Experience-dependent information routing through the basolateral amygdala shapes behavioral outcomes.

It is well established that the basolateral amygdala (BLA) is an emotional processing hub that governs a diverse repertoire of behaviors. Selective engagement of a heterogeneous cell population in the BLA is thought to contribute to this flexibility in behavioral outcomes. However, whether this process is impacted by previous experiences that influence emotional processing remains unclear. Here we demonstrate that previous positive (enriched environment [EE]) or negative (chronic unpredictable stress [CUS]) experiences differentially influence the activity of populations of BLA principal neurons projecting to either the nucleus accumbens core or bed nucleus of the stria terminalis. Chemogenetic manipulation of these projection-specific neurons can mimic or occlude the effects of CUS and EE on behavioral outcomes to bidirectionally control avoidance behaviors and stress-induced helplessness. These data demonstrate that previous experiences influence the responsiveness of projection-specific BLA principal neurons, biasing information routing through the BLA, to drive divergent behavioral outcomes.

Single nucleus RNA-sequencing integrated into risk variant colocalization discovers 17 cell-type-specific abdominal obesity genes for metabolic dysfunction-associated steatotic liver disease.

BACKGROUND: Abdominal obesity increases the risk for non-alcoholic fatty liver disease (NAFLD), now known as metabolic dysfunction-associated steatotic liver disease (MASLD). METHODS: To elucidate the directional cell-type level biological mechanisms underlying the association between abdominal obesity and MASLD, we integrated adipose and liver single nucleus RNA-sequencing and bulk cis-expression quantitative trait locus (eQTL) data with the UK Biobank genome-wide association study (GWAS) data using colocalization. Then we used colocalized cis-eQTL variants as instrumental variables in Mendelian randomization (MR) analyses, followed by functional validation experiments on the target genes of the cis-eQTL variants. FINDINGS: We identified 17 colocalized abdominal obesity GWAS variants, regulating 17 adipose cell-type marker genes. Incorporating these 17 variants into MR discovers a putative tissue-of-origin, cell-type-aware causal effect of abdominal obesity on MASLD consistently with multiple MR methods without significant evidence for pleiotropy or heterogeneity. Single cell data confirm the adipocyte-enriched mean expression of the 17 genes. Our cellular experiments across human adipogenesis identify risk variant -specific epigenetic and transcriptional mechanisms. Knocking down two of the 17 genes, PPP2R5A and SH3PXD2B, shows a marked decrease in adipocyte lipidation and significantly alters adipocyte function and adipogenesis regulator genes, including DGAT2, LPL, ADIPOQ, PPARG, and SREBF1. Furthermore, the 17 genes capture a characteristic MASLD expression signature in subcutaneous adipose tissue. INTERPRETATION: Overall, we discover a significant cell-type level effect of abdominal obesity on MASLD and trace its biological effect to adipogenesis. FUNDING: NIH grants R01HG010505, R01DK132775, and R01HL170604; the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant No. 802825), Academy of Finland (Grants Nos. 333021), the Finnish Foundation for Cardiovascular Research the Sigrid Jusélius Foundation and the Jane and Aatos Erkko Foundation; American Association for the Study of Liver Diseases (AASLD) Advanced Transplant Hepatology award and NIH/NIDDK (P30DK41301) Pilot and Feasibility award; NIH/NIEHS F32 award (F32ES034668); Finnish Diabetes Research Foundation, Kuopio University Hospital Project grant (EVO/VTR grants 2005-2021), the Academy of Finland grant (Contract no. 138006); Academy of Finland (Grant Nos 335443, 314383, 272376 and 266286), Sigrid Jusélius Foundation, Finnish Medical Foundation, Finnish Diabetes Research Foundation, Novo Nordisk Foundation (#NNF20OC0060547, NNF17OC0027232, NNF10OC1013354) and Government Research Funds to Helsinki University Hospital; Orion Research Foundation, Maud Kuistila Foundation, Finish Medical Foundation, and University of Helsinki.

Needle-scalpel therapy inhibits the apoptosis of nucleus pulposus cells via the SDF-1/CXCR4 axis in a rat degenerative cervical intervertebral disc model.

As a common disease, cervical spondylosis (CS) results from the degeneration of the cervical intervertebral disc. However, there are still no effective clinical strategies for the treatment of this disease. Needle-scalpel (Ns), a therapy guided by traditional Chinese medicine theory, alleviates intervertebral disc degradation and is widely used in the clinic to treat CS. Stromal cell-derived factor-1 (SDF-1) and its receptor CXC receptor 4 (CXCR4) in nucleus pulposus cells play an important role in CS onset and development. This study aimed to explore whether Ns can relieve pain and regulate the SDF-1/CXCR4 axis in nucleus pulposus cells to inhibit apoptosis, thereby delaying cervical intervertebral disc degradation in a rat model of CS. It was found that the Ns-treated groups exhibited higher mechanical allodynia scores than the model group, and H&E staining, MRI, and scanning electron microscopy revealed that Ns therapy inhibited intervertebral disc degeneration. Additionally, Ns therapy significantly inhibited increases in the RNA and protein expression levels of SDF-1 and CXCR4. Furthermore, these treatments alleviated the apoptosis of nucleus pulposus cells, which manifested as a decline in the proportion of apoptotic nucleus pulposus cells and inhibition of the decrease in the levels of Bcl-2/Bax. These findings indicated that Ns mitigated CS-induced pain, inhibited the apoptosis of nucleus pulposus cells, and alleviated intervertebral disc degeneration in CS rats. These effects may be mediated by specifically regulating the SDF-1/CXCR4 signaling axis. Based on these findings, we conclude that Ns might serve as a promising therapy for the treatment of CS.

POSTN knockdown suppresses IL-1ß-induced inflammation and apoptosis of nucleus pulposus cells via inhibiting the NF-κB pathway and alleviates intervertebral disc degeneration.

The aim of this study is to investigate the effects of POSTN on IL-1ß induced inflammation, apoptosis, NF-κB pathway and intervertebral disc degeneration (IVDD) in Nucleus pulposus (NP) cells (NPCs). NP tissue samples with different Pfirrmann grades were collected from patients with different degrees of IVDD. Western blot and immunohistochemical staining were used to compare the expression of POSTN protein in NP tissues. Using the IL-1ß-induced IVDD model, NPCs were transfected with lentivirus-coated si-POSTN to down-regulate the expression of POSTN and treated with CU-T12-9 to evaluate the involvement of NF-κB pathway. Western blot, immunofluorescence, and TUNEL staining were used to detect the expression changes of inflammation, apoptosis and NF-κB pathway-related proteins in NPCs. To investigate the role of POSTN in vivo, a rat IVDD model was established by needle puncture of the intervertebral disc. Rats were injected with lentivirus-coated si-POSTN, and H&E staining and immunohistochemical staining were performed. POSTN expression is positively correlated with the severity of IVDD in human. POSTN expression was significantly increased in the IL-1ß-induced NPCs degeneration model. Downregulation of POSTN protects NPCs from IL-1ß-induced inflammation and apoptosis. CU-T12-9 treatment reversed the protective effect of si-POSTN on NPCs. Furthermore, lentivirus-coated si-POSTN injection partially reversed NP tissue damage in the IVDD model in vivo. POSTN knockdown reduces inflammation and apoptosis of NPCs by inhibiting NF-κB pathway, and ultimately prevents IVDD. Therefore, POSTN may be an effective target for the treatment of IVDD.

Single-nucleus multi-omics of Parkinson's disease reveals a glutamatergic neuronal subtype susceptible to gene dysregulation via alteration of transcriptional networks.

The genetic architecture of Parkinson's disease (PD) is complex and multiple brain cell subtypes are involved in the neuropathological progression of the disease. Here we aimed to advance our understanding of PD genetic complexity at a cell subtype precision level. Using parallel single-nucleus (sn)RNA-seq and snATAC-seq analyses we simultaneously profiled the transcriptomic and chromatin accessibility landscapes in temporal cortex tissues from 12 PD compared to 12 control subjects at a granular single cell resolution. An integrative bioinformatic pipeline was developed and applied for the analyses of these snMulti-omics datasets. The results identified a subpopulation of cortical glutamatergic excitatory neurons with remarkably altered gene expression in PD, including differentially-expressed genes within PD risk loci identified in genome-wide association studies (GWAS). This was the only neuronal subtype showing significant and robust overexpression of SNCA. Further characterization of this neuronal-subpopulation showed upregulation of specific pathways related to axon guidance, neurite outgrowth and post-synaptic structure, and downregulated pathways involved in presynaptic organization and calcium response. Additionally, we characterized the roles of three molecular mechanisms in governing PD-associated cell subtype-specific dysregulation of gene expression: (1) changes in cis-regulatory element accessibility to transcriptional machinery; (2) changes in the abundance of master transcriptional regulators, including YY1, SP3, and KLF16; (3) candidate regulatory variants in high linkage disequilibrium with PD-GWAS genomic variants impacting transcription factor binding affinities. To our knowledge, this study is the first and the most comprehensive interrogation of the multi-omics landscape of PD at a cell-subtype resolution. Our findings provide new insights into a precise glutamatergic neuronal cell subtype, causal genes, and non-coding regulatory variants underlying the neuropathological progression of PD, paving the way for the development of cell- and gene-targeted therapeutics to halt disease progression as well as genetic biomarkers for early preclinical diagnosis.

Chst9 marks a spatially and transcriptionally unique population of Oprm1-expressing neurons in the nucleus accumbens.

Opioids produce addictive, analgesic, and euphoric effects via actions at mu opioid receptors (µORs). The µOR is encoded by the Oprm1 gene and is expressed in multiple brain regions that regulate reward and motivation, such as the nucleus accumbens (NAc). Oprm1 expression in NAc medium spiny neurons (MSNs) mediates opioid place preference, seeking, and consumption. However, recent single nucleus RNA sequencing (snRNA-seq) studies have revealed that multiple subpopulations of NAc neurons express Oprm1 mRNA, making it unclear which populations mediate diverse behaviors resulting from µOR activation. Using published snRNA-seq datasets from the rat NAc, we identified a novel population of MSNs that express the highest levels of Oprm1 of any NAc cell type. Here, we show that this population is selectively marked by expression of Chst9, a gene encoding a carbohydrate sulfotransferase. Notably, Chst9+ neurons exhibited more abundant expression of Oprm1 as compared to other cell types, and formed discrete cellular clusters along the medial and ventral borders of the NAc shell subregion. Moreover, CHST9 mRNA was also found to mark specific MSN populations in published human and primate snRNA-seq studies, indicating that this unique population may be conserved across species. Together, these results identify a spatially and transcriptionally distinct NAc neuron population characterized by the expression of Chst9. The abundant expression of Oprm1 in this population and the conservation of these cells across species suggests that they may play a key functional role in opioid response and identify this subpopulation as a target for further investigation.

Anterior thalamic nucleus local field potentials during focal temporal lobe epileptic seizures.

Objective: To analyze the local field potentials (LFPs) in patients with focal drug-resistant epilepsy (DRE) from the anterior nucleus of the thalamus (ANT) during inter-ictal state and seizure state. Method: ANT stereotactic EEG (SEEG) recordings were studied in four patients with focal temporal lobe epilepsy. SEEG data was classified as inter-ictal and ictal state and sub-categorized into electrographic (ESz), focal aware seizure (FAS), focal with impaired awareness (FIA), or focal to bilateral tonic-clonic seizure (FBTC). LFP was analyzed at 4 Hz, 8 Hz, 16 Hz, 32 Hz, high gamma (100 Hz), and ripples (200 Hz) using spectrogram analysis and a statistical comparison of normalized power spectral density (PSD) averaged during seizures versus pre-ictal baseline segments. Result: The LFP recordings were analyzed for 162 seizures (127 ESz, 23 FAS, 6 FIA, and 6 FBTC). Based on time-frequency data (spectrogram), a broad band of activity, occurring between 2 and 6 Hz and centered at 4 Hz, and thin-band activity occurring specifically at 8 Hz on the frequency spectrogram were observed during the inter-ictal state. Statistically significant changes in LFP-PSD were seen for FAS, FIA, and FBTC. We observed a significant gain in LFP at the lower frequency band during FAS at 4 Hz, FIA, and FBTC at 4, 8, and 16 Hz while also observing increases at higher frequencies during FBTC at 100 and 200 Hz and a decrease during FAS seizures at 32 Hz. In contrast, no significant change in LFP power was seen for electrographic seizures. Interpretation: Our observations from a limited dataset indicate that all clinical seizure types, but not electrographic seizures, caused a change in ANT-LFP based on the magnitude of the associated power spectral density (PSD). Future work will be needed to validate the use of ANT-LFP at these frequencies as accurate measurements of seizure occurrence and severity. This work represents a first step toward understanding ANT thalamic LFP patterns during focal seizures and developing adaptive DBS strategies.

Impact of ovariectomy on neurotransmitter receptors BDNF/TrkB and endoplasmic reticulum molecular chaperones in rat hypoglossal nucleus.

Currently hypoglossal nerve-genioglossus axis is the major research core of OSA pathogenesis. The pathogenesis of OSA incidence changes before and after menopause needs to be clarified further. Little is known about the influences of ovariectomy on hypoglossal motoneurons. In the research, we utilized a rat ovariectomy model to evaluate the expression changes of 5-HT2A and α1-Adrenergic receptors in the hypoglossal nucleus and to explore the involvement of BDNF/TrkB signaling and endoplasmic reticulum molecular chaperones in the hypoglossal nucleus. Results indicated that the expression of 5-HT2A and α1-Adrenergic receptors reduced dramatically in the hypoglossal nucleus of ovariectomized rats. The apoptosis level of hypoglossal motor neurons increased markedly in the OVX groups. The up-regulated expression of BDNF and down-regulated expression of TrkB were found in the OVX groups. Ovarian insufficiency resulted in the activation of UPR and the loss of CANX-CALR cycle. Estrogen replacement could restore these changes partially. Estrogen level influences the expression of neurotransmitter receptors, and regulates BDNF/TrkB signaling compensation and endoplasmic reticulum homeostasis, which might be one of the pathogenesis of menopausal female OSA. The results reveal a new perspective for studying female OSA from the view of hypoglossal nerve and hormonal changes and attempt to propel 17ß-estradiol toward a feasible therapy for female OSA. Supplementary Information: The online version contains supplementary material available at 10.1007/s41105-024-00520-5.

Mouse Models in Circadian Rhythm and Melatonin Research.

This contribution reviews the role of inbred and transgenic mouse strains for deciphering the mammalian melatoninergic and circadian system. It focusses on the pineal organ as melatonin factory and two major targets of the melatoninergic system, the suprachiasmatic nuclei (SCN) and the hypophysial pars tuberalis (PT). Mammalian pinealocytes sharing molecular characteristics with true pineal and retinal photoreceptors synthesize and secrete melatonin into the blood and cerebrospinal fluid night by night. Notably, neuron-like connections exist between the deep pinealocytes and the habenular/pretectal region suggesting direct pineal-brain communication. Control of melatonin biosynthesis in rodents involves transcriptional regulation including phosphorylation of CREB and upregulation of mPer1. In the SCN, melatonin acts upon MT1 and MT2 receptors. Melatonin is not necessary to maintain the rhythm of the SCN molecular clockwork, but it has distinct effects on the synchronization of the circadian rhythm by light, facilitates re-entrainment of the circadian system to phase advances in the level of the SCN molecular clockwork by acting upon MT2 receptors and plays a stabilizing role in the circadian system as evidenced from locomotor activity recordings. While the effects in the SCN are subtle, melatonin is essential for PT functions. Via the MT1 receptor it drives the PT-intrinsic molecular clockwork and the retrograde and anterograde output pathways controlling seasonal rhythmicity. Although inbred and transgenic mice do not show seasonal reproduction, the pathways from the PT are fully intact if the animals are melatonin proficient. Thus, only melatonin-proficient strains are suited to investigate the circadian and melatoninergic systems.

Neuronal network controlling REM sleep.

Rapid eye movement sleep is a state characterized by concomitant occurrence of rapid eye movements, electroencephalographic activation and muscle atonia. In this review, we provide up to date knowledge on the neuronal network controlling its onset and maintenance. It is now accepted that muscle atonia during rapid eye movement sleep is due to activation of glutamatergic neurons localized in the pontine sublaterodorsal tegmental nucleus. These neurons directly project and excite glycinergic/γ-aminobutyric acid-ergic pre-motoneurons localized in the ventromedial medulla. The sublaterodorsal tegmental nucleus rapid eye movement-on neurons are inactivated during wakefulness and non-rapid eye movement by rapid eye movement-off γ-aminobutyric acid-ergic neurons localized in the ventrolateral periaqueductal grey and the adjacent dorsal deep mesencephalic reticular nucleus. Melanin-concentrating hormone and γ-aminobutyric acid-ergic rapid eye movement sleep-on neurons localized in the lateral hypothalamus would inhibit these rapid eye movement sleep-off neurons initiating the state. Finally, the activation of a few limbic cortical structures during rapid eye movement sleep by the claustrum and the supramammillary nucleus as well as that of the basolateral amygdala would be involved in the function(s) of rapid eye movement sleep. In summary, rapid eye movement sleep is generated by a brainstem generator controlled by forebrain structures involved in autonomic control.

Distinct Thalamo-Subcortical Circuits Underlie Painful Behavior and Depression-Like Behavior Following Nerve Injury.

Clinically, chronic pain and depression often coexist in multiple diseases and reciprocally reinforce each other, which greatly escalates the difficulty of treatment. The neural circuit mechanism underlying the chronic pain/depression comorbidity remains unclear. The present study reports that two distinct subregions in the paraventricular thalamus (PVT) play different roles in this pathological process. In the first subregion PVT posterior (PVP), glutamatergic neurons (PVPGlu) send signals to GABAergic neurons (VLPAGGABA) in the ventrolateral periaqueductal gray (VLPAG), which mediates painful behavior in comorbidity. Meanwhile, in another subregion PVT anterior (PVA), glutamatergic neurons (PVAGlu) send signals to the nucleus accumbens D1-positive neurons and D2-positive neurons (NAcD1→D2), which is involved in depression-like behavior in comorbidity. This study demonstrates that the distinct thalamo-subcortical circuits PVPGlu→VLPAGGABA and PVAGlu→NAcD1→D2 mediated painful behavior and depression-like behavior following spared nerve injury (SNI), respectively, which provides the circuit-based potential targets for preventing and treating comorbidity.

Integrating single-nucleus RNA sequencing and spatial transcriptomics to elucidate a specialized subpopulation of astrocytes, microglia and vascular cells in brains of mouse model of lipopolysaccharide-induced sepsis-associated encephalopathy.

BACKGROUND: Understanding the mechanism behind sepsis-associated encephalopathy (SAE) remains a formidable task. This study endeavors to shed light on the complex cellular and molecular alterations that occur in the brains of a mouse model with SAE, ultimately unraveling the underlying mechanisms of this condition. METHODS: We established a murine model using intraperitoneal injection of lipopolysaccharide (LPS) in wild type and Anxa1-/- mice and collected brain tissues for analysis at 0-hour, 12-hour, 24-hour, and 72-hour post-injection. Utilizing advanced techniques such as single-nucleus RNA sequencing (snRNA-seq) and Stereo-seq, we conducted a comprehensive characterization of the cellular responses and molecular patterns within the brain. RESULTS: Our study uncovered notable temporal differences in the response to LPS challenge between Anxa1-/- (annexin A1 knockout) and wild type mice, specifically at the 12-hour and 24-hour time points following injection. We observed a significant increase in the proportion of Astro-2 and Micro-2 cells in these mice. These cells exhibited a colocalization pattern with the vascular subtype Vas-1, forming a distinct region known as V1A2M2, where Astro-2 and Micro-2 cells surrounded Vas-1. Moreover, through further analysis, we discovered significant upregulation of ligands and receptors such as Timp1-Cd63, Timp1-Itgb1, Timp1-Lrp1, as well as Ccl2-Ackr1 and Cxcl2-Ackr1 within this region. In addition, we observed a notable increase in the expression of Cd14-Itgb1, Cd14-Tlr2, and Cd14-C3ar1 in regions enriched with Micro-2 cells. Additionally, Cxcl10-Sdc4 showed broad upregulation in brain regions containing both Micro-2 and Astro-2 cells. Notably, upon LPS challenge, there was an observed increase in Anxa1 expression in the mouse brain. Furthermore, our study revealed a noteworthy increase in mortality rates following Anxa1 knockdown. However, we did not observe substantial differences in the types, numbers, or distribution of other brain cells between Anxa1-/- and wildtype mice over time. Nevertheless, when comparing the 24-hour post LPS injection time point, we observed a significant decrease in the proportion and distribution of Micro-2 and Astro-2 cells in the vicinity of blood vessels in Anxa1-/- mice. Additionally, we noted reduced expression levels of several ligand-receptor pairs including Cd14-Tlr2, Cd14-C3ar1, Cd14-Itgb1, Cxcl10-Sdc4, Ccl2-Ackr1, and Cxcl2-Ackr1. CONCLUSIONS: By combining snRNA-seq and Stereo-seq techniques, our study successfully identified a distinctive cellular colocalization, referred to as a special pathological niche, comprising Astro-2, Micro-2, and Vas-1 cells. Furthermore, we observed an upregulation of ligand-receptor pairs within this niche. These findings suggest a potential association between this cellular arrangement and the underlying mechanisms contributing to SAE or the increased mortality observed in Anxa1 knockdown mice.

GSK3 regulation Wnt/ß-catenin signaling affects adipogenesis in bovine skeletal muscle fibro/adipogenic progenitors.

Clarifying the cellular origin and regulatory mechanisms of intramuscular fat (IMF) deposition is crucial for improving beef quality. Here, we used single-nucleus RNA sequencing to analyze the structure and heterogeneity of skeletal muscle cell populations in different developmental stages of Yanbian cattle and identified eight cell types in two developmental stages of calves and adults. Among them, fibro/adipogenic progenitors (FAPs) expressing CD29 (ITGA7)pos and CD56 (NCAM1)neg surface markers were committed to IMF deposition in beef cattle and expressed major Wnt ligands and receptors. LY2090314/XAV-939 was used to activate/inhibit Wnt/ß-catenin signal. The results showed that the blockade of Glycogen Synthase Kinase 3 (GSK3) by LY2090314 promoted the stabilization of ß-catenin and reduced the expression of genes related adipogenic differentiation (e.g., PPARγ and C/EBPα) in bovine FAPs, confirming the anti-adipogenic effect of GSK3. XAV-939 inhibition of the Wnt/ß-catenin pathway promoted the lipid accumulation capacity of FAPs. Furthermore, we found that blocking GSK3 enhanced the paracrine effects of FAPs-MuSCs and increased myotube formation in muscle satellite cells (MuSCs). Overall, our results outline a single-cell atlas of skeletal muscle development in Yanbian cattle, revealed the role of Wnt/GSK3/ß-catenin signaling in FAPs adipogenesis, and provide a theoretical basis for further regulation of bovine IMF deposition.

Whole-brain inputs and outputs of Phox2b and GABAergic neurons in the nucleus tractus solitarii.

The nucleus tractus solitarii (NTS) plays a critical role in the homeostatic regulation of respiration, blood pressure, sodium consumption and metabolic processes. Despite their significance, the circuitry mechanisms facilitating these diverse physiological functions remain incompletely understood. In this study, we present a whole-brain mapping of both the afferent and efferent connections of Phox2b-expressing and GABAergic neurons within the NTS. Our findings reveal that these neuronal populations not only receive monosynaptic inputs primarily from the medulla oblongata, pons, midbrain, supra-midbrain and cortical areas, but also mutually project their axons to these same locales. Moreover, intense monosynaptic inputs are received from the central amygdala, the paraventricular nucleus of the hypothalamus, the parasubthalamic nucleus and the intermediate reticular nucleus, along with brainstem nuclei explicitly engaged in respiratory regulation. In contrast, both neuronal groups extensively innervate brainstem nuclei associated with respiratory functions, although their projections to regions above the midbrain are comparatively limited. These anatomical findings provide a foundational platform for delineating an anatomical framework essential for dissecting the specific functional mechanisms of these circuits.

Dimerization of hub protein DYNLL1 and bZIP transcription factor CREB3L1 enhances transcriptional activation of CREB3L1 target genes like arginine vasopressin.

bZIP transcription factors can function as homodimers or heterodimers through interactions with their disordered coiled-coil domain. Such dimer assemblies are known to influence DNA-binding specificity and/or the recruitment of binding partners, which can cause a functional switch of a transcription factor from being an activator to a repressor. We recently identified the genomic targets of a bZIP transcription factor called CREB3L1 in rat hypothalamic supraoptic nucleus by ChIP-seq. The objective of this study was to investigate the CREB3L1 protein-to-protein interactome of which little is known. For this approach, we created and screened a rat supraoptic nucleus yeast two-hybrid prey library with the bZIP region of rat CREB3L1 as the bait. Our yeast two-hybrid approach captured five putative CREB3L1 interacting prey proteins in the supraoptic nucleus. One interactor was selected by bioinformatic analyses for more detailed investigation by co-immunoprecipitation, immunofluorescent cellular localisation, and reporter assays in vitro. Here we identify dimerisation hub protein Dynein Light Chain LC8-Type 1 as a CREB3L1 interacting protein that in vitro enhances CREB3L1 activation of target genes.

Restraint stress-induced antinociceptive effects in acute pain: Involvement of orexinergic system in the nucleus accumbens.

The complicated relevance between stress and pain has been identified. Neurotransmitters and neuropeptides of various brain areas play a role in this communication. Pain inhibitory response is known as stress-induced analgesia (SIA). The studies demonstrated that the nucleus accumbens (NAc) is critical in modulating pain. As a neuropeptide, orexin is crucially involved in initiating behavioral and physiological responses to threatening and unfeeling stimuli. However, the role of the orexin receptors of the NAc area after exposure to restraint stress (RS) as acute physical stress in the modulation of acute pain is unclear. One hundered twenty adult male albino Wistar rats (230-250g) were used. Animals were unilaterally implanted with cannulae above the NAc. The SB334867 and TCS OX2 29 were used as antagonists for OX1r and OX2r, respectively. Different doses of the antagonists (1, 3, 10, and 30 nmol/0.5µl DMSO) were microinjected intra-NAc five minutes before exposure to RS (3hours). Then, the tail-flick test as a model of acute pain was performed, and the nociceptive threshold (Tail-flick latency; TFL) was measured in 60-minute time set intervals. According to this study's findings, the antinociceptive effects of RS in the tail-flick test were blocked during intra-NAc administration of SB334867 or TCS OX2 29. The RS as acute stress increased TFL and deceased pain-like behavior responses. The 50% effective dose values of the OX1r and OX2r antagonists were 12.82 and 21.64 nmol, respectively. The result demonstrated contribution of the OX1r into the NAc was more remarkable than that of the OX2r on antinociceptive responses induced by the RS. Besides, in the absence of RS, the TFL was attenuated. The current study's data indicated that OX1r and OX2r into the NAc induced pain modulation responses during RS in acute pain. In conclusion, the findings revealed the involvement of intra-NAc orexin receptors in improving SIA.

Atypical Course of the Habenulo-Interpeduncular Tract in Chick Embryos.

Classical studies of the avian diencephalon hardly mention the habenulo-interpeduncular tract (a.k.a. retroflex tract), although both the habenula (HB) (its origin) and the interpeduncular nuclear complex (its target) are present. Retroflex tract fibers were described at early embryonic stages but seem absent in the adult in routine stains. However, this tract is a salient diencephalic landmark in all other vertebrate lineages. It typically emerges out of the caudal HB, courses dorsoventrally across thalamic alar and basal plates just in front of the thalamo-pretectal boundary, and then sharply bends 90° caudalwards at paramedian basal plate levels (this is the "retroflexion"), to approach longitudinally via paramedian pretectum and midbrain the rostralmost hindbrain, specifically the prepontine median interpeduncular complex across isthmus and rhombomere 1. We systematize this habenulo-interpeduncular course into four parts named subhabenular, retrothalamic, tegmental, and interpeduncular. We reexamined the chicken habenulo-interpeduncular fibers at stages HH30 and HH35 (6.5- and 9-day incubation) by mapping them specifically with immunoreaction for BEN protein, a well-known marker. We found that only a small fraction of the stained retroflex tract fibers approaches the basal plate by coursing along the standard dorsoventral pathway in front of the thalamo-pretectal boundary. Many other habenular fibers instead diverge into atypical dispersed courses across the thalamic cell mass (implying alteration of the first subhabenular part of the standard course) before reaching the basal plate; this dispersion explains their invisibility. A significant number of such transthalamic habenular fibers cross orthogonally the zona limitans (ZLI) (the rostral thalamic boundary) and invade the caudal alar prethalamus. Here, they immediately descend dorsoventrally, just rostrally to the ZLI, until reaching the prethalamic basal plate, where they bend (retroflex) caudalwards, entering the thalamic basal paramedian area. These atypical fibers gradually fasciculate with the other groups of habenular efferent fibers in their final longitudinal approach to the hindbrain interpeduncular complex. We conclude that the poor visibility of this tract in birds is due to its dispersion into a diversity of atypical alternative routes, though all components eventually reach the interpeduncular complex. This case merits further analysis of the diverse permissive versus nonpermissive guidance mechanisms called into action, which partially correlate distinctly with successive diencephalic, mesencephalic, and hindbrain neuromeric fields and their boundaries.

Real-time imaging of intracellular deformation dynamics in vibrated adherent cell cultures.

Mechanical vibration has been shown to regulate cell proliferation and differentiation in vitro and in vivo. However, the mechanism of its cellular mechanotransduction remains unclear. Although the measurement of intracellular deformation dynamics under mechanical vibration could reveal more detailed mechanisms, corroborating experimental evidence is lacking due to technical difficulties. In this study, we aimed to propose a real-time imaging method of intracellular structure deformation dynamics in vibrated adherent cell cultures and investigate whether organelles such as actin filaments connected to a nucleus and the nucleus itself show deformation under horizontal mechanical vibration. The proposed real-time imaging was achieved by conducting vibration isolation and making design improvements to the experimental setup; using a high-speed and high-sensitivity camera with a global shutter; and reducing image blur using a stroboscope technique. Using our system, we successfully produced the first experimental report on the existence of the deformation of organelles connected to a nucleus and the nucleus itself under horizontal mechanical vibration. Furthermore, the intracellular deformation difference between HeLa and MC3T3-E1 cells measured under horizontal mechanical vibration agrees with the prediction of their intracellular structure based on the mechanical vibration theory. These results provide new findings about the cellular mechanotransduction mechanism under mechanical vibration.

The Neural Representation of Binaural Sound Localization Cues Across Different Subregions of the Chicken's Inferior Colliculus.

The sound localization behavior of the nocturnally hunting barn owl and its underlying neural computations is a textbook example of neuroethology. Differences in sound timing and level at the two ears are integrated in a series of well-characterized steps, from brainstem to inferior colliculus (IC), resulting in a topographical neural representation of auditory space. It remains an important question of brain evolution: How is this specialized case derived from a more plesiomorphic pattern? The present study is the first to match physiology and anatomical subregions in the non-owl avian IC. Single-unit responses in the chicken IC were tested for selectivity to different frequencies and to the binaural difference cues. Their anatomical origin was reconstructed with the help of electrolytic lesions and immunohistochemical identification of different subregions of the IC, based on previous characterizations in owl and chicken. In contrast to barn owl, there was no distinct differentiation of responses in the different subregions. We found neural topographies for both binaural cues but no evidence for a coherent representation of auditory space. The results are consistent with previous work in pigeon IC and chicken higher-order midbrain and suggest a plesiomorphic condition of multisensory integration in the midbrain that is dominated by lateral panoramic vision.

Nucleus accumbens ghrelin signaling controls anxiety-like behavioral response to acute stress.

BACKGROUND: Anxiety disorders are one of the most common mental disorders. Ghrelin is a critical orexigenic brain-gut peptide that regulates food intake and metabolism. Recently, the ghrelin system has attracted more attention for its crucial roles in psychiatric disorders, including depression and anxiety. However, the underlying neural mechanisms involved have not been fully investigated. METHODS: In the present study, the effect and underlying mechanism of ghrelin signaling in the nucleus accumbens (NAc) core on anxiety-like behaviors were examined in normal and acute stress rats, by using immunofluorescence, qRT-PCR, neuropharmacology, molecular manipulation and behavioral tests. RESULTS: We reported that injection of ghrelin into the NAc core caused significant anxiolytic effects. Ghrelin receptor growth hormone secretagogue receptor (GHSR) is highly localized and expressed in the NAc core neurons. Antagonism of GHSR blocked the ghrelin-induced anxiolytic effects. Moreover, molecular knockdown of GHSR induced anxiogenic effects. Furthermore, injection of ghrelin or overexpression of GHSR in the NAc core reduced acute restraint stress-induced anxiogenic effects. CONCLUSIONS: This study demonstrates that ghrelin and its receptor GHSR in the NAc core are actively involved in modulating anxiety induced by acute stress, and raises an opportunity to treat anxiety disorders by targeting ghrelin signaling system.