Animal learning can be analyzed on two timescales: task acquisition across training sessions and motivation fluctuations within training sessions. How do variations in motor and neurophysiologic activity relate to task performance over these timescales? Here, this question was examined in head-fixed mice performing a whisker-based sensory discrimination task. Male mice were trained for 12-14 daily sessions on a go/no-go task, each lasting approximately one hour to capture spontaneous performance fluctuations over minutes. Simultaneous to task performance, "non-performance variables" were tracked, including wheel running, pupil size, eyelid aperture and sensory cortical activity. First, motivation states were defined based on performance tendencies over minutes, leading to three state categories: persistent, disengaged, or attentive Non-performance variables were found to predict these states independent of task correctness. Then, when further parsing these states by the go/no-go outcomes of hit, miss, false alarm or correct rejection, learning-like changes were detected in wheel running, eye movements and brain activity. Thus, learning over days and motivation fluctuations over minutes form a continuum, as evidenced by changes in motor and physiologic activity variables not directly controlled by task contingencies, even during periods of suboptimal performance in well-trained subjects. These findings improve the understanding of performance variations and implicit learning, in addition to contributing a framework for the analysis of task performance indirectly from motor and neurophysiologic activity.Significance statement Task performance is typically measured by correctness percentages over daily training sessions but can also correlate with motivation state fluctuations within each session. Thus, while aggregating correctness percentages per session may reveal a learning curve, accounting for within-session state fluctuations can reveal variability in that curve, even among well-trained subjects. In this head-fixed mouse study, task acquisition across sessions and motivation fluctuations within sessions were categorized into three states: persistent, disengaged, or attentive Subsequently, metrics not directly controlled by the task, including locomotion, pupil dilation, eyelid aperture and brain activity, were found to predict both learning and state changes. Therefore, task performance can be tracked more comprehensively from brain and body activity metrics, adding nuance to correctness percentages.

Vocal communication is essential for social behavior, yet the distributed brain networks underlying vocal production remain elusive. Male mice produce ultrasonic vocalizations (USVs) during courtship, providing a rodent model for investigating neural circuits underlying innate vocal communication. Here, we used a double-labeling strategy combining genetic activity tagging (TRAP2) and c-Fos immunohistochemistry to generate an unbiased, whole-brain map of neuronal ensembles activated during courtship-induced USV production in adult male mice. By tracking neuronal activity across 25 brain regions during two independent courtship sessions, we identified populations consistently recruited during social vocalization. Quantitative analyses revealed robust activation in the caudal periaqueductal gray, confirming its established role as a hub for vocal motor control. Importantly, correlation analyses between neuronal activity and USV count distinguished regions specifically linked to vocal output from those associated with general social interaction. The medial prefrontal cortex, particularly the prelimbic and cingulate cortices, exhibited strong positive correlations with USV production, suggesting an integrative role in translating the social and motivational context into vocal commands. Notably, the rostral striatum showed a strong correlation with USV count, with vocalization-related activity preferentially localized within striosome compartments of the medial striatum. Given that striosomes receive limbic cortical input and are implicated in motivational processing, these findings suggest that corticostriatal limbic–motor circuits modulate innate vocalization contingent on emotional and social contexts. Collectively, our study proposes a comprehensive neuroanatomical framework linking cortical motivation centers, striatal limbic–motor pathways, and brainstem vocal motor circuits, providing insights into the distributed neural architecture underlying mammalian social communication.

Stressful events are a leading factor in the development of depression. The medial prefrontal cortex (mPFC) is strongly associated with depression etiology and exposure to uncontrollable stressors results in synaptic dysfunction and loss. Learned helplessness is a behavioral paradigm that measures effects of repeated exposure to uncontrollable, inescapable stress on later responses to escapable stress. We therefore performed a proteomic analysis of mPFC synaptosomes in a mouse learned helplessness model to identify molecular changes that could contribute to functional consequences of inescapable stress. Male and female mice were evaluated at baseline and following exposure to escapable or inescapable stress followed by an active avoidance test. Label-free mass spectrometry followed by pathway and protein–protein interaction network analyses identified alterations in signaling pathways involved in energy metabolism, neurotransmitter signaling, and protein shuttling. Furthermore, phosphoproteomics revealed alterations related to synaptic function, neurotransmitter signaling and protein internalization, as well as changes in activity of kinases previously identified as mediators of antidepressant efficacy (GSK3B) and receptor internalization (ADRBK1). We more deeply examined alterations in the acetylcholine receptor signaling pathway and identified muscarinic receptor proteins (Chrm1, Chrm2, Chrm4) and key proteins involved in their translocation to and from the membrane. These results identify substantial changes in the mPFC proteome following exposure to inescapable stressors. In addition, mPFC muscarinic cholinergic signaling is well placed to mediate responses to an inescapable stressor. This proteomic study will be useful in guiding studies of human mPFC relevant to depression. Data are available via ProteomeXchange with identifier PXD073765.

Multi-electrode recording of neuronal activity in cultures offer opportunities for understanding how the structure of a network gives rise to function. Neuronal cultures derived from human induced pluripotent stem cells (iPSCs) from male and female individuals are often plated at highly variable cell densities across studies, but its impact on neuronal activity remains poorly understood. We found that properties such as the mean firing rate of the individual cells, the pairwise correlations between cells, and the entropy of the population all changed significantly with changes in culture density. We used a maximum entropy model to capture the structure of the population activity using only the firing rates and correlations, and we found that the model performed best at the highest densities, suggesting that changes in activity reflected differences in structure of interactions between neurons across scales of complexity. Our work thus shows that culture density is an important experimental parameter that impacts neuronal activity in human iPSC-derived cultures. Additionally, our findings provide an analytical framework to study population activity in neuronal cultures including those from patient populations where a disease process may impact network activity.

Elucidating the neural substrates of pavlovian reward learning requires reliable behavioral readouts. In conditioned magazine approach studies, rodents express reward expectancy by approaching the food magazine during cues that predict reward. This behavior is typically quantified using one of three measures: number of head entries, percentage of time in the magazine, or latency to respond. Yet these measures often diverge within the same discrimination task, making reliance on a single metric problematic. At the individual level, some animals express discrimination learning most clearly in one measure while showing little or no learning in the others, and animals may even switch their preferred measure across training. Reporting only one measure therefore risks underestimating the ability of a subset of animals. At the group level, sampling error can produce apparent differences across replications of the same design, limiting replicability. Moreover, brain manipulations can alter response topography, such that choosing one measure over another may lead to conflicting interpretations of neural function. To address this issue, we recommend reporting all raw behavioral measures and supplementing them with a dimensionality-reduction approach such as principal component analysis. Across multiple discrimination tasks in rats from both sexes, we show that subject-specific first principal component scores provide a composite index that more consistently reflects discrimination learning than any single raw measure. This approach enhances statistical power, improves reproducibility, and helps distinguish true learning deficits from changes in response topography. However, its broader application will require continued validation and careful consideration of its inherent methodological tradeoffs.

The tail suspension test (TST) is a well-known rodent behavioral test that assesses stress and depressive-like behavior. While several automatic tail suspension test scoring programs have emerged, many researchers still prefer a manual scoring method for accuracy and reliability. However, manual scoring can introduce significant errors. Thus, in this work, we present a novel graphical user interface that assists in the manual scoring process to minimize possibility for errors. The GUI, which we refer to as "TST Score Helper," minimizes errors through consolidation of the TST scoring procedure into a single cohesive program. Further, a rescore mode enhances rigor by enabling comparison of two different scorers' mobility status timelines and rereview of periods of disagreement. In a cohort of 64 male and 45 female mice subject to closed head injury or sham injury, we demonstrate the challenges with manual scoring and we characterize performance of the TST Score Helper program. The results show how this program can reduce sources of manual scoring error and improve the fidelity of results.

Sparse labeling techniques are essential for morphological analysis of the central nervous system. Various sophisticated strategies have been developed, but conventional methods, such as Golgi-Cox staining and biocytin injection, remain widely used. Recent advances in adeno-associated virus (AAV) technology have enabled sparse neuronal labeling in rodents, either through postnatal AAV injection or by coinjecting a dilute Cre recombinase-expressing driver with a Cre-dependent amplifier. However, postnatal AAV injection requires prior preparation, and Cre-dependent systems are incompatible with Cre-expressing or floxed genetic backgrounds. Here, we present a Cre-orthogonal AAV-based sparse labeling method that utilizes Supernova technology in male and female mice. We employed Flpe recombinase to achieve Cre-independent labeling. A retro-orbital injection of PHP.eB AAVs failed to label neurons, whereas local injection enabled the bright and sparse labeling of multiple neuronal types, including cerebellar neurons. The labeled neurons were sufficiently bright to visualize fine structures, such as dendritic spines, without the need for immunostaining. This method is compatible with both Cre-expressing and floxed genetic backgrounds. Furthermore, we demonstrated that the labeled neurons can be manipulated by the coinjection of a Flpe-dependent third virus. This straightforward and flexible approach is applicable across various genetic backgrounds and can be readily integrated into existing experimental systems.

Neurite outgrowth is essential for neural circuit formation and is tightly regulated by secreted factors and their receptors. The secreted extracellular domain of the amyloid precursor protein (sAPPα) has been shown to modulate neurite outgrowth. Recently, the gamma amino butyric acid receptor type-B subunit 1a (GABABR1a) was identified as an sAPPα binding partner that mediates its effects on synaptic transmission. Here, we investigated whether this interaction also regulates neurite outgrowth. In mouse primary hippocampal neurons of either sex, the GABABR agonist baclofen reduced axon length; whereas its antagonist CGP54626 increased axon length in primary hippocampal neurons. Moreover, GABABR1a knock-out increased axon length and abolished the effect of baclofen. Application of sAPPα reduced axon length, an effect that required the presence of both GABABR1a and the extension domain of sAPPα, which mediates its binding to GABABR1a. Similarly, the APP 17mer peptide, which is sufficient to bind GABABR1a and mimic the effects of sAPP on synaptic transmission, reduced axon outgrowth in wild-type but not in GABABR1a-deficient neurons. Together, these findings indicate that the 1a isoform contributes to GABABR-dependent suppression of neurite outgrowth and mediates the inhibitory effect of sAPPα on neurite outgrowth.

Age-related vascular changes accompany or precede the development of Alzheimer's disease (AD) pathology. The comorbidity of AD and arterial stiffening suggests that vascular changes have a pathogenic role. Carotid artery mechanics and hemodynamics have been associated with age-related cognitive decline. However, the impact of hemodynamics and vascular mechanics on regional vulnerability within the brain has not been thoroughly explored. Compared with the arterial system, brain venous circulation in cognitive impairment is less understood despite the venous system's role in transport. To study vasculature impact on biochemistry in AD models, we must first establish the differences in vasculature mechanics and hemodynamics in a common AD model compared with healthy controls. With this baseline data, future studies on manipulating vasculature integrity in mice become feasible. Young and aged female 3xTg mice and age-matched controls were imaged using a combination of ultrasound and mass spectrometry. Wall shear stress varied across age and AD models. Mean velocity and pulsatility index varied across age and AD. Liquid chromatography-mass spectrometry of brain tissue revealed several lipids that were statistically different between age and AD, and matrix-assisted laser desorption/ionization MS imaging revealed region-specific differences between groups. Combining both ultrasound and mass spectrometry, we were able to detect significant changes in the vascular biomechanics of neck vasculature prior to observing significant changes in the brain biochemistry. Our work revealed significant vascular differences in the 3xTg compared with controls and, to our knowledge, is the first to study vascular biomechanics via ultrasound in the 3xTg AD mouse model.

Neurexins (Nrxns) are presynaptic cell adhesion molecules essential for synapse development and function. Of the many neurexin isoforms, only β-Nrxns contain the histidine-rich domain (HRD). While the HRD has been implicated in several pathological contexts, its normal physiological role remains unclear. To address this, we used a CRISPR-Cas9 method to generate a new mouse line expressing in-frame truncated Nrxn1β lacking the HRD. We found that HRD deletion did not affect mouse viability, gross brain development, or general behavior of either sex. However, loss of the HRD significantly altered neuroligin-1-dependent excitatory, but not inhibitory, presynaptic differentiation in primary cultured neurons. Moreover, this deletion affected presynaptic short-term plasticity, but not basal synaptic transmission, at hippocampal Schaffer collateral→CA1 synapses. These findings identify the Nrxn1β HRD as a potential contributor to excitatory presynaptic organization and function, providing new insight into the molecular diversity and specialization of Nrxns.