Gene expression analysis in individual neuronal types helps in understanding brain function. Genetic methods expressing fluorescent proteins are widely used to label specific neuronal populations. However, because cell type specificity of genetic labeling is often limited, it is advantageous to combine genetic labeling with additional methods to select specific cell/neuronal types. Laser capture microdissection is one of such techniques with which one can select a specific cell/neuronal population based on morphological observation. However, a major issue is the disappearance of fluorescence signals during the tissue processing that is required for high-quality sample preparation. Here, we developed a simple, novel method in which fluorescence signals are preserved. We use genetic labeling with fluorescence proteins fused to transmembrane proteins, which shows highly stable fluorescence retention and allows for the selection of fluorescent neurons/cells based on morphology. Using this method in mice, we la...

Bilateral convergence of external stimuli is a common feature of vertebrate sensory systems. This convergence of inputs from the bilateral receptive fields allows higher order sensory perception, such as depth perception in the vertebrate visual system and stimulus localization in the auditory system. The functional role of such bilateral convergence in the olfactory system is unknown. To test whether each olfactory bulb (OB) contributes a separate piece of olfactory information, and whether information from the bilateral OB is integrated, we synchronized the activation of OBs with blue light in mice expressing ChIEF in the olfactory sensory neurons (OSNs) and behaviorally assessed the relevance of dual OBs in olfactory perception. Our findings suggest that each OB contributes separate components of olfactory information, and the mice integrate the bilaterally synchronized olfactory information for olfactory identity.

Information theoretic metrics have proven useful in quantifying the relationship between behaviorally relevant parameters and neuronal activity with relatively few assumptions. However, these metrics are typically applied to action potential (AP) recordings and were not designed for the slow timescales and variable amplitudes typical of functional fluorescence recordings (e.g., calcium imaging). The lack of research guidelines on how to apply and interpret these metrics with fluorescence traces means the neuroscience community has yet to realize the power of information theoretic metrics. Here, we used computational methods to create mock AP traces with known amounts of information. From these, we generated fluorescence traces and examined the ability of different information metrics to recover the known information values. We provide guidelines for how to use information metrics when applying them to functional fluorescence and demonstrate their appropriate application to GCaMP6f population recordings fro...

Psychostimulants such as amphetamine (AMPH) target dopamine (DA) neuron synapses to engender drug-induced plasticity. While DA neurons modulate the activity of striatal (Str) cholinergic interneurons (ChIs) with regional heterogeneity, how AMPH affects ChI activity has not been elucidated. Here, we applied quantitative fluorescence imaging approaches to map the dose-dependent effects of a single dose of AMPH on ChI activity at 2.5 and 24 h after injection across the mouse Str using the activity-dependent marker phosphorylated ribosomal protein S6 (p-rpS6240/244). AMPH did not affect the distribution or morphology of ChIs in any Str subregion. While AMPH at either dose had no effect on ChI activity after 2.5 h, ChI activity was dose dependently reduced after 24 h specifically in the ventral Str/nucleus accumbens (NAc), a critical site of psychostimulant action. AMPH at either dose did not affect the spontaneous firing of ChIs. Altogether this work demonstrates that a single dose of AMPH has delayed regional...

The opioid epidemic led to an increase in the number of neonatal opioid withdrawal syndrome (NOWS) cases in infants born to opioid-dependent mothers. Hallmark features of NOWS include weight loss, severe irritability, respiratory problems, and sleep fragmentation. Mouse models provide an opportunity to identify brain mechanisms that contribute to NOWS. Neonatal outbred Swiss Webster Cartworth Farms White (CFW) mice were administered morphine (15 mg/kg, s.c.) twice daily from postnatal day 1 (P1) to P14, an approximation of the third trimester of human gestation. Female and male mice underwent behavioral testing on P7 and P14 to determine the impact of opioid exposure on anxiety and pain sensitivity. Ultrasonic vocalizations (USVs) and daily body weights were also recorded. Brainstems containing pons and medulla were collected during morphine withdrawal on P14 for RNA sequencing. Morphine induced weight loss from P2 to P14, which persisted during adolescence (P21) and adulthood (P50). USVs markedly increase...

Convincing evidence of blood-spinal cord barrier (BSCB) alterations has been demonstrated in amyotrophic lateral sclerosis (ALS) and barrier repair is imperative to prevent motor neuron dysfunction. We showed benefits of human bone marrow-derived CD34+ cells (hBM34+) and endothelial progenitor cells (hBM-EPCs) intravenous transplantation into symptomatic G93A SOD1 mutant mice on barrier reparative processes. These gains likely occurred by replacement of damaged endothelial cells, prolonging motor neuron survival. However, additional investigations are needed to confirm the effects of administered cells on integrity of the microvascular endothelium. The aim of this study was to determine tight junction protein levels, capillary pericyte coverage, microvascular basement membrane, and endothelial filamentous actin (F-actin) status in spinal cord capillaries of G93A SOD1 mutant mice treated with human bone marrow-derived stem cells. Tight junction proteins were detected in the spinal cords of cell-treated vers...

Visual representations can be generated via feedforward or feedback processes. The extent to which these processes result in overlapping representations remains unclear. Previous work has shown that imagined stimuli elicit similar representations as perceived stimuli throughout the visual cortex. However, while representations during imagery are indeed only caused by feedback processing, neural processing during perception is an interplay of both feedforward and feedback processing. This means that any representational overlap could be because of overlap in feedback processes. In the current study, we aimed to investigate this issue by characterizing the overlap between feedforward- and feedback-initiated category representations during imagined stimuli, conscious perception, and unconscious processing using fMRI in humans of either sex. While all three conditions elicited stimulus representations in left lateral occipital cortex (LOC), significant similarities were observed only between imagery and consci...

Atypical sensory processing is currently included within the diagnostic criteria of autism. The cerebellum is known to integrate sensory inputs of different modalities through its connectivity to the cerebral cortex. Interestingly, cerebellar malformations are among the most replicated features found in postmortem brain of individuals with autism. We studied sensory processing in the cerebellum in a mouse model of autism, knock-out (KO) for the Cntnap2 gene. Cntnap2 is widely expressed in Purkinje cells (PCs) and has been recently reported to regulate their morphology. Further, individuals with CNTNAP2 mutations display cerebellar malformations and CNTNAP2 antibodies are associated with a mild form of cerebellar ataxia. Previous studies in the Cntnap2 mouse model show an altered cerebellar sensory learning. However, a physiological analysis of cerebellar function has not been performed yet. We studied sensory evoked potentials in cerebellar Crus I/II region on electrical stimulation of the whisker pad in a...

The ability to learn dexterous motor skills is a fundamental aspect of human behavior. However, the underlying neural circuit mechanisms for dexterous skill learning are unclear. Advancing our understanding of motor skill learning requires the integration of modern neuroscientific techniques with a rigorously characterized dexterous task. The development of automated rodent skilled reaching with paw tracking allows detailed analysis of how reach-to-grasp kinematics evolve during learning. We assessed how both “gross” forelimb and “fine” digit kinematics changed as rats learned skilled reaching. Rats whose success rates increased (learners) consistently reduced the variability in their reach trajectories. Refinement of fine digit control generally continued after consistency in gross hand transport to the pellet plateaued. Interestingly, most rats whose success rates did not increase (non-learners) also converged on consistent reach kinematics. Some non-learners, however, maintained substantial variability ...

The V-ATPase is a highly conserved enzymatic complex that ensures appropriate levels of organelle acidification in virtually all eukaryotic cells. While the general mechanisms of this proton pump have been well studied, little is known about the specific regulations of neuronal V-ATPase. Here, we studied CG31030, a previously uncharacterized Drosophila protein predicted from its sequence homology to be part of the V-ATPase family. In contrast to its ortholog ATP6AP1/VhaAC45 which is ubiquitous, we observed that CG31030 expression is apparently restricted to all neurons, and using CRISPR/Cas9-mediated gene tagging, that it is mainly addressed to synaptic terminals. In addition, we observed that CG31030 is essential for fly survival and that this protein co-immunoprecipitates with identified V-ATPase subunits, and in particular ATP6AP2. Using a genetically-encoded pH probe (VMAT-pHluorin) and electrophysiological recordings at the larval neuromuscular junction, we show that CG31030 knock-down induces a major...