MAB377 Sigma-AldrichAnti-NeuN Antibody, clone A60

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.

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.

MicroRNAs (miRNAs) are involved in the neuroplastic changes which induce and maintain neuropathic pain. However, it is unknown whether nerve injury leads to altered miRNA expression and modulation of pain relevant target gene expression within peripheral nerves. In the present study, expression profiles of miR-1 and the pain-relevant targets, brain derived neurotrophic factor (BDNF) and Connexin 43 (Cx43), were studied in peripheral neuropathic pain, which was induced by chronic constriction injury (CCI) of the sciatic nerve in rats. The expression of miR-1 was investigated in the sciatic nerve, dorsal root ganglion (DRG) and the ipsilateral spinal cord by qPCR. Changes of BDNF and Cx43 expression patterns were studied using qPCR, Western blot analysis, ELISA and immunohistochemistry.In sciatic nerves of naïve rats, expression levels of miR-1 were more than twice as high as in DRG and spinal cord. In neuropathic rats, CCI lead to a time-dependent downregulation of miR-1 in the sciatic nerve but not in DRG and spinal cord. Likewise, protein expression of the miR-1 targets BDNF and Cx43 was upregulated in the sciatic nerve and DRG after CCI. Immunohistochemical staining revealed an endoneural abundancy of Cx43 in injured sciatic nerves which was absent after Sham operation.This study demonstrates that CCI leads to a regulation of miRNAs (miR-1) in the peripheral nervous system. This regulation is associated with alterations in the expression and localization of the miR-1 dependent pain-relevant proteins BDNF and Cx43. Further studies will have to explore the function of miRNAs in the context of neuropathic pain in the peripheral nervous system.

Mutations, duplication and triplication of α-synuclein genes are linked to familial Parkinson's disease (PD), and aggregation of α-synuclein (α-syn) in Lewy bodies (LB) is involved in the pathogenesis of the disease. The targeted overexpression of α-syn in the substantia nigra (SN) mediated by viral vectors may provide a better alternative to recapitulate the neurodegenerative features of PD. Therefore, we overexpressed human wild-type α-syn using rAAV2/1 vectors in the bilateral SN of mouse and examined the effects for up to 12 weeks. Delivery of rAAV-2/1-α-syn caused significant nigrostriatal degeneration including appearance of dystrophic striatal neurites, loss of nigral dopaminergic (DA) neurons and dissolving nigral neuron bodies in a time-dependent manner. In addition, the α-syn overexpressed mice also developed significant deficits in motor function at 12 weeks when the loss of DA neurons exceeded a threshold of 50%. To investigate the sensitivity to neurotoxins in mice overexpressing α-syn, we performed an MPTP treatment with the subacute regimen 8 weeks after rAAV injection. The impact of the combined genetic and environmental insults on DA neuronal loss, striatal dopamine depletion, dopamine turnover and motor dysfunction was markedly greater than that of either alone. Moreover, we observed increased phosphorylation (S129), accumulation and nuclear distribution of α-syn after the combined insults. In summary, these results reveal that the overexpressed α-syn induces progressive nigrostriatal degeneration and increases the susceptibility of DA neurons to MPTP. Therefore, the targeted overexpression of α-syn and the combination with environmental toxins may provide valuable models for understanding PD pathogenesis and developing related therapies.

Deficits in impulse control are often observed in psychiatric disorders in which abnormalities of the prefrontal cortex are observed, including attention-deficit/hyperactivity disorder and bipolar disorder. We recently found that milnacipran, a serotonin/noradrenaline reuptake inhibitor, could suppress impulsive action in normal rats. However, whether milnacipran could suppress elevated impulsive action in rats with lesions of the ventromedial prefrontal cortex, which is functionally comparable with the human prefrontal cortex, remains unknown.Selective lesions of the ventromedial prefrontal cortex were made using quinolinic acid in rats previously trained on a 3-choice serial reaction time task. Sham rats received phosphate buffered saline. Following a period of recovery, milnacipran (0 or 10mg/kg/d × 14 days) was orally administered 60 minutes prior to testing on the 3-choice task. After 7 days of drug cessation, Western blotting, immunohistochemistry, electrophysiological analysis, and morphological analysis were conducted.Lesions of the ventromedial prefrontal cortex induced impulsive deficits, and repeated milnacipran ameliorated the impulsive deficit both during the dosing period and after the cessation of the drug. Repeated milnacipran remediated the protein levels of mature brain-derived neurotrophic factor and postsynaptic density-95, dendritic spine density, and excitatory currents in the few surviving neurons in the ventromedial prefrontal cortex of ventromedial prefrontal cortex-lesioned rats.The findings of this study suggest that milnacipran treatment could be a novel strategy for the treatment of psychiatric disorders that are associated with a lack of impulse control.

The brain is composed of many different types of neurons. Therefore, analysis of brain activity with single-cell resolution could provide fundamental insights into brain mechanisms. However, the electrical signal of an individual neuron is very small, and precise isolation of single neuronal activity from moving subjects is still challenging. To measure single-unit signals in actively behaving states, establishment of technologies that enable fine control of electrode positioning and strict spike sorting is essential. To further apply such a single-cell recording approach to small brain areas in naturally behaving animals in large spaces or during social interaction, we developed a compact wireless recording system with a motorized microdrive. Wireless control of electrode placement facilitates the exploration of single neuronal activity without affecting animal behaviors. Because the system is equipped with a newly developed data-encoding program, the recorded data are readily compressed almost to theoretical limits and securely transmitted to a host computer. Brain activity can thereby be stably monitored in real time and further analyzed using online or offline spike sorting. Our wireless recording approach using a precision motorized microdrive will become a powerful tool for studying brain mechanisms underlying natural or social behaviors.

Various GABAergic neuron types of the amygdala cooperate to control principal cell firing during fear-related and other behaviors, and understanding their specialized roles is important. Among GABAergic neurons, the so-called intercalated cells (ITCcs) are critically involved in the expression and extinction of fear memory. Tightly clustered small-sized spiny neurons constitute the majority of ITCcs, but they are surrounded by sparse, larger neurons (L-ITCcs) for which very little information is known. We report here a detailed neurochemical, structural and physiological characterization of rat L-ITCcs, as identified with juxtacellular recording/labeling in vivo. We supplement these data with anatomical and neurochemical analyses of nonrecorded L-ITCcs. We demonstrate that L-ITCcs are GABAergic, and strongly express metabotropic glutamate receptor 1α and GABAA receptor α1 subunit, together with moderate levels of parvalbumin. Furthermore, L-ITCcs are innervated by fibers enriched with metabotropic glutamate receptors 7a and/or 8a. In contrast to small-sized spiny ITCcs, L-ITCcs possess thick, aspiny dendrites, have highly branched, long-range axonal projections, and innervate interneurons in the basolateral amygdaloid complex. The axons of L-ITCcs also project to distant brain areas, such as the perirhinal, entorhinal, and endopiriform cortices. In vivo recorded L-ITCcs are strongly activated by noxious stimuli, such as hindpaw pinches or electrical footshocks. Consistent with this, we observed synaptic contacts on L-ITCc dendrites from nociceptive intralaminar thalamic nuclei. We propose that, during salient sensory stimulation, L-ITCcs disinhibit local and distant principal neurons, acting as "hub cells," to orchestrate the activity of a distributed network.

Microglia are the brain's resident immune cells and function as the main defense against pathogens or injury. However, in the absence of disease, microglia have other functions in the normal brain. For example, previous studies showed that microglia contribute to circuit refinement and synaptic plasticity in the developing and adult brain, respectively. Thus, microglia actively participate in regulating neuronal excitability and function. Here, we report that in the cortex, but not other brain regions, a subset of microglia extend a single process that specifically associates and overlaps with the axon initial segment (AIS), the site where action potentials are generated. Similar associations were not observed with dendrites or distal axons. Microglia-AIS interactions appear early in development, persist throughout adulthood, and are conserved across species including mice, rats, and primates. However, these interactions are lost after microglial activation following brain injury, suggesting that such interactions may be part of healthy brain function. Loss of microglial CX3CR1 receptors, or the specialized extracellular matrix surrounding the AIS, did not disrupt the interaction. However, loss of AIS proteins by the neuron-specific deletion of the master AIS scaffold AnkyrinG disrupted microglia-AIS interactions. These results reveal a unique population of microglia that specifically interact with the AIS in the adult cortex.