Magnetic resonance imaging (MRI) is a critical tool for translational neuroscience, but preclinical studies frequently rely on anesthesia, which alters neural activity and limits comparison with human studies. Awake rodent functional MRI (fMRI) enables investigation of brain function under physiologically relevant conditions; however, its implementation is constrained by the need for anesthesia during restraint setup. We developed and evaluated a restraint system and habituation protocol for awake rat fMRI. Ten rats were studied: an awake group and an anesthetized group (three males and two females per group). The protocol included head post implantation and an 11 d habituation period. T2-weighted anatomical and functional scans were acquired. Head motion and functional connectivity were analyzed using the RABIES pipeline and compared between groups. The modular 3D-printed restraint system developed can be assembled in under 5 min; eliminates the need for anesthesia, ear bars, and bite bars; and supports several behavioral paradigms. High-quality anatomical and functional images were obtained for awake rats. Anesthetized rats exhibited significantly lower translation, rotation, and framewise displacement. Functional connectivity differed between awake and anesthetized rats, with some region pairs showing higher (e.g., left-right primary somatosensory cortex and hypothalamus-insula) and lower (e.g., cingulate-prelimbic cortex and retrosplenial-motor cortex) correlations in awake rats. However, these differences did not survive network-based statistics correction. This work presents a scalable, reproducible, and animal-friendly platform for awake rat fMRI that enables high-quality, behaviorally enriched imaging without anesthesia, while highlighting the effects of anesthesia on functional connectivity.

The chemokine CXCL12 plays critical roles in the development of the hippocampus dentate gyrus during both embryogenesis and adulthood. While multiple cell types in the hippocampus express Cxcl12, their individual contributions to the dentate gyrus development and function remain unclear. Here, using Cxcl12 reporter mice of both sexes, we characterize Cxcl12 expression in Cajal–Retzius (CR) cells—neurons that guide dentate gyrus morphogenesis and influence hippocampal circuitry. We show that CR cells prominently express Cxcl12 during early postnatal development, although both the number and proportion of Cxcl12-expressing CR cells decline significantly in adulthood. Notably, partial deletion of Cxcl12 from hippocampal CR cells in male and female mice does not result in detectable changes in dentate gyrus architecture, adult neurogenesis, or specific behaviors. These findings suggest that CR cell-derived CXCL12 may be less critical for dentate gyrus development than previously assumed and underscore the complexity and potential redundancy of CXCL12 signaling in the hippocampus.

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.

Dopamine release in the nucleus accumbens is classically linked to associative learning, signaling relationships between predictive cues and outcomes. Yet, dopamine is also strongly modulated by novelty, a nonassociative factor that has received comparatively little attention. Here, we used optical dopamine sensors in awake, behaving male and female mice to define how novelty alters the temporal dynamics of dopamine release during aversive learning. We manipulated novelty in three ways: (1) omitting expected footshocks, (2) introducing novel neutral cues concurrently with shock-predictive stimuli, and (3) presenting novel stimuli in an unpaired fashion within a context. Across all conditions, manipulations robustly increased dopamine release and in some cases altered the directionality of cue-evoked dopamine responses. Notably, these effects extended beyond the immediate stimulus window, altering subsequent responses to both conditioned cues and footshocks. Together, these findings demonstrate that changes in the environment that extend beyond prediction-based learning can exert a powerful and sustained influence on dopamine signaling, reshaping how aversive cues and outcomes are represented in the brain.

RNA localization to neuronal axons and dendrites provides spatiotemporal control over gene expression to support synapse function. Neuronal messenger RNAs (mRNAs) localize as ribonucleoprotein particles (RNPs), commonly known as RNA granules, the composition of which influences when and where proteins are made. High-throughput sequencing has revealed thousands of mRNAs that localize to the hippocampal neuropil. Whether these mRNAs are spatially organized into common RNA granules or distributed as independent mRNAs for proper delivery to synapses is debated. Here, using highly multiplexed single-molecule fluorescence in situ hybridization (HiPlex smFISH) and colocalization analyses, we investigate the subcellular spatial distribution of 15 synaptic neuropil localized mRNAs in the male and female rodent hippocampus. We observed that these mRNAs are present in the neuropil as heterogeneously sized fluorescent puncta with spatial colocalization patterns that generally scale by neuropil mRNA abundance. Indeed, differentially expressed mRNAs across cell types displayed colocalization patterns that scaled by abundance, as did simulations that reproduce cell-specific differences in abundance. Thus, the probability of these mRNAs colocalizing in the neuropil is best explained by stochastic interactions based on abundance, which places constraints on the mechanisms mediating efficient transport to synapses.

Exposure to early life stress (ELS) can exert long-lasting impacts on emotional regulation. The corticolimbic system including the basolateral amygdala (BLA), ventral hippocampus (vHIP), and the medial prefrontal cortex (mPFC) plays a key role in fear learning. Using the limited bedding paradigm (LB), we examined the functional consequences of ELS on excitatory and inhibitory tone in the prelimbic (PL) mPFC after fear conditioning in rats. In adults, LB exposure enhanced in vivo glutamate release in the PL mPFC during fear conditioning in male, but not female offspring. In contrast, the glutamate response to fear conditioning was diminished in LB-exposed pre-adolescent males, but not females. We investigated whether reduced glutamatergic inputs and/or elevated inhibitory tone might contribute to the diminished glutamate response in the mPFC following LB in pre-adolescent male rats. Indeed, we found that LB exposure specifically increased the activation of PV, but not SST interneurons in layer V, but not layer II/III of the PL mPFC in fear-exposed pre-adolescent males. Presynaptic glutamate release probability was reduced by LB exposure in layer V, but increased in layer II/III of the PL mPFC. These functional changes might be related to the LB-induced alterations in the bilaminar distribution of BLA and vHIP projections to the PL mPFC we observed in pre-adolescent males. Overall, our findings suggest that ELS modifies glutamate release and PL mPFC function during fear conditioning in a sex- and age-dependent fashion, likely through layer-specific shifts in excitation/inhibition balance.

The ability to inhibit and adapt our behavior in response to changing stimuli is a critical component of everyday life. Individuals with Parkinson’s disease (PD) may struggle to inhibit behavior, particularly in the presence of dopaminergic therapy, which can result in impulsive behavior. Impulse control disorders are often operationalized in the laboratory using motor inhibition tasks. However, deficits of motor inhibition tasks are not always observed in PD, perhaps because of the nature of the motor inhibition that is engaged in typical tasks (e.g., suppression of incipient movement such as a button press). We employed a novel continuous movement stop task to investigate planned and unplanned motor inhibition during ongoing movement. EEG was recorded during task performance from individuals with PD (OFF and ON dopaminergic medication) and age-matched healthy controls (HC). Participants were of any sex. We found that the time it took for participants to stop a continuous movement was impaired (i.e., longer) in PD patients ON medication compared with both patients OFF medication and HC. This finding was accompanied by diminished midfrontal theta power following the stop signal in PD (ON and OFF) compared with HC. Additionally, an increase in midfrontal beta power was observed, which was higher in unplanned stopping compared with planned for all groups. However, this increase in beta occurred late—after the time of outright stopping. Together, these findings demonstrate that stopping ongoing movements was impaired in PD patients ON medication and theta and beta power play distinct roles in inhibition of movement.

Aberrant dopamine transmission is a hallmark of several psychiatric disorders. Dopamine neurons in the ventral tegmental area (VTA) display distinct activity states that are regulated by discrete afferent inputs. For example, burst firing requires excitatory input from the mesopontine tegmentum, while dopamine neuron population activity, defined as the number of spontaneously active dopamine neurons, is thought to be dependent on inhibitory drive from the ventral pallidum (VP). Rodent models used to study psychiatric disorders, such as psychosis, consistently exhibit elevated dopamine neuron population activity, due to decreased tonic inhibition from the VP. However, it remains unclear whether the VP can modulate all dopamine neurons or if only a specific subset of VTA dopamine neurons receive innervation from the VP to be recruited as required. This knowledge is critical for understanding dopamine regulation in normal and pathological conditions. Here, we used in vivo electrophysiology in male and female rats to record VTA dopamine neurons inhibited by electrical stimulation of the VP. Specifically, VP stimulation inhibited ∼22% of spontaneously active dopamine neurons; however, activation of the ventral hippocampus, a modulator of VTA population activity, increased the proportion to ∼48%. This increase suggests that VP selectively modulates a subset of dopamine neurons that can be recruited by afferent activation. Anterograde monosynaptic tracing revealed that approximately half of the VTA dopamine neurons receive input from the VP. Taken together, we demonstrate that a subset of VTA dopamine neurons receives monosynaptic input from the VP, providing valuable information regarding the regulation of VTA neuron activity.

Social interactions are fundamental to human cognition, with the right temporoparietal junction (rTPJ) playing a key role in integrating motor coordination and social inference. While transcranial random noise stimulation (tRNS) is a promising technique for modulating cortical excitability in real time, its effect on dynamic social processes remains largely unexplored. This study applied high-definition tRNS (HD-tRNS) over the rTPJ during an interactive task to modulate motor coordination and social inference. Eighty neurotypical adults (49 female) were equally distributed across two experiments: Experiment 1, a block design with randomized active and sham stimulation blocks; or Experiment 2, a trial-by-trial design with intermixed stimulation protocols. Participants performed a coordination task with a covert virtual partner programmed to behave cooperatively or competitively. Kinematic data and self-reported attributions of humanness and cooperativeness were analyzed. The results showed that HD-tRNS over the rTPJ did not affect motor coordination or overall task performance in either experiment. However, in Experiment 1, active stimulation progressively reduced attributed humanness and cooperativeness toward the competitive virtual partner, suggesting enhanced detection of antagonistic intent. This gradual modulation of social inference was absent in Experiment 2, where frequent protocol switching likely disrupted the buildup of stimulation effects. Together, these findings highlight the rTPJ's causal role in self–other distinction, underscore the importance of stimulation protocol design in shaping social cognition, and support the exploration of targeted neuromodulation in clinical and developmental populations with atypical social cognition.

The parabrachial (PB) nucleus participates in taste processing and integration with other senses. PB neurons that express the Calca gene support sensory-integrative responses, albeit only limited data have addressed their influence on taste. Here we investigated how chemogenetic dampening of PB-Calca neurons affected mouse orosensory preferences for diverse taste stimuli in brief-access fluid exposure tests, which capture oral sensory/tongue control of licking behavior. Intracranial delivery of Cre-dependent viruses in female and male CalcaCre/+ mice induced expression of the inhibitory designer receptor hM4Di:mCherry (hM4Di mice) or fluorophore mCherry alone (mCherry control mice) in PB-Calca neurons. Several weeks later, hM4Di and mCherry mice entered brief-access tests where they could lick solutions during discrete, seconds-long trials. Stimuli included concentration series of the behaviorally avoided bitter taste stimuli quinine and cycloheximide, the appetitive sugar sucrose, and mildly cool water and less preferred innocuous warm water. Blinded experimenters administered the hM4Di ligand clozapine-N-oxide (CNO) to all hM4Di and mCherry mice prior to daily tests. With CNO, hM4Di mice displayed greater average licking (i.e., less avoidance) of quinine than mCherry mice (p < 0.05). Moreover, male hM4Di mice selectively showed reduced mean licking preferences for sucrose under CNO (p < 0.05). These data suggest that PB-Calca neurons participate in both aversive and appetitive taste-guided behaviors, with their role in appetitive taste dependent on sex. Finally, orosensory responses to cycloheximide and thermal-controlled water did not differ (p > 0.05) between hM4Di and mCherry mice under CNO. Results are discussed considering functional differences among stimuli and study limitations.