. The purpose of this study is to characterize the regional and temporal expression of IL -18 and related factors in the brain following SE in a rat GD seizure model followed by localization of IL-18 to specific cell types.The protein levels of IL-18, vascular endothelial growth factor and interferon gamma was quantified in the lysates of injured brain regions up to 72 h following GD-induced SE onset using bead multiplex immunoassays. IL-18 was localized to various cell types using immunohistochemistry and transmission electron microscopy. In addition, macrophage appearance scoring and T-cell quantification was determined using immunohistochemistry. Micro-hemorrhages were identified using hematoxylin and eosin staining of brain sections.Significant increases in IL-18 occurred in the piriform cortex, hippocampus and thalamus following SE. IL-18 was primarily expressed by endothelial cells and astrocytes associated with the damaged neurovascular unit. The increase in IL-18 was not related to macrophage accumulation, neutrophil infiltration or T-cell appearance in the injured tissue.These data show that IL-18 is significantly upregulated following GD-induced SE and localized primarily to endothelial cells in damaged brain vasculature. IL-18 upregulation occurred following leukocyte/lymphocyte infiltration and in the absence of other IL-18-related cytokines, suggesting another function, potentially for angiogenesis related to GD-induced micro-hemorrhage formation. Further studies at more chronic time points may help to elucidate this function.

Inhibitory neurons make up a substantial fraction of the neurons in the preBötzinger complex (preBötC), a site that is critical for mammalian eupneic breathing. We investigated the role of glycinergic preBötC neurons in respiratory rhythmogenesis in mice using optogenetically targeted excitation and inhibition. Channelrhodopsin-2 (ChR2) or Archaerhodopsin (Arch) were expressed in glycinergic preBötC neurons of glycine transporter 2 (Glyt2, also known as Slc6a5)-Cre mice. In ChR2-transfected mice, brief inspiratory-phase bilateral photostimulation targeting the preBötC prematurely terminated inspiration, whereas expiratory-phase photostimulation delayed the onset of the next inspiration. Prolonged photostimulation produced apneas lasting as long as the light pulse. Inspiratory-phase photoinhibition in Arch-transfected mice during inspiration increased tidal volume without altering inspiratory duration, whereas expiratory-phase photoinhibition shortened the latency until the next inspiration. During persistent apneas, prolonged photoinhibition restored rhythmic breathing. We conclude that glycinergic preBötC neurons modulate inspiratory pattern and are important for reflex apneas, but that the rhythm can persist after substantial dampening of their activity.

Autophagy is a homeostatic degradative process essential for basal turnover of long-lived proteins and organelles as well as for removal of dysfunctional cellular components. Dysregulation of the autophagic machinery has been recently associated to several conditions including neurodegenerative diseases and cancer, but only very few studies have investigated its role in pain processing.We previously described autophagy impairment at the spinal cord in the experimental model of neuropathic pain induced by spinal nerve ligation (SNL). In this study, we characterized the main autophagic markers in two other common experimental models of neuropathic pain, the chronic constriction injury (CCI) and the spared nerve injury (SNI). The different modulation of LC3-I, Beclin 1 and p62 suggested that autophagy is differentially affected in the spinal dorsal horn depending on the type of peripheral injury. Confocal analysis of p62 distribution in the spinal dorsal horn indicated its presence mainly in NeuN-positive cell bodies and occasionally in glial processes, thus suggesting a predominant expression in the neuronal compartment. Finally, we investigated the consequences of autophagy impairment on pain behaviour by using the autophagy blocker cloroquine. Intrathecal chloroquine injection in naïve mice induced spinal accumulation of LC3 and p62 paralleled by significant mechanical hypersensitivity thus confirming the block in autophagosome clearance and suggesting the participation of the autophagic process in spinal mechanisms of pain processing. Altogether, our data indicate that spinal autophagy is differentially altered in different experimental pain models of neuropathic pain and that this process may be relevant for pain control.

In vivo functional investigation of oncogenes using somatic gene transfer has been successfully exploited to validate their role in tumorigenesis. For tumour suppressor genes this has proven more challenging due to technical aspects. To provide a flexible and effective method for investigating somatic loss-of-function alterations and their influence on tumorigenesis, we have established CRISPR/Cas9-mediated somatic gene disruption, allowing for in vivo targeting of TSGs. Here we demonstrate the utility of this approach by deleting single (Ptch1) or multiple genes (Trp53, Pten, Nf1) in the mouse brain, resulting in the development of medulloblastoma and glioblastoma, respectively. Using whole-genome sequencing (WGS) we characterized the medulloblastoma-driving Ptch1 deletions in detail and show that no off-targets were detected in these tumours. This method provides a fast and convenient system for validating the emerging wealth of novel candidate tumour suppressor genes and the generation of faithful animal models of human cancer.