Brain Research

Multipotent mesenchymal stem cells (MSCs) including bone marrow stromal cells (BMSCs) have shown analgesic efficacy in recent years. Studies suggested that the therapeutic effect of MSCs was mediated by their secreted small extracellular vesicles (sEVs) mainly exosomes. The present study evaluated the antihyperalgesic effect of BMSC-related sEVs in a mouse model of neuropathic pain involving chronic constriction injury of the infraorbital nerve (CCI-ION). Our separation protocol generated EV particles mostly sized in the range of exosomes (30-170 nm) and express exosome marker proteins CD9, CD81, and Tsg101, suggesting their endosome origin. We show that intravenous injection of BMSC-related sEVs attenuated pain hypersensitivity induced by CCI-ION as indicated by decreased mechanical hypersensitivity (von Frey test) and reduced aversion to noxious stimulation (conditioned place avoidance test). The antihyperalgesic effect of sEVs was observed in both female and male animals, and the effect was dose-dependent. sEVs from NAIVE serum-treated BMSC cultures produced short-lasting antihyperalgesia in male but not female mice, suggesting a subtle sex difference. The antihyperalgesia of sEVs from BMSC culture was blocked by the pretreatment of the culture with GM4869, the antagonist of exosome secretion, suggesting that the effect was not related to other co-isolated soluble mediators but mediated by MSC-derived exosomes. Interestingly, the prior injury condition in which sEVs were isolated favors the pain-relieving effect of sEVs. sEVs isolated from the serum of BMSC-treated animals receiving tendon ligation (TL) injury attenuated hyperalgesia for 24 h, while sEVs from the serum of BMSC-treated NAIVE animals only attenuated hyperalgesia at 3 h after injection. sEVs from the BMSC culture treated with the serum of TL rats were antihyperalgesic, but sEVs from the BMSC culture treated with the serum of naive animals were ineffective. Our results indicate that BMSC-related sEVs produced antihyperalgesia similar to that produced by BMSCs. The results suggest that the interactions between BMSCs and injury conditions are crucially important for producing efficacious sEVs/exosomes and support that the effect of sEVs could be optimized by priming BMSCs with injury-related conditions.

Dorsal (PMd) and ventral (PMv) premotor cortices can modulate contralateral primary motor cortex (M1) excitability, but their distinct interhemispheric influence via transcranial magnetic stimulation (TMS) remains unclear. Single-pulse TMS over PMd, PMv and M1 assessed transcallosal inhibition via the ipsilateral silent period (iSP). Dual-site TMS examined short-(10 ms inter-stimulus interval [ISI]), long-(50 ms ISI) and non-callosal-(0 ms ISI) interhemispheric inhibition (IHI). An iSP was elicited from PMd, PMv, and M1, with distinctly evoked iSP parameters. The iSP magnitude was greatest from M1, followed by PMd and then PMv, while iSP duration was greatest for M1 and showed no differences between PMd and PMv. Dual-site TMS revealed that PMd and M1 inhibited contralateral M1 excitability across all ISIs, while PMv showed inhibition at 0-and 50-ms ISIs. PMd and M1 demonstrated greater short-IHI compared to PMv, all demonstrating similar long-IHI, and PMd demonstrating greater non-callosal-IHI than M1. PMv displayed distinct IHI across ISIs, PMd showed differences across most ISIs and M1 demonstrated the fewest differences across ISIs. Longer iSP duration related to greater long-IHI magnitude elicited from PMd and PMv. Our findings demonstrate differential IHI from PMd and PMv on contralateral M1, which may inform neuromodulation strategies in rehabilitation contexts.

Stem cell therapy represents a promising strategy for the replacement and functional restoration of damaged neural tissue in neurodegenerative conditions. Human dental pulp stem cells (hDPSCs) have emerged as potential candidates for neuroregeneration due to their ease of isolation, neural crest origin, neurotrophic and anti-inflammatory capacity, and demonstrated ability to differentiate in vitro into neuronal-like cells exhibiting electrophysiological activity. Although the immunomodulatory and neuroprotective properties of hDPSCs have been reported in multiple models of brain disease, their capacity to functionally integrate into host neuronal circuits remain poorly understood. In this study, we have grafted green fluorescent protein (GFP)-transduced, neural preconditioned hDPSCs into the CA1 region of the hippocampus of C57BL/6J mice. One month after transplantation, GFP+-hDPSCs survived in the brains of non-immunosuppressed mice and remained localized within the grafted area. Notably, the transplanted cells underwent in situ differentiation and exhibited a neuroblast-like phenotype, characterized by positive doublecortin expression and immature neuronal-like electrophysiological properties, like high membrane input resistance, low capacitance, and the ability to generate single action potentials after stimulation. Together, these findings provide the first evidence that hDPSCs can survive and integrate into the hippocampal network of the mouse brain at one-month post graft, supporting their potential use for future therapeutic applications in acute brain lesions and neurodegenerative disorders.

Objective: Aftereffects of 1 Hz repetitive transcranial magnetic stimulation (rTMS) often differ within and between subjects and thus show low reliability. In this study we investigated the mean and individual aftereffects of 1 Hz rTMS using two opposing current directions, their reliability and potential influences of current direction, participants' sex and state on cortical excitability modulations. Methods: Thirteen healthy, right-handed participants underwent four experimental sessions separated by at least 7 days receiving 2000 pulses of suprathreshold 1 Hz rTMS over the primary motor cortex per session. Two sessions were conducted with an induced current direction of anterior-posterior - posterior-anterior (AP-PA) and two sessions with a PA-AP current direction. Before and after rTMS, 100 single TMS pulses were administered with the respective current direction and electromyography was recorded from the first dorsal interosseous. Questionnaires on demographic data and subjective ratings were completed during the experiment. Results: Linear mixed effect model analysis revealed that 1 Hz rTMS induced an excitatory aftereffect when applied with the PA-AP current direction, and no aftereffect with AP-PA. There was a substantial interindividual variability with only three subjects showing an inhibition to 1 Hz rTMS overall. Also, current direction was the only predictor of rTMS aftereffect. Reliability values of these aftereffects were in the poor to moderate range. Conclusions: Current direction plays a crucial role in determining 1 Hz rTMS aftereffects. Reliability was found to be moderate at best. Additional to current direction, more factors need to be considered to tailor the 1 Hz rTMS aftereffects individually.

Parkinsons disease is a progressive neurodegenerative disorder associated with mitochondrial dysfunction, oxidative stress, impaired autophagy, and dysregulated cellular signaling pathways. Although Glycyrrhiza glabra has been reported to exhibit neuroprotective properties, the early phosphorylation-mediated signaling mechanisms underlying its protective effects remain poorly understood. In this study, we employed a Tandem Mass Tag (TMT)-based temporal quantitative phosphoproteomic approach to investigate early signaling events associated with Glycyrrhiza glabra-mediated neuroprotection in a rotenone-induced in vitro PD model. Differentiated IMR-32 neuronal cells were treated with rotenone alone or in combination with Glycyrrhiza glabra extract, and phosphoproteomic alterations were analyzed at 2, 5, 15, and 30 minutes using liquid chromatography coupled with tandem mass spectrometer. Temporal phosphoproteomic analysis identified 6,424 phosphopeptides corresponding to 2,368 phosphoproteins and 5,468 phosphorylation sites. Comparative analysis revealed extensive phosphorylation rewiring induced by rotenone and restoration of several dysregulated phosphorylation events following Glycyrrhiza glabra co-treatment. More than 130 phosphoproteins and multiple kinase-associated signaling pathways were dynamically regulated across the temporal conditions. Kinase enrichment analysis identified restoration of several critical kinases, including AKT1, MTOR, MAPK1/3, PRKACA, PRKCD, and GSK3A/B, which are associated with neuronal survival, stress adaptation, and autophagy. Integrated pathway and kinase-substrate interaction analyses further revealed enrichment of AMPK signaling, FOXO signaling, receptor tyrosine kinase signaling, RNA processing, and cell-cycle regulatory pathways. Notably, several spliceosome-associated phosphoproteins demonstrated dynamic phosphorylation changes during the early neuroprotective response. Collectively, this study provides a detailed temporal phosphoproteomic landscape of early signaling events associated with Glycyrrhiza glabra-mediated neuroprotection and highlights kinase-driven signaling pathways that may represent potential therapeutic targets in Parkinsons disease.

Spinal cord injury (SCI) is a devastating neurological condition with limited regenerative capacity. Stem cell-based approaches have emerged as promising strategies due to their neuroprotective and immunomodulatory properties, largely mediated by small extracellular vesicles (sEVs) and their molecular cargo, including miRNAs. In this study, we aimed to evaluate the neuroprotective and anti-apoptotic potential of sEVs derived from SPC-01 and iMR-90 neural stem cell sources using an in vitro rat model of SCI. sEVs were isolated from conditioned media and characterized by multi-angle dynamic light scattering and Western blot analysis. Organotypic spinal cord slices (SCS) were used as an in vitro SCI model, with injury induced at 18-20 days, followed by immediate sEV application. After 72 h, tissue samples were collected and tissue was analyzed for markers of apoptosis, cytoskeletal integrity, and survival-related signaling pathways. Results show that SCI induced cytoskeletal disruption and increased apoptotic markers. Treatment with sEVs mitigated these changes, reducing injury-associated protein levels toward baseline. Both SPC-01- and iMR-90-derived sEVs exerted comparable neuroprotective effects, accompanied by decreased PTEN expression, enhanced STAT3 phosphorylation, and increased levels of the anti-apoptotic protein Bcl-xL. In parallel, reduced Nogo-A expression and normalization of RhoA suggested improved cytoskeletal stability and attenuation of inhibitory signaling. Together, these findings demonstrate that neural stem cell-derived sEVs promote early neuroprotective responses in vitro by modulating key signaling pathways, reducing apoptosis, and stabilizing cytoskeletal dynamics, supporting their potential as a cell-free therapeutic strategy for SCI.

Background. Pain influences motor function and has been proposed to reduce corticospinal and intracortical excitability. At the same time, performance can be maintained during pain, at the cost of increased perceived effort, a centrally generated signal reflecting resource engagement. Here, we tested whether contralateral thermal heat pain-related changes in corticospinal and intracortical excitability contribute to increased effort perception. Methods. In this preregistered transcranial magnetic stimulation (TMS) study, twenty-one healthy participants received single and paired pulse TMS at rest and during submaximal isometric right wrist flexions performed at 20% maximal peak force. Trials were conducted under a control condition or during contralateral thermal stimulation (painful or non-painful warm) applied to the left forearm. After each contraction, participants rated the intensity of their perceived effort. Corticospinal and intracortical excitability of the right wrist flexor was assessed at rest and during submaximal contractions. Results. Contralateral heat pain significantly increased perceived effort compared with the control and warm conditions. Contralateral heat pain did not reduce corticospinal or intracortical excitability. Conversely, contralateral heat pain increased corticospinal excitability, reflected primarily in decreased cortical silent period duration. Perceived effort was associated with the subjective experience of pain rather than with TMS-derived variables. Conclusions. These findings suggest that increased effort during contralateral heat pain cannot be attributed to inhibition of the primary motor cortex or the corticospinal pathway. The higher perceived effort in the presence of contralateral heat pain likely reflects the cognitive cost of pain rather than alterations in the transmission of the motor command.

PurposeTranscranial direct current stimulation (tDCS) can noninvasively modulate activity in targeted brain regions. It is well established that the excitability of motor-related regions can increase when the target region is located beneath the anode (anodal tDCS), suggesting its potential to increase motor performance. Although such attempts have been widely examined, the results remain inconclusive. The purpose of this study was to assess the conditions under which anodal tDCS may improve motor performance in healthy adults. MethodsWe conducted a systematic review of studies on the use of anodal tDCS for improving motor performance in healthy adults. A computerized search was performed using the Web of Science, Scopus, PubMed, JDreamIII, and Ichushi-Web to identify relevant studies published between January 1, 1990 and May 25, 2022. ResultsTwenty-five studies were included in the qualitative synthesis. For the meta-analysis, 25 trials (N=885) were extracted from 23 studies. There were significant effects of anodal tDCS on motor performance improvement, but with evidence of publication bias and substantial heterogeneity among the trials. Post-hoc analysis revealed that motor performance 24 hours after the application of anodal tDCS may benefit from stimulation. There was no marked effect related to stimulation intensity, duration, or whether stimulation was provided during motor performance. ConclusionsOur study clarified the current state of anodal tDCS use for motor performance enhancement and indicates that there is currently no reliable evidence to support its effectiveness. Further studies, particularly randomized controlled trials, are necessary to establish the reliability of these effects for future applications.

The amplitude of motor-evoked potentials (MEPs) elicited using transcranial magnetic stimulation (TMS) has been shown to decrease in the short interval prior to response initiation. The cause of this premovement MEP suppression is currently unclear and has been attributed to various processes such as preparation-related inhibition preventing the premature release of planned action or increasing signal-to-noise ratio to facilitate rapid response initiation. The present study explored whether the decrease in MEP amplitude is affected by the task requirements, using reaction time (RT) paradigms that differ in the timeline of preparation and initiation of a motor response. Participants completed simple RT (SRT), choice RT (CRT), and go/no-go (GNG) tasks, while TMS was applied at various times between the warning signal and go-signal. It was hypothesized that if MEP suppression relates to preparation level, the greatest suppression would be observed during the SRT and GNG tasks, as these paradigms encourage advance preparation and response inhibition. Conversely, if the reduction in corticospinal excitability is associated with facilitating response initiation processes, then suppression would be expected for all tasks, including the CRT paradigm in which preparation does not occur until presentation of the go-signal. Results showed MEP amplitudes decreased for all tasks as the go-signal approached; however, both the SRT and GNG had significantly greater MEP suppression 50 ms prior to, and coincident with the go-signal. These results indicate that the nature and origin of the suppression is likely multifactorial and relates to both preparatory and initiation-related processes, with the timeline and magnitude of suppression dependent on the nature of the task being executed. Impact StatementTranscranial magnetic stimulation was used to elicit motor-evoked potentials to examine the timeline of corticospinal activation during the instructed delay period for choice, simple and go/no-go reaction time tasks. For all tasks, corticospinal excitability was initially elevated compared to baseline, followed by a similar magnitude of early suppression. However, just prior to the go-signal, those tasks that allowed advance preparation showed additional suppression, providing novel information linking pre-movement corticospinal suppression to preparatory and inhibition processes.

Voluntary contraction in one limb can facilitate motor output in a distant limb, a phenomenon commonly referred to as the remote effect. However, the neural mechanisms underlying this remote interlimb facilitation remain unclear. This study investigated cortical and spinal contributions to the remote effect in able-bodied participants. Transcranial magnetic stimulation (TMS) was applied over the hand area of the primary motor cortex using posterior-anterior (PA) and anterior-posterior (AP) current directions, which are sensitive to different cortical inputs. Cortical excitability was assessed using single- and paired-pulse paradigms to measure short-interval intracortical inhibition (SICI), short-interval intracortical facilitation (SICF), and short-latency afferent inhibition (SAI). Spinal motoneuron excitability was assessed from F-waves elicited by peripheral nerve stimulation. During voluntary lower-limb contractions, single-pulse TMS elicited larger motor evoked potentials in hand muscles across current directions, indicating a broad increase in net corticospinal output. However, only AP-sensitive paired-pulse measures showed reduced SICI and enhanced SICF during contraction, whereas PA-sensitive SICI and SICF were not significantly altered, suggesting that cortical modulation during the remote effect is expressed more clearly in AP-sensitive measures. SAI with PA stimulation was less consistently expressed during contraction, suggesting that afferent-related inhibitory modulation may also be influenced during the remote effect. In parallel, F-wave amplitude and persistence increased, consistent with enhanced spinal motoneuron excitability. Together, these results provide converging evidence that the remote effect in humans involves broad corticospinal and spinal facilitation, accompanied by current direction-dependent modulation of cortical excitability measures. KEY POINTS SUMMARYO_LIVoluntary contraction in one limb can facilitate motor output in a distant limb, but the mechanisms underlying this remote interlimb facilitation remain unclear. C_LIO_LIWe tested whether remote lower-limb contraction modulates corticospinal output, intracortical excitability, and spinal motoneuron excitability in a resting hand muscle. C_LIO_LISingle-pulse transcranial magnetic stimulation showed that motor evoked potentials in the hand were facilitated during remote lower-limb contraction across multiple current directions, indicating a broad increase in net corticospinal output. C_LIO_LIPaired-pulse measures were modulated preferentially with anterior-posterior stimulation, with reduced short-interval intracortical inhibition and increased short-interval intracortical facilitation, suggesting current direction-dependent modulation of cortical excitability measures. C_LIO_LIF-wave amplitude and persistence were also enhanced during remote lower-limb contraction, indicating increased spinal motoneuron excitability. These findings provide converging evidence that the remote effect involves both cortical and spinal contributions. C_LI

PurposeTo investigate whether rapamycin can regulate microglial activation and polarization via mTOR and its downstream signals via autophagy both in vivo and in vitro. MethodsThe in vivo study used wild type C57BL/6 mice that were intraperitoneally injected with rapamycin (2 mg/kg) plus ONC. The BV2 cell line was used in the in vitro study and the cells were incubated with rapamycin (50 nM) or transfected with a specific mTOR-targeting small interfering RNA (si-mTOR). Immunohistochemical staining was used to observe the changes in the morphology and cell surface area of microglia and Weste blotting analysis was used for detection of the changes in the proteins related autophagy, microglia polarization and mTOR pathway after the retinal tissue or the cell samples were collected. ResultsThese results indicate that rapamycin increases autophagy and M2 polarization by inhibiting p-mTOR in wild-type C57BL/6 mice in vivo. In the BV2 cell line, rapamycin and si-mTOR can enhance autophagy and promote M2 polarization by inhibiting the p-mTOR/p-Unc-51-like kinase 1 (p-ULK1) pathway. ConclusionsIn conclusion, this work contributes to the understanding of the complex interplay among rapamycin, autophagy and microglial activation/polarization, highlights the downstream signaling pathway of mTOR, and highlights the potential therapeutic effects of autophagy-modulating drugs in retinal neuroinflammation and neurodegeneration after TON.

The success of stem cell-derived brain repair for Parkinsons is limited by the variable survival and poor maturation of dopaminergic progenitors after transplantation into the Parkinsonian brain. One approach that has been developed to improve this is engraftment of the cells within a neurotrophin-enriched collagen hydrogel. Although this has been shown to improve progenitor survival and maturation in athymic nude rats, the same beneficial effects of the hydrogel were not seen in cyclosporine immunosuppressed rats. To determine the reasons for these differences, the aim of this study was to assess the local and systemic immune responses to progenitor transplantation in these two recipient groups. To do so, human induced pluripotent stem cell-derived dopaminergic progenitors were transplanted into 6-hydroxydopamine-lesioned striatum of athymic or cyclosporine immunosuppressed rats. The cells were transplanted either alone, with the neurotrophins GDNF and BDNF, in an unloaded collagen hydrogel, or in a neurotrophin-loaded collagen hydrogel. Post-mortem assessment included both graft site and blood analysis of immune cell populations. As expected, nude rats showed a pronounced innate immune cell response at the graft site but no T-cell recruitment or activation locally or systemically. In contrast, while the immunosuppressed rats also showed the expected innate immune cells response to the transplant, there was also infiltration of CD4+ and CD8+ T cells at the site of transplantation as well as circulating activated T-cells. Thus, this study suggests that the benefits of the hydrogel that were seen in the athymic nude rats did not manifest in the cyclosporine immunosuppressed rats due to incomplete immunosupression. This study shows the importance of careful optimisation of the immunosuppressive regime chosen before xenotransplantation experiments.

The paraventricular nucleus (PVN) plays a central role in neuroendocrine and autonomic regulation, including male sexual behavior. Dopaminergic and nitrergic signaling within the PVN are functionally linked, but their cellular relationship remains unclear. We examined the colocalization of dopamine D1 and D2 receptors with neuronal nitric oxide synthase (nNOS) in adult male rats using double-label immunohistochemistry. nNOS-immunoreactive neurons were widely distributed in PVN, with subsets co-expressing D1R or D2R. Approximately 45% of nNOS-positive neurons expressed dopaminergic receptors. These findings provide structural evidence for dopaminergic-nitrergic interaction in the PVN and support the possibility of direct dopaminergic modulation of nitrergic neurons.

Fast periodic visual stimulation (FPVS) has recently demonstrated an ability to index multiple cognitive domains such as facial expression processing, working memory, and more recently, semantic categorization in just a few minutes of recording time. The present work investigates the effects of low semantic distance and how this modulates semantic categorization responses. Twenty-seven healthy young adults completed an FPVS oddball paradigm in order to determine whether comparing fruits and vegetables of the same color would elicit semantic categorization responses. Strong oddball responses were observed up to the 10th frequency harmonic, and responses showed statistically significant right occipito-temporal lateralization that was not present in a low-level color change condition completed by participants and is opposite the pattern observed in recent word-based semantic categorization FPVS studies. The findings suggest that image-based FPVS paradigms should be investigated further as candidate tools to study conditions that affect semantic categorization such as Alzheimers disease.

Neural stem cell (NSC) transplantation is a promising strategy for repairing the injured spinal cord, but transplanted cells typically require immunosuppressive therapy to prevent rejection, even for induced pluripotent stem cell (iPSC)-derived autologous grafts. However, the effects of immunosuppressive drugs on neurite outgrowth and axonal regeneration, processes critical for neural circuit reconstruction, have not been fully characterized. In this study, we tested nine clinically relevant immunosuppressants on human iPSC-derived neurons and primary human spinal cord NSCs in vitro at concentrations approximating clinical exposure levels. The drug panel included FK-506 (tacrolimus), cyclosporine A (CsA), rapamycin, belatacept (Nulojix), etanercept (Enbrel), mycophenolate mofetil (CellCept), cyclophosphamide (Cytoxan), prednisone, and azathioprine (Imuran). Neurite outgrowth was quantified via automated high-content imaging. Multiple agents, including CsA, Imuran, Nulojix, and CellCept, induced significant reductions in neurite outgrowth in a cell type- and dose-dependent manner, with CsA producing the most robust and consistent inhibition across both cell lines. In contrast, FK-506 showed no significant effect on neurite extension at clinically relevant concentrations. Consistent with the in vitro results, human neural progenitor cell grafts in a rodent spinal cord injury model exhibited significantly reduced graft-derived axon extension in the host spinal cord when hosts were treated with CsA rather than FK-506. These findings demonstrate that immunosuppressant choice can profoundly influence neural graft integration and axonal regeneration. Our study underscores the importance of preclinical evaluation of immunosuppressive regimens and suggests that selecting agents such as FK-506 over CsA may improve outcomes in future stem cell-based therapeutic trials for spinal cord injury and related disorders of the central nervous system.

Spinal cord injury (SCI) results in dysfunction of both motor and sensory systems, which can be characterized by neuropathic pain, hypersensitivity, muscular spasticity and rigidity. Most SCIs result from incidents such as vehicle accidents or falls, resulting in polytraumatic SCI that includes peripheral injuries in addition to direct CNS damage. Recent findings suggest that spinal cord synaptic plasticity plays a crucial role in neuropathic pain pathophysiology, specifically in association with spinal sensitization and the consequent onset of AMPA-related maladaptive plasticity. Further findings have demonstrated that nociceptive peripheral stimulation in the acute phase of SCI results in maladaptive spinal synaptic plasticity by overdriving GluA2-lacking calcium-permeable AMPARs (CP-AMPARs). Here, we investigated the effect of a spared nerve injury (SNI) in conjunction with SCI to determine the effect of polytraumatic SCI on maladaptive plasticity in the spinal cord. Near-IR quantitative Western blot analysis demonstrated that SCI+SNI increases spinal GluA1 expression, but not GluA2. Patch-clamp confirmed that AMPAR currents in spinal motorneurons increase after SCI with SNI, and decrease after the administration of NASPM, a CP-AMPAR antagonist. Data-driven analysis using non-linear principal components analysis (NL-PCA) also demonstrated that SCI with SNI produces a multivariate signature of AMPAR plasticity that is observed in other forms of nociceptive peripheral input, indicating a general mechanism for maladaptive plasticity in spinal motor systems in response to polytraumatic SCI.

The addictive properties of opioids are due in part to these drugs ability to alter ventral tegmental area (VTA) activity via activation of mu opioid receptors (MORs) on local and distal inputs. Prior studies have identified numerous opioid-modulated afferents to the VTA, some of which show differing levels of functional modulation by opioids, but the degree to which this parallels differences in receptor expression is not known. Hence, we used retrograde labeling combined with RNAscope to examine oprm1 mRNA expression in VTA-projecting afferents arising from a variety of distal brain regions. Because opioids are thought to be particularly influential on GABAergic afferents to the VTA, we also examined colocalization of oprm1 with GABAergic markers in VTA-projecting neurons. Interestingly, we found that oprm1 mRNA is present in both GABAergic and non-GABAergic VTA-projecting neurons. However, many (though not all) GABAergic afferents expressed higher levels of oprm1 compared to most non-GABAergic afferents (especially those arising from the cortex). These results complement previous anatomical studies that had examined oprm1 expression in these regions but in a non-quantitative way and without regard to their efferent targets. Our findings encourage future work to examine the functional implications of MOR sensitivity within these afferent pathways.

Curiosity, a key driver of exploration and learning, is reinforced by reward-related neurochemical systems, yet the role of the opioidergic system in modulating this behavior remains unclear. Music, as a highly rewarding stimulus, offers a unique context to investigate the neurochemical basis of curiosity, particularly the unexplored role of opioids in music-driven exploration. To fill this gap, we performed a double-blind within-subject pharmacological design, in which 26 participants received, in two different sessions, either a placebo or the opioid antagonist naltrexone. During each session, participants engaged in a music exploration/exploitation trade-off paradigm designed to assess their willingness to pay for exploring unfamiliar electronic music. Using logistic regression mixed-effects models, we found that while naltrexone did not affect overall curiosity ratings, it significantly reduced exploratory behavior in states of heightened curiosity. These findings suggest that the opioidergic system plays a critical role in regulating the relationship between curiosity and exploration, particularly in the context of novel and rewarding stimuli like music. Overall, the present research provides new and compelling evidence on the important relationship between curiosity and exploration and its regulation with the opioidergic neurotransmitter subsystem. Significance StatementThe present research aimed to advance our understanding of the neurochemical mechanisms underlying curiosity and information seeking. In our study, we employed a pharmacological design to examine the role of the opioidergic system in music-related exploration. Using a novel music exploration/exploitation paradigm, we found that while naltrexone, an opioid antagonist, did not affect baseline curiosity ratings, it markedly reduced exploratory behavior during high-curiosity states in the presence of potential monetary losses. These results provide new evidence that opioidergic modulation plays a critical role in regulating curiosity-driven exploration. This new evidence might be relevant in the future for better understanding how neurochemical systems shape learning, motivation, and affective responses in complex cognitive domains such as music.

Facial color is associated with the perceptual evaluation of emotions, and compared with faces with original facial or greenish color, reddish angry faces are often judged as having higher emotion intensity. Although perceptual modulation by the relationship between anger and red has also been reported from the perspective of electroencephalography (EEG), how variations in perceived emotion intensity are reflected in brain activity remains unclear. This study investigated whether EEG activity associated with face and facial expression processing is modulated as a function of the interaction between facial color and perceived emotion intensity. In the experiment, we recorded EEGs while participants evaluated emotion intensity using facial stimuli created by combining morph continua from neutral to angry expressions with three types of facial color conditions (original, red, and green). The results revealed that, in the red facial color condition, the early posterior negativity (EPN) amplitude significantly increased as a function of emotion intensity compared with those in the original and green facial color conditions. These findings suggest that the early, automatic affective processing of facial expressions, reflected in the EPN, is modulated by the combination of facial color and emotion intensity. Our findings provide new evidence that early, automatic affective processing of facial expressions, as indexed by the EPN, is modulated by the congruency between high anger intensity and a reddish facial color. HighlightO_LIReddish angry faces increase the ERP component associated with emotion evaluation. C_LIO_LIThe relationship between anger and red is evident in the left hemisphere. C_LIO_LIThe interaction between facial expression and color occurs at a later cognitive processing stage than facial expression or facial color processing alone. C_LI

The Dorsal Vagal Complex (DVC) is a critical brainstem relay for visceral sensory information, sympathetic regulation, and gut-brain communication. Current weight-reducing pharmacotherapies are reported to target this brainstem region to elicit their main satiety actions. Despite its importance, no published step-by-step protocol exists for stereotaxic targeting of this region in rodents. Here, we present a detailed protocol for bilateral administration of substances into the DVC of mice using the atlanto-occipital membrane approach. We describe the surgical access, obex-referenced coordinate system, injection parameters, and we provide a histological validation. This protocol is useful for the study of DVC cells and efferent and afferent neuronal DVC circuits using common neuroscience tools such as tracings, optogenetics or chemogenetics. For complete details on the use and execution of this protocol, please refer to Martinez de Morentin et al.(Martinez De Morentin et al., 2024)