The purpose of this study was to investigate the anxiety-like behaviors, circadian rhythms and sleep, and to elucidate the possible underlying mechanisms of the abnormal sleep behavior in Shank3 gene knockout (Shank3-KO) mice. The anxiety-like behaviors were detected by elevated plus-maze (EPM) test, open field test (OFT) and tail suspension test (TST). The circadian rhythms were detected by running wheel test. The electroencephalogram (EEG)/electromyogram (EMG) recordings were performed synchronically by polysomnograph. The distribution of SHANK3 in anterior cingulate cortex (ACC), paraventricular thalamus (PVT), nucleus accumbens (NAc), basolateral amygdala (BLA) and hippocampal CA2 region in wild type (WT) mice was detected by immunofluorescence assay. The protein expression of c-Fos in PVT, ACC and NAc was also detected by immunofluorescence assay during light cycle. The colocalization of c-Fos and vesicular glutamate transporter 2 (Vglut2, a marker for glutamatergic neurons) in the PVT was detected by immunofluorescence double labeling experiment. The results of EPM test showed that, compared with the WT mice, the Shank3-KO mice showed less time in open arms and less number of open arm entries. The results of OFT showed that the Shank3-KO mice showed less time in central area and less number of central area entries. The immobility time of Shank3-KO mice was increased in the TST. The results of running wheel rhythm test showed that the phase shift time of Shank3-KO mice in the continuous dark period was increased. The results of EEG/EMG recording showed that, compared with the WT mice, the duration of wakefulness in Shank3-KO mice was increased and the duration of non-rapid eye movement (NREM) sleep was decreased during light phase; The bout number of wakefulness was increased, the bout number of NREM sleep was decreased, NREM-wake transitions were increased, and wake-NREM transitions were decreased during light phase. SHANK3 was expressed in ACC, PVT, NAc and BLA in the WT mice. The expression of c-Fos in the PVT of Shank3-KO mice was up-regulated 2 h after entering the light phase, and majority of c-Fos was co-localized with Vglut2. These results suggest that the anxiety level of Shank3-KO mice is increased, the regulation of the internal rhythms is decreased, and the bout number of wakefulness is increased during light phase. The glutamatergic neurons in PVT may be involved in the regulation of abnormal sleep behavior in Shank3-KO mice during the light phase.
Animals ; Mice, Knockout ; Mice ; Neurons/metabolism* ; Nerve Tissue Proteins/physiology* ; Male ; Midline Thalamic Nuclei/cytology* ; Circadian Rhythm/physiology* ; Sleep/physiology* ; Anxiety/physiopathology* ; Proto-Oncogene Proteins c-fos/metabolism* ; Vesicular Glutamate Transport Protein 2/metabolism* ; Mice, Inbred C57BL ; Microfilament Proteins

OBJECTIVE: To determine whether the glutamatergic neurons in the paraventricular nucleus of the thalamus (PVT) is involved in the change of consciousness induced by propofol through a combination of behavioral and electroencephalography (EEG) recordings. METHODS: Healthy male VGluT2-IRES-Cre mice aged 8-12 weeks were used in this experiment. (1) The glutamatergic neurons in the PVT was selectively damaged, and its effect on propofol anesthesia induction and recovery times as well as the energy of EEG in different frequency bands were observed. (2) Optogenetics was utilized to selectively activate or inhibit glutamatergic neurons in the PVT to assess their influence on anesthesia induction and recovery times under propofol as well as the energy of EEG in different frequency bands. RESULTS: (1) Selective ablation of glutamatergic neurons in the PVT significantly delayed recovery from propofol anesthesia with statistical difference as compared with the control group (s: 409.43±117.49 vs. 273.71±51.52, P < 0.05), but had no significant effect on anesthesia induction time. During the recovery phase of propofol, selective ablation of glutamatergic neurons in the PVT exhibited higher α-wave (1-4 Hz) power and reduced β-wave (12-15 Hz) power as compared with the control group. (2) Optogenetic activation of glutamatergic neurons in the PVT significantly prolonged anesthesia induction time under propofol (s: 161.67±29.09 vs. 119.33±18.98, P < 0.05) while significantly shortening the recovery time from propofol anesthesia (s: 208.67±57.19 vs. 288.83±34.52, P < 0.05). During the induction phase of propofol, activation of glutamatergic neurons in PVT reduced α-wave and α-wave (8-12 Hz) power, while during the recovery phase, α-wave power significantly increased as compared with the control group. (3) Optogenetic inhibition of glutamatergic neurons in the PVT delayed recovery from propofol anesthesia (s: 403.50±129.06 vs. 252.83±45.31, P < 0.05), but had no significant effect on induction time. During both the induction phase and recovery phase of propofol, the optogenetic inhibition of glutamatergic neurons in the PVT exhibited increased α-wave power. CONCLUSION Glutamatergic neurons in the PVT are involved in the regulation of propofol anesthesia recovery process.
Animals ; Propofol/pharmacology* ; Mice ; Neurons/physiology* ; Male ; Electroencephalography ; Wakefulness ; Midline Thalamic Nuclei ; Optogenetics

At present, the problem of drug addiction treatment mainly lies in the high relapse rate of drug addicts. Addictive drugs will bring users a strong sense of euphoria and promote drug seeking. Once the drug is withdrawn, there will be withdrawal symptoms such as strong negative emotions and uncomfortable physical reactions. The recurrence of context-induced withdrawal memory is an important reason for drug relapse. Our previous study has shown increased c-Fos expression in prelimbic cortex (PrL) neurons projecting to paraventricular nucleus of the thalamus (PVT) (PrL^-PVT) during conditioned context-induced retrieval of morphine withdrawal memory. However, whether PrL^-PVT neurons are involved in withdrawal memory retrieval and the underlying molecular mechanisms remain unknown. In this study, we used conditioned place aversion (CPA) model combined with in vivo calcium signal recording, chemogenetics and nucleus drug injection methods to investigate the role and molecular mechanism of PrL^-PVT neurons in retrieval of morphine withdrawal memory. The results showed that the calcium signals of PrL^-PVT neurons were significantly enhanced by withdrawal-related context; Inhibition of PrL^-PVT neurons blocked the conditioned context-induced morphine withdrawal memory retrieval; Activation of PrL^-PVT neurons caused animals to escape from the context; After the inhibition of NMDA receptors in the PrL, withdrawal-related context failed to increase c-Fos and Arc expressions in PrL^-PVT neurons. The above results suggest that NMDA receptors in PrL^-PVT neurons are associated with retrieval of morphine withdrawal memory. This study is of great significance for further understanding the neural circuit mechanism of withdrawal memory retrieval as well as the intervention and prevention of drug relapse.
Animals ; Substance Withdrawal Syndrome/physiopathology* ; Morphine/adverse effects* ; Neurons/physiology* ; Receptors, N-Methyl-D-Aspartate/metabolism* ; Male ; Rats ; Paraventricular Hypothalamic Nucleus/metabolism* ; Memory ; Rats, Sprague-Dawley ; Morphine Dependence/physiopathology* ; Midline Thalamic Nuclei/physiology* ; Neural Pathways/metabolism*

Sleep deprivation has been shown to exacerbate pain sensitivity and may contribute to the onset of chronic pain, yet the precise neural mechanisms underlying this association remain elusive. In our study, we explored the contribution of cholinergic neurons within the medial habenula (MHb) to hyperalgesia induced by sleep deprivation in rats. Our findings indicate that the activity of MHb cholinergic neurons diminishes during sleep deprivation and that chemogenetic stimulation of these neurons can mitigate the results. Interestingly, we did not find a direct response of MHb cholinergic neurons to pain stimulation. Further investigation identified the interpeduncular nucleus (IPN) and the paraventricular nucleus of the thalamus (PVT) as key players in the pro-nociceptive effect of sleep deprivation. Stimulating the pathways connecting the MHb to the IPN and PVT alleviated the hyperalgesia. These results underscore the important role of MHb cholinergic neurons in modulating pain sensitivity linked to sleep deprivation, highlighting potential neural targets for mitigating sleep deprivation-induced hyperalgesia.
Animals ; Habenula/physiology* ; Sleep Deprivation/physiopathology* ; Cholinergic Neurons/physiology* ; Male ; Hyperalgesia/physiopathology* ; Rats, Sprague-Dawley ; Rats ; Interpeduncular Nucleus/physiology* ; Pain Threshold/physiology* ; Midline Thalamic Nuclei/physiology* ; Neural Pathways/physiopathology*

The incidence rate of anxiety and depression is significantly higher in patients with inflammatory bowel diseases (IBD) than in the general population. The mechanisms underlying dextran sulfate sodium (DSS)-induced depressive-like behaviors are still unclear. We clarified that IBD mice induced by repeated administration of DSS presented depressive-like behaviors. The paraventricular thalamic nucleus (PVT) was regarded as the activated brain region by the number of c-fos-labeled neurons. RNA-sequencing analysis showed that lipocalin 2 (Lcn2) was upregulated in the PVT of mice with DSS-induced depressive behaviors. Upregulating Lcn2 from neuronal activity induced dendritic spine loss and the secreted protein induced chemokine expression and subsequently contributed to microglial activation leading to blood-brain barrier permeability. Moreover, Lcn2 silencing in the PVT alleviated the DSS-induced depressive-like behaviors. The present study demonstrated that elevated Lcn2 in the PVT is a critical factor for DSS-induced depressive behaviors.
Mice ; Humans ; Animals ; Lipocalin-2/genetics* ; Midline Thalamic Nuclei ; Brain ; Inflammatory Bowel Diseases ; Proto-Oncogene Proteins c-fos ; Mice, Inbred C57BL

BDNF is a protein that allows the survival and differentiation of the central nervous system. In the present study, we have examined the postnatal development of BDNF -immunoreactive (IR) neuron system in the forebrain and the upper brain stem of the rat using immunohistochemistry. In the piriform cortex, claustrum, CA2 and 3, anterodorsal and paraventricular thalamic nucleus (nu.), ventromedial hypothalamic nu. and substantia nigra, BDNF-IR neurons were detected at postnatal day 1. BDNF-IR neurons in the anterior olfactory n., layers V and VI of the neocortex, claustrum, dentate gyrus, basolateral amygdaloid nu., paraventricular hypothalamic nu., mammillary nu. first appeared at postnatal 1 week of age and tended to increase in number as the rats grew. BDNF -IR neurons in ventromedial and paraventricular hypothalamic nu., mammillary nu., and substantia nigra decreased in number and none or only a few BDNF-IR neurons were seen in these areas of the adult rats. However, after treatment of colchicine, these areas showed numerous BDNF-IR neurons. BDNF-IR axon terminals were found in the septal nu., central amygdaloid nu., bed nu., of the stria terminalis, anterior ventral, anterior medial, interanteromedial and paravertricular thalamic nu., at postnatal day 1 and in dentate gyrus and paraventricular hypothalamic nu., at 1 week of age, respectively. These terminals in general continued to increase in number as the rats grew. Our results showed that BDNF immunoreactivity increased in various regions of the postnatally developing rat brain and suggest that BDNF might play an important role in neuronal maturation.
Adult ; Animals ; Basal Ganglia ; Brain Stem ; Brain* ; Brain-Derived Neurotrophic Factor ; Central Nervous System ; Colchicine ; Dentate Gyrus ; Humans ; Immunohistochemistry ; Midline Thalamic Nuclei ; Neocortex ; Neurons* ; Presynaptic Terminals ; Prosencephalon ; Rats* ; Substantia Nigra

This experimental studies was to investigate the location of CNS labeled neurons following injection of pseudorabies virus (PRV), Bartha strain, into the rat thymus. After survival times of 96~120 hours following injection of PRV, the rats were perfused, and their spinal cord and brain were frozen sectioned(30micrometer). These sections were stained by PRV immunohistochemical staining method, and observed with light microscope The results were as follows: 1. The PRV labeled spinal cord segments projecting to the rat thymus were founded in cervical and thoracic segments. Densely labeled areas of each spinal cord segment were founded in lamina V, VII, X, intermediolateral nucleus and dorsal nucleus. 2. In the rhombencephalon, PRV labeled neurons projecting to the thymus were founded in the A1 noradrenalin cells/C1 adrenalin cells/caudoventrolateral reticular nucleus, rostroventro-lateral reticular nucleus, medullary reticular nucleus, area postrema, nucleus solitary tract, nucleus raphe obscurus, nucleus raphe pallidus, nucleus raphe magnus, gigantocellular reticular nucleus, lateral paragigantocellular nucleus and spinal trigeminal nucleus. 3. In the mesencephalon, PRV labeled neurons were founded in parabrachial nucleus, Kolliker-Fuse nucleus, central gray matter, substantia nigra, nucleus dorsal raphe, A8 dopamin cells of retrorubral field, Edinger-Westphal nucleus, locus coeruleus, subcoeruleus nucleus and A5 noradrenalin cells. 4. In the prosencephalon, PRV labeled neurons were founded in reuniens thalamic nucleus, paraventricular thalamic nucleus, precommissural nucleus, paraventricular hypothalamic nucleus, anterior hypothalamic nucleus, lateral hypothalamic nucleus, preoptic hypothalamic nucleus, retrochiasmatic area, arcuate nucleus, dorsomedial hypothalamic nucleus and ventromedial hypothalamic nucleus. These results suggest that PRV labeled neurons of the spinal cord projecting to the rat thymus might be the neurons related to the viscero-somatic sensory and sympathetic preganglionic neurons, and PRV labeled neurons of the brain may be the neurons response to the movement of smooth muscle in blood vessels. These PRV labeled neurons may be central autonomic center related to the integration and modulation of reflex control linked to the sensory system monitoring the internal environment. These observations provide evidence for previously unknown projections from spinal cord and brain to the thymus which may be play an important role in the regulation of thymic function.
Animals ; Anterior Hypothalamic Nucleus ; Arcuate Nucleus ; Area Postrema ; Blood Vessels ; Brain ; Dorsomedial Hypothalamic Nucleus ; Herpesvirus 1, Suid* ; Hypothalamic Area, Lateral ; Immunohistochemistry ; Locus Coeruleus ; Mesencephalon ; Midline Thalamic Nuclei ; Muscle, Smooth ; Neurons* ; Paraventricular Hypothalamic Nucleus ; Prosencephalon ; Pseudorabies* ; Rats* ; Reflex ; Rhombencephalon ; Spinal Cord ; Substantia Nigra ; Thymus Gland* ; Trigeminal Nucleus, Spinal ; Ventromedial Hypothalamic Nucleus

Widespread brain-derived neurotrophic factor (BDNF) mRNA and protein expression has been detected in the brain. Despite substantial overlap between BDNF mRNA and protein expression, there is general anatomical regions, where there is discordance of these expression. We performed, therefore, immunohistochemistry after colchicine treatment into the ventricle to evaluate the possible presence of BDNF-immunoreactive (IR) in the regions where BDNF mRNA was expressed, but not BDNF-IR. The results obtained were as follows; There was substantial increase in the number of BDNF-IR neurons in the anterior olfactory nucleus, the piriform cortex, the cerebral cortex, the claustrum, the stratum pyramidale of the CA2 and the CA3, the granule cell layer of the dentate gyrus, the basolateral amygdaloid nucleus, the lateral geniculate nucleus, the anteromedial thalamic nucleus, the anterodorsal thalamic nucleus, the paraventricular thalamic nucleus, the paraventricular hypothalamic nucleus and the ventromedial hypothalamus nucleus, compared to the same brain area of non-colchicine treated rat. We detected many new BDNF-IR neurons in the stratum pyramidale of the CA1, A1, A2, A4-A10 cell groups, C1-C3 cell groups, the raphe magnus nucleus, the lateral paragigantocellular nucleus and the spinal vestibular nucleus. The results show that the localization of BDNF-IR neurons after colchicine treatment is consistant with that of BDNF mRNA containing neurons in the brain.
Animals ; Anterior Thalamic Nuclei ; Basal Ganglia ; Brain* ; Brain-Derived Neurotrophic Factor ; Cerebral Cortex ; Colchicine* ; Dentate Gyrus ; Hypothalamus ; Immunohistochemistry ; Midline Thalamic Nuclei ; Neurons* ; Paraventricular Hypothalamic Nucleus ; Rats* ; RNA, Messenger

BACKGROUND: Recently there have been many experiences regarding systemic effects of stellate ganglion block(SGB). During sympathetic hyperactivation, the SGB can be helpful to impaired microcirculation in brain-stem and hypothalamus. However, the exact mechanism and possible central action sites of SGB have not yet been investigated. In the present study, we traced central neural pathways following superior cervical ganglion block using the protein product(Fos) of c-fos protooncogene as a metabolic marker in a rat's brain. Method: The animals were divided into a superior cervical ganglion block group(n=5) using Marcaine 0.2 ml and a control block group(n=5) using saline 0.2ml. Medulla oblongata, pons, midbrain and hypothalamus were sectioned transversely with a sliding microtome. After imunohistochemical staining using rabbit polyclonal antibody we observed the distribution and grade of Fos expression under a light microscope. RESULTS: A blockade of superior cervical ganglion in rat led to the induction of c-fos in areas related to pain modulation sites and the autonomic nervous system; such as the parabrachial nucleus and central gray of the pons including dorsal raphe nucleus, as well as the substatia nigra of the midbrain, paraventricular hypothalamic nucleus, paraventricular thalamic nucleus posterior and habenular nucleus. CONCLUSIONS: Expressions of c-fos-like protein as a marker for neuronal activity following the blockade of the superior cervical ganglion in the rat have a part in the transcriptional control of the neurons which are related to the autonomic nervous system and endogenous pain modulation sites in the brain-stem.
Animals ; Autonomic Nervous System ; Brain ; Bupivacaine ; Habenula ; Hypothalamus* ; Medulla Oblongata ; Mesencephalon ; Microcirculation ; Midline Thalamic Nuclei ; Neural Pathways ; Neurons ; Paraventricular Hypothalamic Nucleus ; Pons ; Raphe Nuclei ; Rats* ; Staphylococcal Protein A ; Stellate Ganglion ; Superior Cervical Ganglion*