Neurons couple to various degrees to the activity level of the local neighboring population whereby strongly coupled 'choristers' and weakly coupled 'soloists' have been identified as two extremes of a continuous spectrum. At the same time neuronal populations undergo coordinated ON and OFF cortical state activity fluctuations, which are locally modulated by attention. The population coupling of soloists and choristers suggests that soloists should show limited alignment with cortical state fluctuations, while choristers should exhibit profound alignment. To test this, we recorded neurons across cortical layers in macaque areas V1 and V4 (n=2 males), while animals performed a feature based spatial attention task. As expected, we found a wide range of population coupling strength of neurons. In line with our prediction, coupling of choristers to cortical state changes (ON-OFF transitions) was generally stronger than that of soloists. The strength of population coupling of neurons was similar during spontaneous and stimulus driven activity. Allocation of attention to the receptive field reduced the population coupling strength. Attentional modulation of neurons was positively correlated with population coupling strength. While neurons on average retained their coupling strengths across conditions, some neurons change coupling strength condition dependent, thereby potentially enhancing the coding abilities of cortical circuits. Significance statement Cortical neurons can be categorized according to the level with which they couple their activity to the surrounding population, with extremes being referred to as soloists and choristers, respectively. Here we test to what extent this coupling is a constant feature across cortical states, stimulus and attention conditions. We find that at the population level the coupling is modulated by the conditions under which they are investigated. Critically, individual cells can behave as soloists (or choristers) on average while acting like choristers (soloists) during e.g. cortical state ON-OFF transitions. Thus, neurons can align with the surrounding population under some conditions, and behave independently under different conditions, thereby potentially enhancing the coding abilities of cortical circuits.

Synaptic physiology experiments are fundamental to neuroscience research. Consequently, accurate detection of synaptic currents is crucial for conducting high quality experiments. Traditionally, detecting inhibitory and excitatory postsynaptic currents (sIPSCs / sEPSCs) relied on hand-counting individual events, and while sEPSCs and sIPSCs are clear to the trained eye, hand analysis is time and labor intensive. Recent advances in applied machine learning promise faster, superior event detectors that may improve data quality and reduce or even completely negate the need for hand curation. While many strategies for sIPSC and sEPSC detection exist, rarely have they been quantitatively compared for accuracy within an experiment. Our study aims to establish practical ground truth event detection in a large experimental dataset through meticulous hand counting, and to assess variance in detection results across different laboratories, analysis techniques, and cell-types. Using thoroughly hand-counted data as our ground-truth comparison we will benchmark current popular detection methods, including a modern supervised deep learning approach. Our results suggest that current analysis strategies vary widely in their results, and that a supervised machine learning approach rivals manual event counting performed by expert electrophysiologists better than other automated approaches.Significance Statement Our study aims to establish a practical ground truth to measure inter-lab variability and to benchmark specific inhibitory and excitatory synaptic event detection techniques, including hand counting and the main automated approaches used in the field of slice electrophysiology.

We describe a novel method for adapting a two-photon scanning microscope to enable simultaneous detection of two-photon-generated visible fluorescence and single-photon-generated near-infrared (nIR) fluorescence. In this configuration, nIR fluorescence is routed through a single-mode optical fiber before detection by a photomultiplier tube. This fiber coupling offers two advantages: first, the optical fiber functions as a pinhole aperture, allowing for improved optical sectioning of the nIR signal; second, it minimizes nIR background fluorescence. To validate the effectiveness of this design, we conducted two sets of experiments in male and female C57B/6 mice. First, we compare two fluorescence indicators of the neurotransmitter dopamine: the genetically encoded indicator GRABDA and single walled carbon nanotube based optical nanosensors (nIRCats). Although nIRCats exhibit lower affinity for dopamine than GRABDA, this property allows for identification of high concentration release sites in the striatum. Second, we simultaneously imaged depolarization-induced calcium changes and dopamine release in the retina. Together, these results demonstrate the utility of integrating confocal nIR detection into a two-photon platform for simultaneous functional imaging across complementary spectral channels.Significance Statement Dual-color, real-time imaging is a powerful technique in biomedical imaging, including neuroscience. Here, we present a widely applicable modification to a standard two-photon scanning microscope that adds a near-infrared detection capability, a wavelength range that minimizes photon scattering and autofluorescence from biological samples. Using this microscope, we demonstrate the first direct comparison of two dopamine sensors: the genetically encoded sensor GRABDA3m detected in the visible channel and carbon-nanotube-based sensors detected in the near-infrared channel. We further demonstrate simultaneous imaging calcium activity and dopamine signaling in the developing retina. While we focused on dopamine sensors in this study, this platform is broadly applicable to a wide range of fluorophores and can be implemented on existing two-photon microscopes.

The refinement of gross motor skills, such as locomotion, during development is conserved across vertebrate species. Our previous work demonstrated, in larval zebrafish, that dopaminergic signaling through the dopamine D2-like family of receptors, specifically the dopamine 4 receptor subtype, was necessary for the developmental transformation of behaviorally relevant locomotor activity from an immature to a mature pattern between 3- and 4-days post-fertilization. In this study, we used a complement of tools, including electrophysiology, pharmacology, in vivo calcium imaging, liquid chromatography-mass spectrometry, and quantitative reverse transcription polymerase chain reaction to characterize the functional and molecular mechanisms responsible for this dopaminergic-mediated refinement of spinal locomotor activity. The results demonstrate that the dopamine 4 receptor subtype is functional in, at least, a subset of immature larvae. Further, gene expression of all D2-like receptor subtypes, levels of dopamine, and activity of diencephalic dopaminergic neurons are significantly greater in mature larvae compared to immature larvae. The integration of these results provides correlative evidence for the developmental role of dopaminergic signaling, specifically the dopamine receptor 4 subtype, in the refinement of locomotor activity in vertebrates.Significance Statement Throughout life, all vertebrates acquire and improve gross motor skills. This is particularly evident in the locomotor system where motor output is initially coarse and becomes progressively more refined during development. Previously, we demonstrated that dopaminergic signaling was a factor in the developmental refinement of locomotor activity. However, an understanding of the molecular and functional mechanisms underlying the dopaminergic-mediated refinement of spinal locomotor activity remains elusive. This study demonstrates, in larval zebrafish, that increased expression of all D2-like dopamine receptor subtypes, levels of dopamine, and activity of diencephalic dopaminergic neurons correlate with the refinement of locomotor activity.

Cortical neurons in sensory areas undergo a protracted process of postnatal maturation that includes changes in membrane properties, synaptic drive and connectivity. The completion of this process is associated with the closure of critical periods for experience-dependent plasticity in visual, auditory and somatosensory cortices. Whether these findings extend to the postnatal development of cortical circuits for taste is currently unknown. Taste receptor cells in the taste buds reliably fire action potentials in response to taste stimuli by the third postnatal week and show extended refinement of membrane excitability into adulthood. Taste responsive neurons in the nucleus of the solitary tract show reorganization of peripheral nerve terminals (NTS) over a timeline comparable to taste buds. However, no study to date investigated the postnatal development of neurons in the gustatory cortex (GC). Here, we focused on pyramidal neurons in the deep layers of GC in acute slices from male and female mice and compared their membrane properties from the third to the eighth postnatal week. We report changes in intrinsic excitability and a shift of the excitation/inhibition (E/I) balance toward inhibition as pyramidal neurons reach their young adult properties. The increase in inhibitory drive accompanied a protracted process of postnatal maturation of inhibitory circuits mediated by parvalbumin expressing neurons (PV+ neurons) that showed an increase in their association with perineuronal nets (PNNs) as well as refinement of their connectivity onto pyramidal neurons. Together, our results indicate that GC neurons undergo protracted postnatal maturation that may influence taste response properties.Significance Statement We show that the circuit in the gustatory cortex (GC) undergoes a protracted maturation process extending into adulthood that shifts the excitability of GC toward inhibition through changes in pyramidal neurons membrane properties, increased inhibitory synaptic drive and refinement of parvalbumin neurons connectivity. GC circuit refinement extends beyond the developmental windows previously reported for other sensory cortical circuits and overlaps with the window of maturation for taste receptor cells and with the critical period for the development of taste preferences. As finding nutritious food sources may require the integration of vision, audition, somatosensation and olfaction, an extended maturation of GC may facilitate the integration of sensory information for the identification of food and the decision to ingest it.

Stressful events are a leading factor in 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.Significance Statement The medial prefrontal cortex (mPFC) is strongly associated with depression etiology and exposure to uncontrollable stressors results in synaptic dysfunction and loss. Understanding the molecular changes in the mPFC that occur under stressful conditions that lead to maladaptive behavioral responses is essential for basic characterization of stress responses and identification of novel targets for antidepressant medications.

Environmental enrichment (EE) reduces vulnerability to multiple drugs of abuse, yet its impact on fentanyl use and relapse-like behavior remains unclear. Here, we tested whether long-term, non-social, object-based EE alters fentanyl self-administration, extinction, and stress-induced reinstatement in male and female rats. Rats were individually housed in either standard non-enriched (NE) conditions or in EE cages containing a rotating set of novel objects beginning at least three days prior to self-administration. EE did not impact acquisition of fentanyl self-administration but reduced fentanyl intake during maintenance of self-administration and reduced the persistence of drug-seeking in extinction. Following extinction, yohimbine robustly reinstated drug-seeking behavior in NE rats but reinstatement in EE rats was markedly attenuated, indicating reduced sensitivity to stress-induced relapse triggers. Circulating corticosterone levels were lower in EE rats across the experiment and were positively correlated with reinstatement responding, suggesting that enrichment's protective effects may be mediated in part by reduced hypothalamic-pituitary-adrenal (HPA) axis activation. These findings demonstrate that object-based EE, even in the absence of social enrichment, is sufficient to blunt fentanyl use, facilitate extinction, and constrain stress-induced reinstatement. The results highlight the role of environmental context and stress regulation in fentanyl vulnerability and suggest that enrichment-inspired, non-social interventions may offer a scalable strategy to reduce opioid use and relapse risk.Significance Statement This study demonstrates that long-term, object-based environmental enrichment in the absence of social peers is sufficient to reduce fentanyl intake and blunt stress-induced reinstatement in male and female rats. These findings identify a simple, scalable environmental intervention capable of dampening both drug use and vulnerability to relapse triggers. Given the prevalence of individuals exposed to periods of social isolation or with limited access to support, understanding how non-peer-based enrichment influences maladaptive opioid use and stress reactivity is of timely and translational importance. The ability of enrichment to reduce corticosterone, fentanyl consumption, and drug-cue reactivity highlights the importance of environmental context in shaping opioid misuse liability and implicates novel and practical avenues for relapse prevention.

Olfactory anhedonia and heightened aversion to unpleasant odors are well-documented features of depression in humans, yet the neural mechanisms linking chronic stress to altered olfactory perception remain poorly understood. We used the Unpredictable Chronic Mild Stress (UCMS) paradigm to examine how chronic stress affects olfactory avoidance behavior and glial cell morphology across multiple olfactory brain regions in male and female mice. UCMS-treated mice showed increased avoidance of aversive odorants in an odorized light/dark box assay, consistent with heightened aversive reactivity to odors following chronic stress. Using immunohistochemistry, we assessed microglial morphology and astrocyte density across six olfactory and limbic brain regions. Chronic stress produced region-specific glial remodeling: astrocyte counts were selectively elevated in the medial amygdala, and microglial process complexity was increased in the anterior olfactory nucleus and anterior piriform cortex. Microglial morphological complexity in the anterior piriform cortex was correlated with individual odor avoidance scores. These findings reveal that chronic stress induces regionally specific glial plasticity within olfactory sensory and affective networks and suggest that microglial remodeling in piriform cortex may contribute to stress-related changes in olfactory perception.Significance Statement Changes in sensory perception frequently accompany depression. While previous studies have implicated neuroinflammation in depression-related dysfunction within cortical and limbic structures, little is known about how chronic stress affects glial cells in olfactory processing regions. Here, we show that chronic stress induces glial remodeling in key olfactory areas, including the olfactory bulb, anterior piriform cortex, and medial amygdala, and that these changes correlate with heightened behavioral avoidance of aversive odors. These findings suggest that glial plasticity in sensory networks contributes to affective alterations in olfactory perception, revealing a potential mechanism by which mood disorders can influence sensory experience. This work advances our understanding of the neuroimmune basis of sensory-affective integration.

Extinction-reinstatement paradigms have been used to study reward-seeking for both food and drug rewards. The nucleus accumbens (NAc) is of particular interest in reinstatement due to its ability to energize motivated behavior. Previous work found that suppression of neuronal activity or dopamine signaling in NAc reduces reinstatement of food-seeking. Here we used fiber photometry and sensor multiplexing (red-shifted dopamine sensor and genetically-encoded calcium indicator) to measure dopamine and calcium in NAc core of male and female rats on each day of an extinction-reinstatement paradigm with food reward to determine how signals vary across task phases. During self-administration training, we detected positive dopamine transients that initially followed lever-pressing but moved earlier in time as training progressed. A post-press dopamine decrease also emerged with training. For calcium, a decrease from baseline occurred after the press and became more prominent across training. Both patterns were reduced in the first extinction session, with no deflections from baseline detected in either dopamine or calcium traces in the last extinction session. During reinstatement tests primed with either cue or combined food reward and cue presentation, we observed positive calcium and dopamine responses that differed significantly from the signals measured in the last extinction session. While multiplexing has been validated by prior studies, ours is the first to simultaneously record dopamine and calcium during an extinction-reinstatement task. The results provide new information about changing relationships between these signals across task phases, setting the stage for exploring their behavioral significance and mechanisms that may link the two signals.Significance Statement The nucleus accumbens (NAc) is a critical region in reward seeking behavior with dopamine acting on local neurons to energize behavior. Studies examining reward seeking behavior have typically assessed activity of NAc neurons and dopamine release separately. However, recent studies have validated sensor multiplexing for simultaneous recording of calcium (which is related to neuronal activation state) and dopamine. Here we used sensor multiplexing in rats to assess calcium and dopamine signals in NAc on each day of food self-administration, extinction training, and reinstatement testing. We found changing relationships between calcium and dopamine signaling across phases of this paradigm, providing new information about their interplay and setting the stage for future studies of mechanisms linking these signals and their behavioral significance.

Despite major advances in brain-computer interfaces (BCIs), decoding high-level language representations prior to speech remains challenging. While prior efforts have primarily focused on acoustic or articulatory features, how semantic categories are decoded in time and space remains unclear. Here, we investigated how semantic representations unfold over time by analyzing high-gamma (HG, 70-170 Hz) electrocorticography (ECoG) signals from twenty subjects (7 females and 13 males) performing a word-reading task involving body- and non-body-related words. HG activity was examined from word presentation to 500 ms, capturing the pre-speech window. Group-level time-resolved decoding, pooling features across subjects within each Brodmann area (BA), revealed significant classification accuracy above chance in both hemispheres (p<0.05, FDR-corrected). In the left hemisphere, peak-performing BAs followed a frontal-temporal-occipital-parietal cascade: dorsolateral prefrontal cortex (dlPFC) (50 ms), inferior temporal and fusiform gyri (350-400 ms), and supramarginal gyrus (SMG) (500 ms). In contrast, the right hemisphere exhibited an occipital-temporal-frontal-temporal-parietal sequence: visual and temporal pole (TP) regions (50-100 ms), dlPFC (200 ms), fusiform gyrus (FG) (400 ms), and angular gyrus (450 ms). This progression contrasts with the frontal-initiated cascade of the left hemisphere, underscoring hemispheric differences in the timing of peak decoding loci. Cross-temporal regression revealed predictive interregional engagement. In the left hemisphere, early dlPFC activity (0-150 ms) predicted later SMG responses (300-350 ms). In the right, a strong but brief predictive link emerged from the TP to the angular gyrus (200-300 ms; peak R² ≈ 0.70). These findings demonstrate that semantic category decoding relies on temporally structured interregional interactions, revealing distinct hemispheric patterns.Significance statement This study investigates spatiotemporal dynamics in decoding semantic categories during the pre-speech interval using high-resolution intracranial EEG. We reveal a left-hemisphere cascade beginning in frontal areas and extending to temporal, occipital, and parietal regions, and a distinct right-hemisphere cascade involving early occipital and temporal pole activity. Cross-temporal regression reveals sustained left-lateral predictive temporal pattern and a brief but high-precision right-hemisphere link. These findings advance our understanding of how semantic categories are constructed in the brain over time and may inform future efforts to develop neural decoding frameworks that operate before speech output.