Neuronal Populations Research Articles

12237 Monosynaptic Gabaergic Signaling From Suprachiasmatic Nucleus Neuromedin S Neurons To Preoptic Area Kisspeptin Neurons In Mice

Abstract Disclosure: W.I. Abdulmajeed: None. B.B. Jamieson: None. S.X. Thomas: None. Y. Rim: None. A.G. Novak: None. R.E. Campbell: None. R. Piet: None. In female rodents, projections from suprachiasmatic nucleus (SCN) neurons relay timing cues to the gonadotropin-releasing hormone (GnRH) neuronal network for the preovulatory surge. Kisspeptin (KISS1) neurons of the rostral periventricular area of the third ventricle (RP3V) are thought to integrate these cues with estradiol positive feedback to drive GnRH neuron activity, the gonadotropin surge and ovulation. We recently reported that SCN neuron projections release arginine vasopressin (AVP), thereby stimulating female RP3VKiss[1] neuron action potential firing in an estrous cycle stage-dependent manner. In the SCN, AVP is expressed in a subset of neuromedin S (NMS) neurons, a neuronal population essential for circadian rhythms. Moreover, a previous report indicates that exogenous NMS stimulates LH secretion in female rodents. SCNNMS neurons might, therefore, play a role in timing the preovulatory surge. However, whether SCNNMS neurons project to and regulate the activity of RP3VKISS[1] neurons is currently unknown. To address this question, we combined anterograde viral tract-tracing and channelrhodopsin-2 (ChR2)- assisted circuit mapping. Female mice expressing Cre recombinase in NMS cells (NMS-Cre) received stereotaxic injections in the SCN of adeno-associated viral vectors (AAVs) carrying the Cre-dependent sequence for mCherry. Fluorescence immunohistochemistry revealed that mCherry-expressing fibers come in close apposition with the great majority (>80%) of KISS1-immunoreactive neurons in the RP3V, suggesting a SCNNMS-to-RP3VKISS[1] neuron circuit. To interrogate this circuit, we next injected AAVs carrying Cre-dependent channelrhodopsin (ChR2) in the SCN of female NMS-Cre mice that also express the green fluorescent protein in KISS1 cells. Blue LED light (460-487 nm) activation of ChR2-expressing SCNNMS neuronal fibers in the RP3V evoked postsynaptic currents (PSCs) in most KISS1 neurons recorded in whole-cell voltage clamp (-60 mV) in brain slices. Evoked PSCs had short latencies and were blocked by bath application of 0.5 µM tetrodotoxin (TTX), indicating that they were generated by action potential-dependent release. Addition of 100 µM 4-Aminopyridine (4-AP, a potassium channel blocker) to TTX rescued these responses, indicating the existence of a monosynaptic SCNNMS-to-RP3VKISS[1] connection. In addition, evoked PSCs were suppressed by 5 µM gabazine, a GABAA receptor antagonist, but not by 20 µM NBQX and 50 µM D-AP5, AMPA- and NMDA-type glutamate receptor antagonists, respectively, revealing that SCNNMS projections release GABA onto RP3VKISS[1] neurons. Together, our findings are consistent with SCNNMS neurons extending monosynaptic GABAergic projections to RP3VKISS[1] neurons. Further studies are needed to determine the impact of these projections on RP3VKISS[1] neuron activity and the role they might play in the preovulatory surge. Presentation: 6/1/2024

7996 Neuroanatomic Characterization of Mouse Kisspeptin Neurons in the Bed Nucleus of the Stria Terminalis

Abstract Disclosure: S.T. Zdon: None. M.S. Silva: None. V.M. Navarro: None. Kisspeptin (Kiss1) neurons are essential for reproductive function as they are strong regulators of the hypothalamic-pituitary-gonadal axis (HPG). Mammalian Kiss1 neurons are mainly located in the hypothalamus, controlling gonadotropin-releasing hormone (GnRH) secretion, where the anteroventral periventricular nucleus (AVPV) population is morphologically and functionally sexually differentiated. Kiss1 neurons are also found in limbic areas, such as the bed nucleus of the stria terminalis (BNST). This nucleus is involved in the integration of the HPG axis with sex-dependent social behaviors and metabolic functions. However, it remains unclear whether these populations show sexually dimorphic features. The present study focused on the neuroanatomical characterization of Kiss1BNST neurons in mice on both sexes. A Kiss1cre;salsa6f mouse strain carrying the expression of tdTomato specifically in Kiss1 neurons was used to study the distribution of Kiss1BNST neurons. The BNST is divided into the anterior division of the BNST (aBNST), and a more posterior division called the principal component of the BNST (prBNST). Results indicate that male mice have significantly more Kiss1prBNST neurons than females (Male: 139.4 ± 23.32 vs. Female: 43.70 ± 5.33 neurons; **P < 0.01). This sex-biased distribution was absent in Kiss1aBNST neurons. Additionally, stereotaxically injecting a Cre-dependent recombinant adenovirus carrying the expression of mCherry reporter into the aBNST or prBNST in adult mice was performed to observe neuronal projections. We observed fibers from aBNST-injected Kiss1 neurons projecting widely to areas that include the AVPV, medial preoptic area (MPO), lateral preoptic area (LPO), and periventricular hypothalamic nucleus (PVH). Moreover, fibers and cell bodies from male prBNST-injected Kiss1 neurons projected to dorsal lateral BNST (dlBNST), juxta-periventricular lateral hypothalamic area (JPLH), and the posterolateral stria terminalis (STMPL). This study observed a sexual dimorphic Kiss1 neuronal population in the prBNST, with males having more Kiss1prBNST neurons present and prospective postsynaptic areas in the brain where these neurons project to, helping to aid future studies on characterizing the functional role of these neurons. Presentation: 6/1/2024

8302 A Novel Tool to Map Estrogen Responsiveness Within Estrogen Sensitive Hypothalamic Neurons in Mice

Abstract Disclosure: A.L. Cara: None. A.L. Hall: None. K.K. Soma: None. J. van Veen: None. S.M. Correa: None. Identification of hormone sensitive cells has been traditionally evaluated by hormone receptor expression. Receptor positive cells can be labeled via expression of mRNA transcript via in situ hybridization, presence of protein via immunohistology, or via conditionally-dependent Cre recombinase driven reporter expression. However, these methods used to label and manipulate cells only indicate which cells are capable of sensing hormones, and not necessarily which cells are responding to a hormonal signal in a specific physiologic or behavioral context. Furthermore, broad labeling of all receptor positive cells may not accurately reflect the heterogeneous nature of certain cell populations, particularly in the brain. The hypothalamus is a brain region with well characterized hormone receptor expression, including estrogen receptors, such as ERα (encoded by the Esr1 gene). Estrogens have a broad array of physiologic and behavioral functions which are mediated through hypothalamic nuclei, including thermoregulation, metabolism, and reproduction. Neuronal populations mediating these functions have been characterized, targeted, and manipulated based on ERα expression, but the dynamic response to estrogens in these highly heterogeneous populations has yet to be uncovered. We developed a custom reporter virus that fluorescently labels estrogen-sensitive and responsive cells. The virus contains a flip-excision switch (FLEX) to drive Cre-dependent expression of a FusionRed fluorescent reporter, eight tandem estrogen response elements (ERE), and a destabilized enhanced green fluorescent protein (EGFP) with a 2-hour half-life. The Cre-dependent expression of FusionRed identifies all Esr1-Cre cells at the time of transduction, while the ERE-EGFP identifies a subset of Esr1-Cre+ cells that are actively responding to estrogens via nuclear receptor signaling. We stereotactically injected the ERE-EGFP virus to distinct hypothalamic nuclei of Esr1-Cre female mice. Following transduction, mice were sacrificed during different stages of the estrous cycle. Spatial analysis of ERE-EGFP expression revealed dynamic changes across the estrous cycle, with responses differing based on hypothalamic nucleus. Reporter expression dynamics were compared to estrogen levels measured by LC-MS/MS in ovary, serum, and microdissected brain tissue. A separate group of mice were ovariectomized and given estradiol benzoate (EB) or vehicle control, and neurons were flow sorted for bulk RNA sequencing. We discovered transcriptomic differences in FusionRed+ cells with high or low ERE-EGFP expression, including upregulation of estrogen target genes (e.g., Pgr, encoding progesterone receptor) with EB treatment in ERE-EGFP+ cells. In summary, this new viral reporter reveals divergent responses to estrogens across subsets of estrogen sensitive neurons, brain regions, and hormonal states. Presentation: 6/2/2024

Crucial role of Snf7-3 in synaptic function and cognitive behavior revealed by conventional and conditional knockout mouse models

Snf7-3 is a crucial component of the endosomal sorting complexes required for transport (ESCRT) pathway, playing a vital role in endolysosomal functions. To elucidate the role of Snf7-3 in vivo, we developed conventional-like and conditional Snf7-3 knockout (KO) mouse models using a “Knockout-first” strategy. Conventional-like Snf7-3 KO mice showed significantly reduced Snf7-3 mRNA expression, and older mice (25–40 weeks) exhibited impaired social recognition and increased miniature excitatory postsynaptic currents (mEPSCs). Similarly, conditional KO mice aged 8–24 weeks, with Snf7-3 specifically deleted in forebrain excitatory neurons, displayed impaired object location memory and elevated mEPSC frequency. Consistently, Snf7-3 knockdown in cultured mouse hippocampal neurons led to increased densities of pre- and postsynaptic puncta, supporting the observed increase in mEPSC frequency. In addition, enhanced dendritic complexity was observed in the medial prefrontal cortex of these mice, indicating early synaptic disturbances. Our findings underscore the critical role of Snf7-3 in maintaining normal cognitive functions and social behaviors. The observed synaptic and behavioral deficits in both conventional-like and conditional KO mice highlight the importance of Snf7-3 in specific neuronal populations, suggesting that early synaptic changes could precede more pronounced cognitive impairments.

Two-photon all-optical neurophysiology for the dissection of larval zebrafish brain functional and effective connectivity

One of the most audacious goals of modern neuroscience is unraveling the complex web of causal relations underlying the activity of neuronal populations on a whole-brain scale. This endeavor, which was prohibitive only a couple of decades ago, has recently become within reach owing to the advancements in optical methods and the advent of genetically encoded indicators/actuators. These techniques, applied to the translucent larval zebrafish have enabled recording and manipulation of the activity of extensive neuronal populations spanning the entire vertebrate brain. Here, we present a custom two-photon optical system that couples light-sheet imaging and 3D excitation with acousto-optic deflectors for simultaneous high-speed volumetric recording and optogenetic stimulation. By employing a zebrafish line with pan-neuronal expression of both the calcium reporter GCaMP6s and the red-shifted opsin ReaChR, we implemented a crosstalk-free, noninvasive all-optical approach and applied it to reconstruct the functional and effective connectivity of the left habenula.

Neural circuit mechanisms underlying context-specific halting in Drosophila

Walking is a complex motor programme involving coordinated and distributed activity across the brain and the spinal cord. Halting appropriately at the correct time is a critical component of walking control. Despite progress in identifying neurons driving halting1–6, the underlying neural circuit mechanisms responsible for overruling the competing walking state remain unclear. Here, using connectome-informed models7–9 and functional studies, we explain two fundamental mechanisms by which Drosophila implement context-appropriate halting. The first mechanism (‘walk-OFF’) relies on GABAergic neurons that inhibit specific descending walking commands in the brain, whereas the second mechanism (‘brake’) relies on excitatory cholinergic neurons in the nerve cord that lead to an active arrest of stepping movements. We show that two neurons that deploy the walk-OFF mechanism inhibit distinct populations of walking-promotion neurons, leading to differential halting of forward walking or turning. The brake neurons, by constrast, override all walking commands by simultaneously inhibiting descending walking-promotion neurons and increasing the resistance at the leg joints. We characterized two behavioural contexts in which the distinct halting mechanisms were used by the animal in a mutually exclusive manner: the walk-OFF mechanism was engaged for halting during feeding and the brake mechanism was engaged for halting and stability during grooming.

The fly connectome reveals a path to the effectome

A goal of neuroscience is to obtain a causal model of the nervous system. The recently reported whole-brain fly connectome1–3 specifies the synaptic paths by which neurons can affect each other, but not how strongly they do affect each other in vivo. To overcome this limitation, we introduce a combined experimental and statistical strategy for efficiently learning a causal model of the fly brain, which we refer to as the ‘effectome’. Specifically, we propose an estimator for a linear dynamical model of the fly brain that uses stochastic optogenetic perturbation data to estimate causal effects and the connectome as a prior to greatly improve estimation efficiency. We validate our estimator in connectome-based linear simulations and show that it recovers a linear approximation to the nonlinear dynamics of more biophysically realistic simulations. We then analyse the connectome to propose circuits that dominate the dynamics of the fly nervous system. We discover that the dominant circuits involve only relatively small populations of neurons—thus, neuron-level imaging, stimulation and identification are feasible. This approach also re-discovers known circuits and generates testable hypotheses about their dynamics. Overall, we provide evidence that fly whole-brain dynamics are generated by a large collection of small circuits that operate largely independently of each other. This implies that a causal model of a brain can be feasibly obtained in the fly.

Is Intraoperative Muscle Motor Evoked Potential Variability due to Fluctuating Lower Motor Neuron Background Excitability?

This pilot study tests the contribution of fluctuating lower motor neuron excitability to motor evoked potential (MEP) variability. In six pediatric patients with idiopathic scoliosis and normal neurologic examination, cascades of 30 intraoperative H-reflexes (HRs) and MEPs were evoked in the soleus muscle using constant-current stimulators and recorded through surface electrodes with a 20-second interstimulus interval. First, HRs were obtained with an intensity capable of evoking the maximum response. Subsequently, MEPs were obtained with double trains and an intensity of 700 to 900 mA. Coefficients of variation (CVs) of amplitude and area under the curve from HRs and MEPs were compared using a paired two-tailed Student t test. Coefficients of correlation between the mean CVs of HR and MEP parameters were also assessed. Pooling the results from the six patients, the mean CV of amplitude from the MEP (24.6 ± 3) was significantly higher than that from the HR (3.5 ± 4.4) (P = 0.000091). The mean CV of the MEP area under the curve (21.8 ± 4.8) was also statistically significantly higher than that from the HR area under the curve (3.4 ± 4.5) (P = 0.00091). The coefficients of correlation of the mean CV of the HR amplitude and area under the curve compared with the corresponding values of the MEP were low (r = 0.29) and very low (r = 0.03), respectively. Our results suggest that fluctuations in lower motor neuron excitability may be less important than previously thought to explain the magnitude of MEP variability. The efficacy of corticospinal volleys to recruit a larger and more stable lower motor neuron population would be critical to obtain reproducible MEPs.

Inter-Strain Differences in the Lumbar Spinal Cord Anatomy and Neuromorphology: Wistar Versus Dark Agouti Rats.

Rat strains differ in physiology, behavior, and recovery after central nervous system injury. To assess these differences, we compared the gross and local anatomy and neuromorphology of the lumbar spinal cord of the Wistar and Dark Agouti (DA) strains. The key findings include (i) distinct spatial relationships between vertebrae and spinal segments in the two strains; (ii) Wistar rats have larger volumes of spinal cord gray and white matter; (iii) DA rats have smaller total neuronal populations, thus indicating an expectation of smaller local neuronal populations; (iv) this expectation was confirmed for interneurons expressing calbindin 28kDa. But contrary to expectations, (v) DA rats had more numerous populations of the interneurons expressing parvalbumin and a population of α-motoneurons. Consequently, these strains displayed divergent ratios in specific spinal neuronal populations. Researchers should consider these inter-strain differences when comparing data across different strains.

Local Contour Features Contribute to Figure-Ground Segregation in Monkey V4 Neural Populations and Human Perception

Figure-ground (FG) segregation is a crucial step towards the recognition of objects in natural scenes. Gestalt psychologists have emphasized the importance of contour features in perception of FG. Recent electrophysiological studies have identified a neural population in V4 that shows FG-dependent modulation (FG neurons). However, whether the contour features contribute to the modulation of the response patterns of the neural population remains unclear. In the present study, we quantified the contour features associated with Gestalt factors in local natural stimuli and examined whether salient contour-features evoked reliable perceptual and neural responses by analyzing response consistency (stability) across trials. The results showed the tendency that the more salient contour-features evoked the greater consistencies in the perceptual FG judgments and population-based neural responses in FG determination; a greater partial correlation for curvature and weaker correlations for closure and parallelism. Multiple linear regression analyses demonstrated that the perceptual consistency depended similarly on curvature and closure, and the neural consistency depended significantly on curvature but weakly on closure. We further observed a strong correlation between the consistencies in the perceptual and neural responses, i.e., stimuli that evoked more stable percepts tended to evoke more stable neural responses. These results indicate that local contour-features modulate the responses of the neural population in V4 and contribute to the perception of FG organization.

A small population of stress-responsive neurons in the hypothalamus-habenula circuit mediates development of depression-like behavior in mice.

Accumulating evidence has shown that various brain functions are associated with experience-activated neuronal ensembles. However, whether such neuronal ensembles are engaged in the pathogenesis of stress-induced depression remains elusive. Utilizing activity-dependent viral strategies in mice, we identified a small population of stress-responsive neurons, primarily located in the middle part of the lateral hypothalamus (mLH) and the medial part of the lateral habenula (LHbM). These neurons serve as "starter cells" to transmit stress-related information and mediate the development of depression-like behaviors during chronic stress. Starter cells in the mLH and LHbM form dominant connections, which are selectively potentiated by chronic stress. Silencing these connections during chronic stress prevents the development of depression-like behaviors, whereas activating these connections directly elicits depression-like behaviors without stress experience. Collectively, our findings dissect a core functional unit within the LH-LHb circuit that mediates the development of depression-like behaviors in mice.

The Spinocerebellar Ataxia 34-Causing W246G ELOVL4 Mutation Does Not Alter Cerebellar Neuron Populations in a Rat Model.

Spinocerebellar ataxia 34 (SCA34) is an autosomal dominant disease that arises from point mutations in the fatty acid elongase, Elongation of Very Long Chain Fatty Acids 4 (ELOVL4), which is essential for the synthesis of Very Long Chain-Saturated Fatty Acids (VLC-SFA) and Very Long Chain-Polyunsaturated Fatty Acids (VLC-PUFA) (28-34 carbons long). SCA34 is considered a neurodegenerative disease. However, a novel rat model of SCA34 (SCA34-KI rat) with knock-in of the W246G ELOVL4 mutation that causes human SCA34 shows early motor impairment and aberrant synaptic transmission and plasticity without overt neurodegeneration. ELOVL4 is expressed in neurogenic regions of the developing brain, is implicated in cell cycle regulation, and ELOVL4 mutations that cause neuroichthyosis lead to developmental brain malformation, suggesting that aberrant neuron generation due to ELOVL4 mutations might contribute to SCA34. To test whether W246G ELOVL4 altered neuronal generation or survival in the cerebellum, we compared the numbers of Purkinje cells, unipolar brush cells, molecular layer interneurons, granule and displaced granule cells in the cerebellum of wildtype, heterozygous, and homozygous SCA34-KI rats at four months of age, when motor impairment is already present. An unbiased, semi-automated method based on Cellpose 2.0 and ImageJ was used to quantify neuronal populations in cerebellar sections immunolabeled for known neuron-specific markers. Neuronal populations and cortical structure were unaffected by the W246G ELOVL4 mutation by four months of age, a time when synaptic and motor dysfunction are already present, suggesting that SCA34 pathology originates from synaptic dysfunction due to VLC-SFA deficiency, rather than aberrant neuronal production or neurodegeneration.

Dependence of Contextual Modulation in Macaque V1 on Interlaminar Signal Flow.

In visual cortex, neural correlates of subjective perception can be generated by modulation of activity from beyond the classical receptive field (CRF). In macaque V1, activity generated by nonclassical receptive field (nCRF) stimulation involves different intracortical circuitry than activity generated by CRF stimulation, suggesting that interactions between neurons across V1 layers differ under CRF and nCRF stimulus conditions. Using Neuropixels probes, we measured border ownership modulation within large, local populations of V1 neurons. We found that neurons in single columns preferred the same side of objects located outside of the CRF. In addition, we found that cross-correlations between pairs of neurons situated across feedback/horizontal and input layers differed between CRF and nCRF stimulation. Furthermore, independent of the comparison with CRF stimulation, we observed that the magnitude of border ownership modulation increased with the proportion of information flow from feedback/horizontal layers to input layers. These results demonstrate that the flow of signals between layers covaries with the degree to which neurons integrate information from beyond the CRF.

Using Machine Learning and Electronic Health Records to Identify Neuropsychiatric Risk Scores for Delirium in ICU and General Hospital Settings.

Delirium is a common and acute neuropsychiatric syndrome that requires timely intervention to prevent its associated morbidity and mortality. Yet, its diagnosis and symptoms are often overlooked due to its variable clinical presentation and fluctuating nature. Thus, in this study, we address the barriers to delirium diagnosis by utilizing a machine learning-based predictive algorithm for incident delirium that relies on archived electronic health records (EHRs) data. We used the Medical Information Mart for Intensive Care (MIMIC) database to create a detailed dataset for identifying delirium in intensive care unit (ICU) patients. Our approach involved training machine learning models on this dataset to pinpoint critical clinical features for delirium detection. These features were then refined and applied to non-ICU patients using EHRs from the American University of Beirut Medical Center (AUBMC). Our study assessed machine learning models like Extreme Gradient Boosting (XGBoost), Categorical Boosting (CatBoost), Classification and Regression Trees (CART), Random Forest (RF), Neural Oblivious Decision Ensembles (NODE), and Logistic Regression (LR), highlighting superior delirium detection in diverse clinical settings. The CatBoost model excelled in ICU environments with an F1 Score of 89.2%, while XGBoost performed best in general hospital settings with a 75.4% F1 Score. Interpretations using Tabular Local Interpretable Model-agnostic Explanations (LIME) revealed critical indicators such as prothrombin time and hematocrit levels, enhancing model transparency and clinical applicability. These clinical insights help differentiate the delirium predictors between ICU patients, who are often sensitive to various factors. The proposed predictive algorithm improves delirium detection rates and streamlines efficiency in hospital electronic systems, thereby enabling prompt interventions to prevent delirium progression and associated complications. The clinical indicators for delirium that we identified in general hospital settings and ICU can greatly help healthcare professionals identify potential causes of delirium and reduce misdiagnosis.

Development, Validation, and Limits of Freezing of Gait Detection Using a Single Waist-Worn Device.

Freezing of Gait (FOG) is prevalent in people with Parkinson's disease (PD) and severely disrupts mobility. Detecting the exact boundaries of FOG episodes may facilitate new technologies in "breaking" FOG in real-time. This study investigates the performance of automatic device-based FOG detection. Eight machine-learning classifiers (including Neural Networks, Ensemble methods, and Support Vector Machines) were developed using (i) accelerometer and (ii) combined accelerometer and gyroscope data from a waist-worn device. While wearing the device, 107 people with PD completed mobility tasks designed to elicit FOG. Two clinicians independently annotated exact FOG episodes using synchronized video and a flowchart algorithm based on international guidelines. Device-detected FOG episodes were compared to annotated episodes using 10-fold cross-validation and Interclass Correlation Coefficients (ICC) for agreement. Development used 50,962 windows of data and annotated activities (>10 hours). Strong agreement between clinicians for precise FOG episodes was observed (90% sensitivity, 92% specificity, and ICC1,1 = 0.97 for total FOG duration). Device performance varied by method, complexity, and cost matrix. The Neural Network using 67 accelerometer features achieved high sensitivity to FOG (89% sensitivity, 81% specificity, and ICC1,1 = 0.83) and stability (validation loss 5%). The waist-worn device consistently reported accurate detection of precise FOG episodes and compared well to more complex systems. The strong clinician agreement indicates room for improvement in future device-based FOG detection. This study may enhance PD care by reducing reliance on visual FOG inspection, demonstrating that high sensitivity in automatic FOG detection is achievable.

Neuropeptide therapeutics to repress lateral septum neurons that disable sociability in an autism mouse model.

Confronting oxytocin and vasopressin deficits in autism spectrum disorders and rare syndromes brought promises and disappointments for the treatment of social disabilities. We searched downstream of oxytocin and vasopressin for targets alleviating social deficits in a mouse model of Prader-Willi syndrome and Schaaf-Yang syndrome, both associated with high prevalence of autism. We found a population of neurons in the lateral septum-activated on termination of social contacts-which oxytocin and vasopressin inhibit as per degree of peer affiliation. These are somatostatin neurons expressing oxytocin receptors coupled to GABA-B signaling, which are inhibited via GABA-A channels by vasopressin-excited GABA neurons. Loss of oxytocin or vasopressin signaling recapitulated the disease phenotype. By contrast, deactivation of somatostatin neurons or receptor signaling alleviated social deficits of disease models by increasing the duration of contacts with mates and strangers. These findings provide new insights into the treatment framework of social disabilities in neuropsychiatric disorders.

Prophylactic (R,S)-ketamine and (2S,6S)-HNK decrease fear expression by differentially modulating fear neural ensembles

We previously reported that a single injection of (R,S)-ketamine or its metabolite (2S,6S)-hydroxynorketamine (HNK) prior to stress attenuates learned fear. However, whether these drugs attenuate learned fear through divergent or convergent effects on neural activity remains to be determined. 129S6/SvEv male mice were injected with saline, (R,S)-ketamine, or (2S,6S)-HNK one week before a 3-shock contextual fear conditioning (CFC) paradigm. Five days later, mice were re-exposed to the aversive context, and euthanized one hour later to quantify active cells. Brains were processed for c-fos immunoreactivity, and neural networks were built with a novel, wide-scale imaging pipeline. We found that (R,S)-ketamine and (2S,6S)-HNK attenuate learned fear. Fear-related neural activity was altered in: dorsal CA3 following (2S,6S)-HNK; ventral CA3 and CA1, infralimbic (IL) and prelimbic (PL) regions, insular cortex (IC), retrosplenial cortex (RSP), piriform cortex (PIR), nucleus reuniens (RE), and periaqueductal grey (PAG) following both (R,S)-ketamine and (2S,6S)-HNK; and in the paraventricular nucleus of thalamus (PVT) following (R,S)-ketamine. Dorsal CA3 and ventral hippocampus activation correlated with freezing in the (R,S)-ketamine group, and RSP activation correlated with freezing in both (R,S)-ketamine and (2S,6S)-HNK groups. (R,S)-ketamine increased connectivity between cortical and subcortical regions while (2S,6S)-HNK increased connectivity within these regions. This work identifies novel nodes in fear networks, involving the RE, PIR, IC, PAG and RSP, that can be targeted with neuromodulatory strategies or pharmaceutical compounds to treat fear-induced disorders. This approach could be used to optimize target engagement and dosing strategies of existing medications.

Endothelial pyroptosis-driven microglial activation in choroid plexus mediates neuronal apoptosis in hemorrhagic stroke rats

BackgroundSpontaneous intracerebral hemorrhage (ICH) is associated with alarmingly high rates of disability and mortality, and current therapeutic options are suboptimal. A critical component of ICH pathology is the initiation of a robust inflammatory response, often termed “cytokine storm,” which amplifies the secondary brain injury following the initial hemorrhagic insult. The precise sources and consequences of this cytokine-driven inflammation are not fully elucidated, necessitating further investigation. MethodsTo address this knowledge gap, our study conducted a comprehensive cytokine profiling using Luminex® assays, assessing 23 key cytokines. We then employed single-cell RNA sequencing and spatial transcriptomics at three critical time points post-ICH: the hyperacute, acute, and subacute phases. Integrating these multimodal analyses allowed us to identify the cellular origins of cytokines and elucidate their mechanisms of action. ResultsLuminex® cytokine assays revealed a significant upregulation of IL-6 and IL-1β levels at the 24-h post-ICH time point. Through the integration of scRNA-seq and spatial transcriptomics in the hemorrhagic hemisphere of rats, we observed a pronounced activation of cytokine-related signaling pathways within the choroid plexus. Initially, immune cell presence was sparse, but it surged 24 h post-ICH, particularly in the choroid plexus, indicating a substantial shift in the immune microenvironment. We traced the source of IL-1β and IL-6 to endothelial cells, establishing a link to pyroptosis. Endothelial pyroptosis post-ICH induced the production of IL-1β and IL-6, which activated microglial polarization characterized by elevated expression of Msr1, Lcn2, and Spp1 via the NF-κB pathway in the choroid plexus. Furthermore, we identified neuronal populations undergoing apoptosis, mediated by the Lcn2-SLC22A17 pathway in response to IL-1β and IL-6 signaling. Notably, the inhibition of pyroptosis using VX-765 significantly mitigated neurological impairments. ConclusionsOur study provides evidence that endothelial pyroptosis, characterized by the release of IL-1β and IL-6, triggers microglial polarization through NF-κB pathway activation, ultimately leading to microglia-mediated neuronal apoptosis in the choroid plexus post-ICH. These findings suggest that targeted therapeutic strategies aimed at mitigating endothelial cell pyroptosis and neutralizing inflammatory cytokines may offer neuroprotection for both microglia and neurons, presenting a promising avenue for ICH treatment.

A multi-network model of Parkinson’s disease tremor: exploring the finger-dimmer-switch theory and role of dopamine in thalamic self-inhibition

Background. Tremor is a cardinal symptom of Parkinson's disease (PD) that manifests itself through complex oscillatory activity across multiple neuronal populations. According to the finger-dimmer-switch (FDS) theory, tremor is triggered by transient pathological activity in the basal ganglia-thalamo-cortical (BTC) network (the finger) and transitions into an oscillatory form within the inner circuitry of the thalamus (the switch). The cerebello-thalamo-cortical (CTC) network (the dimmer) is then involved in sustaining and amplifying tremor amplitude. In this study, we aimed to investigate the generation and progression dynamics of PD tremor oscillations by developing a comprehensive and interacting FDS model that transitions sequentially from healthy to PD to tremor and then to tremor-off state.Methods.We constructed a computational model consisting of 700 neurons in 11 regions of BTC, CTC, and thalamic networks. Transition from healthy to PD state was simulated through modulating dopaminergic synaptic connections; and further from PD to tremor and tremor-off by modulating projections between the thalamic reticular nucleus (TRN), anterior ventrolateral nucleus (VLa), and posterior ventrolateral nucleus (VLp).Results.Sustained oscillations in the frequency range of PD tremor emerged in thalamic VLp (5 Hz) and cerebellar dentate nucleus (3 Hz). Increasing self-inhibition in the thalamus through dopaminergic modulation significantly decreased tremor amplitude.Conclusion/Significance.Our results confirm the mechanistic power of the FDS theory in describing the PD tremor phenomenon and emphasize the role of dopaminergic modulation on thalamic self-inhibition. These insights pave the way for novel therapeutic strategies aimed at reducing the tremor by strengthening thalamic self-inhibition, particularly in dopamine-resistant patients.

Partial chemogenetic inhibition of the locus coeruleus due to heterogeneous transduction of noradrenergic neurons preserved auditory salience processing in wild-type rats.

The acoustic startle reflex (ASR) and prepulse inhibition of the ASR (PPI) assess the efficiency of salience processing, a fundamental brain function that is impaired in many psychiatric conditions. Both ASR and PPI depend on noradrenergic transmission, yet the modulatory role of the locus coeruleus (LC) remains controversial. Clonidine (0.05 mg/kg, i.p.), an alpha2-adrenoreceptor agonist, strongly reduced the ASR amplitude. In contrast, chemogenetic LC inhibition only mildly suppressed the ASR and did affect the PPI in virus-transduced rats. The canine adenovirus type 2 (CAV2)-based vector carrying a gene cassette for the expression of inhibitory receptors (hM4Di) and noradrenergic cell-specific promoter (PRSx8) had high cell-type specificity (94.4±3.1%) but resulted in heterogeneous virus transduction of DbH-positive LC neurons (range: 9.2-94.4%). Clozapine-N-oxide (CNO; 1mg/kg, i.p.), a hM4Di actuator, caused the firing cessation of hM4Di-expressing LC neurons, yet complete inhibition of the entire population of LC neurons was not achieved. Case-based immunohistochemistry revealed that virus injections distal (> 150μm) to the LC core resulted in partial LC transduction, while proximal (< 50 μm) injections caused neuronal loss due to virus neurotoxicity. Neither the ASR nor PPI differed between the intact and virus-transduced rats. Our results suggest that a residual activity of virus-non-transduced LC neurons might have been sufficient for mediating an unaltered ASR and PPI. Our study highlights the importance of a case-based assessment of the virus efficiency, specificity, and neurotoxicity for targeted cell populations and of considering these factors when interpreting behavioral effects in experiments employing chemogenetic modulation.