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Spastin is a conserved microtubule-severing enzyme mutated in hereditary spastic paraplegia. The role that spastin plays in the cell biology of axon regeneration and degeneration has recently been investigated in Drosophila We show that the C. elegans spastin ortholog, spas-1, is expressed in GABA motor neurons, in addition to the known expression in touch receptor neurons (TRNs) and that it is required for axon regeneration in the GABA motor neurons after in vivo laser axotomy. We identified no neuronal developmental defects in the GABA motor neurons and only minor branching variations in the TRNs. However, we show that spas-1 is required for the long-term maintenance of TRN axons in C. elegans, as older spas-1 null C. elegans show a significant increase in specific axonal morphological defects compared with the wild type as identified by confocal microscopy in aged animals. Together, our results suggest that spastin is required for regrowth and maintenance of axons in C. elegans, consistent with previous reports in Drosophila.

There are a wide variety of commercially available antibodies for labeling microglial cells based on different protein targets, as well as antibodies for the same protein target made in different species. While this array of targets and hosts allows for flexibility in immunohistochemical experiments, it is important to validate that different antibodies provide comparable and accurate immunodetection prior to experimental data collection. We found that a commercially available anti-Iba1 antibody, made in goat, produces irregular staining patterns in specific regions of the mouse brain in both sexes, prompting a further investigation into the phenomenon. This Iba1-goat antibody displayed increased numbers of labeled cells when compared with expression of a CX3CR1-GFP reporter and IHC detection of P2RY12, two common microglial markers. Furthermore, immunodetection by other common anti-Iba1 antibodies made in rabbit and chicken did not display the excessive cell labeling when compared with the CX3CR1-GFP reporter. Upon further investigation, this Iba1-goat antibody was observed to highly colocalize with vasopressin neurons in the paraventricular nucleus of the hypothalamus (PVN) and the supraoptic nucleus of the hypothalamus (SON), the two main sites of vasopressin production in the brain. Other anti-Iba1 antibodies made in other species did not show this same colocalization with vasopressin. Finally, this effect was species-specific, as Wistar rats did not display erroneous cell labeling by the Iba1-goat antibody. In sum, the present study employs both qualitative and quantitative data to highlight the importance of validating antibody efficacy and specificity in a region- and species-specific manner.

Nonrapid eye movement (NREM) sleep is characterized by the interaction of multiple oscillations essential for memory consolidation, alongside a dynamic arrhythmic 1/f scale-free background that may also contribute to its functions. Recent spectral parametrization methods, such as fitting oscillation and one-and-over-F and irregular resampling auto-spectral analysis, enable the dissociation of rhythmic and arrhythmic components in the spectral domain; however, they do not resolve these processes in the time domain. Instantaneous measures of frequency, amplitude, and phase-amplitude coupling (PAC) are thus still confounded by fluctuations in arrhythmic activity. This limitation represents a pitfall for studies of NREM sleep relying on instantaneous estimates to investigate oscillatory coupling. To address this limitation, we introduce "Rhythms and Background" (RnB), a novel wavelet-based methodology designed to dynamically denoise time series data of arrhythmic interference. This enables the extraction of purely rhythmic time series, suitable for enhanced time-domain analyses of sleep rhythms. We first validate RnB through simulations, demonstrating it accurately estimates the spectral profiles of individual and multiple oscillations across a range of arrhythmic conditions. We then apply RnB to publicly available intracranial electroencephalogram sleep recordings, showing that it provides an improved spectral and time-domain representation of hallmark NREM rhythms. Finally, we demonstrate that RnB significantly enhances the assessment of PAC between cardinal NREM oscillations, outperforming traditional methods that conflate rhythmic and arrhythmic components. This methodological advance offers a substantial improvement in the analysis of sleep oscillations, providing greater precision in the study of rhythmic activity critical to NREM sleep functions.

There is a critical need for robust and reliable preclinical models for posttraumatic stress disorder (PTSD) to better understand pathophysiological mechanisms and support the development of novel treatments. The single prolonged stress (SPS) model has been previously utilized to investigate various acute behavioral effects and stress hormone changes in rodents. This study paired anxiety-like and social behavioral evaluations with corticosterone assessment as a complementary physiological biomarker to determine the presence of robust and intervenable phenotypes following SPS. Sprague Dawley rats (N = 36, 30 male and 6 female) received SPS model induction (e.g., restraint with odorant, forced-swim, diethyl ether exposure, and isolation) or control handling. Serum corticosterone and behavioral assessments, including the open field test (OFT) and a social motivation test (SMT), were investigated at 1 and 2 weeks following SPS induction. This SPS model did not induce anxiety-like or locomotive differences assessed in the OFT (p's > 0.05). Similarly, SPS did not appear to alter social preference or avoidance in the SMT (p's > 0.05), as groups had similar novel social and novel object interaction levels. SPS-paired cue re-exposure did not unmask group differences in these behaviors. Corticosterone levels were also unaltered between groups in the weeks following SPS (p = 0.178). In the absence of other stressors or modifications, the null behavioral and corticosterone findings in the weeks following SPS suggest that this SPS protocol may not reliably produce adequately robust or intervenable phenotypes.

Alcohol use disorder (AUD) is one of the top behavioral causes of global disease burden in the United States. Repeated cycles of alcohol intoxication and abstinence induce neuroplastic alterations which induce excessive drinking and cognitive impairments. A system deeply dysregulated by chronic drinking is norepinephrine (NE). At moderate levels, NE has beneficial effects on cognition and behavior, mediated by the α2 adrenergic receptor (AR) subtype. Whether α2 AR activation blunts alcohol consumption in models of heavy drinking has not been determined, and whether α2 AR activation improves cognitive performance following chronic alcohol consumption is unknown. Here, we show that the α2 AR agonist clonidine worsens ethanol-induced hypothermia and sedation in male mice, while the more selective α2 AR agonist guanfacine is devoid of these effects. We also observed that, in male and female mice, while both clonidine and guanfacine reduce heavy alcohol drinking, guanfacine does so with higher potency. Furthermore, guanfacine improved cognitive performance in a temporal order test and, partially, in a novel object recognition test but had no effect in a novel spatial location test, in male and female ethanol-experienced mice. Finally, we found that chronic intermittent ethanol drinking increases the number of persistently activated NE neurons in both the locus ceruleus and the nucleus of the tractus solitarius, in both male and female mice. Our results highlight a central role for the α2 AR system in heavy alcohol drinking and associated cognitive deficits, suggesting that α2 AR stimulation may represent a viable pharmacological strategy to treat AUD.

Investigations into the neural basis of behavior frequently employ calcium imaging to measure neuronal activity. Across studies, however, seemingly reasonable but highly diverse methodological choices are typically made to assess the selectivity of individual neurons to task states. Here, we examine systematically the effect of parameter choices, along the pipeline from data acquisition through statistical testing, on the inferred encoding preferences of individual neurons. We use, as an experimental testbed, calcium imaging in the medial prefrontal cortex of freely behaving mice engaged in a classic exploration-avoidance task with animal-controlled state transitions, namely, navigation in the elevated zero maze. We report that most of the key parameters in the pipeline substantially impact the inferred selectivity of neurons and do so in distinct ways. Using novel accuracy and robustness metrics, we directly compare the quality of inference across combinations of parameter levels and discover an optimal combination. We validate its optimality using resampling methods and demonstrate its generality across the two common analytical approaches used to assess neuronal selectivity-average response rate-dependent selectivity indices and continuous time-dependent regression coefficients. Together, our results not only identify an optimal parameter setting for reliably assessing encoding preferences of cortical excitatory neurons using GCaMP6f calcium imaging but also establish a general data-driven procedure for identifying such optimal settings for other cell types, brain areas, and tasks.

The life cycle of the model nematode Caenorhabditis elegans involves a choice between two alternative developmental trajectories. Hermaphroditic larvae can either become reproductive adults or, under conditions of crowding or low food availability, enter a long-term, stress-resistant diapause known as the dauer stage. Chemical signals from a secreted larval pheromone promote the dauer trajectory in a concentration-dependent manner, and their influence can be antagonized by increased availability of a microbial food source. The decision is known to be under neuronal control, involving both sensory and interneurons. However, little is known about the dynamics of the underlying circuit, and the circuit mechanisms by which short-term fluctuations in the ratio of food and pheromone experienced by individual larvae are remembered and averaged over several hours. To investigate this, we quantitatively characterized the neuronal responses to food and pheromone inputs by measuring calcium traces from ASI and AIA neurons, each of which has previously been implicated in regulation of dauer entry. We found that calcium in ASI increases linearly in response to food and similarly decreases in response to pheromone. Notably, the ASI response persists well beyond removal of the food stimulus, thus encoding a memory of recent food exposure. In contrast, AIA reports instantaneous food availability and is unaffected by pheromone. We discuss how these findings may inform our understanding of this long-term decision-making process.

Daughterless (Da), the Drosophila melanogaster homolog of mammalian E-protein transcription factor 4 (TCF4), is well studied in fruit fly embryonic development but its functions in adult nervous system are poorly understood. Mutations in human TCF4 gene lead to intellectual disabilities such as Pitt-Hopkins syndrome and TCF4 has also been linked to schizophrenia. Here, to explore the roles of Da in the Drosophila mature brain, we map Da DNA binding sites and study the transcriptomics of the brains where Da function is inhibited by pan-neuronal Extramacrohaete (Emc) overexpression, in both male and female Drosophila Our transcriptome analyses reveal that in the adult brain Da regulates the expression of genes involved in behavior, memory, synaptic signaling, protein translation, and metabolic processes. Moreover, combining the RNA sequencing data with Da ChIP sequencing results indicates that genes associated with neuronal projection guidance, metabolism, and translation are direct targets of Da. In addition, we validate the involvement of Da in memory formation. Overall, our results provide valuable information about the functions of Da in the adult brain and aid in better understanding the mechanisms of TCF4-related disorders.

Episodic timing refers to the one-shot, automatic encoding of temporal information in the brain, in the absence of attention to time. A previous magnetoencephalography (MEG) study showed that the relative burst time of spontaneous alpha oscillations (α) during quiet wakefulness was a selective predictor of retrospective duration estimation. This observation was interpreted as α embodying the "ticks" of an internal contextual clock. Herein, we replicate and extend these findings using electroencephalography (EEG), assess robustness to time-on-task effects, and test the generalizability in virtual reality (VR) environments. In three EEG experiments, 128 participants of either sex underwent 4 min eyes-open resting-state recordings followed by an unexpected retrospective duration estimation task. Experiment 1 tested participants before any tasks, Experiment 2 after 90 min of timing tasks, and Experiment 3 in VR environments of different sizes. We successfully replicated the original MEG findings in Experiment 1 but did not in Experiment 2. We explain the lack of replication through time-on-task effects (changes in α power and topography) and contextual changes yielding a cognitive strategy based on temporal expectation (supported by a fast passage of time). In Experiment 3, we did not find the expected duration underestimation in VR and did not replicate the correlation between α bursts and retrospective time estimates. Overall, while EEG captures the α burst marker of episodic timing, its reliability depends critically on experimental context. Our findings highlight the importance of controlling experimental context when using α bursts as a neural marker of episodic timing.