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

The functional role of glutamatergic (vGluT2) neurons in the pedunculopontine nucleus (PPN) in modulating motor activity remains controversial. Here, we demonstrated that the activity of vGluT2 neurons in the rostral PPN is correlated with locomotion and ipsilateral head-turning. Beyond these motor functions, we found that these rostral PPN-vGluT2 neurons remarkably respond to salient stimuli. Furthermore, we systematically traced the upstream and downstream projections of these neurons and identified two downstream projections from these neurons to the caudal pontine reticular nucleus/anterior gigantocellular reticular nucleus (PnC/GiA) and the zona incerta (ZI). Our findings indicate that the projections to the PnC/GiA inhibit movement, consistent with 'pause-and-play' behavior, whereas those to the ZI promote locomotion, and others respond to a new 'pause-switch-play' pattern. Collectively, these findings elucidate the multifaceted influence of the PPN on motor functions and provide a robust theoretical framework for understanding its physiological and potential therapeutic implications.
Pedunculopontine Tegmental Nucleus/physiology* ; Animals ; Neural Pathways/physiology* ; Vesicular Glutamate Transport Protein 2/metabolism* ; Locomotion/physiology* ; Glutamic Acid/metabolism* ; Neurons/physiology* ; Male ; Mice ; Motor Activity/physiology* ; Zona Incerta/physiology*

The lateral habenula (LHb), which is a critical neuroanatomical hub and a regulator of midbrain monoaminergic centers, is activated by events resulting in negative valence and contributes to the expression of both appetitive and aversive behaviors. However, whole-brain cell-type-specific monosynaptic inputs to the LHb in both sexes remain incompletely elucidated. In this study, we used viral tracing combined with in situ hybridization targeting vesicular glutamate transporter 2 (vGlut2) and glutamic acid decarboxylase 2 (Gad2) to generate a comprehensive whole-brain atlas of inputs to glutamatergic and γ-aminobutyric acid (GABA)ergic neurons in the LHb. We found >30 ipsilateral and contralateral brain regions that projected to the LHb. Of these, there were significantly more monosynaptic LHb-projecting neurons from the lateral septum, anterior hypothalamus, dorsomedial hypothalamus, and ventromedial hypothalamus in females than in males. More interestingly, we found a stronger GABAergic projection from the medial septum to the LHb in males than in females. Our results reveal a comprehensive connectivity atlas of glutamatergic and GABAergic inputs to the LHb in both sexes, which may facilitate a better understanding of sexual dimorphism in physiological and pathological brain functions.
Animals ; Male ; Mice ; Glutamic Acid/metabolism* ; Habenula/metabolism* ; Hypothalamus/metabolism* ; Neural Pathways/physiology* ; Sex Characteristics ; Vesicular Glutamate Transport Protein 2/metabolism* ; Female

OBJECTIVES: Hearing loss disrupts the balance of auditory-somatosensory inputs in the cochlear nucleus (CN) of the brainstem, which has been suggested to be a mechanism of tinnitus. This disruption results from maladaptive auditory-somatosensory plasticity, which is a form of axonal sprouting. Axonal sprouting is promoted by transforming growth factor (TGF)-β signaling, which can be inhibited by losartan. We investigated whether losartan prevents maladaptive auditory-somatosensory plasticity after hearing loss. METHODS: The study consisted of two stages: determining the time course of auditory-somatosensory plasticity following hearing loss and preventing auditory-somatosensory plasticity using losartan. In the first stage, rats were randomly divided into two groups: a control group that underwent a sham operation and a deaf group that underwent cochlea ablation on the left side. CNs were harvested 1 and 2 weeks after surgery. In the second stage, rats were randomly divided into either a saline group that underwent cochlear ablation on the left side and received normal saline or a losartan group that underwent cochlear ablation on the left side and received losartan. CNs were harvested 2 weeks after surgery. Hearing was estimated with auditory brainstem responses (ABRs). Western blotting was performed for vesicular glutamate transporter 1 (VGLUT1), reflecting auditory input; vesicular glutamate transporter 2 (VGLUT2), reflecting somatosensory input; growth-associated protein 43 (GAP-43), reflecting axonal sprouting; and p-Smad2/3. RESULTS: Baseline ABR thresholds before surgery ranged from 20 to 35 dB sound pressure level. After cochlear ablation, ABR thresholds were higher than 80 dB. In the first experiment, VGLUT2/VGLUT1 ratios did not differ significantly between the control and deaf groups 1 week after surgery. At 2 weeks after surgery, the deaf group had a significantly higher VGLUT2/VGLUT1 ratio compared to the control group. In the second experiment, the losartan group had a significantly lower VGLUT2/VGLUT1 ratio along with significantly lower p-Smad3 and GAP-43 levels compared to the saline group. CONCLUSION: Losartan might prevent axonal sprouting after hearing loss by blocking TGF-β signaling thereby preventing maladaptive auditory-somatosensory plasticity.
Animals ; Axons ; Blotting, Western ; Brain Stem ; Cochlea ; Cochlear Nucleus ; Evoked Potentials, Auditory, Brain Stem ; GAP-43 Protein ; Hearing Loss ; Hearing ; Losartan ; Plastics ; Rats ; Tinnitus ; Transforming Growth Factors ; Vesicular Glutamate Transport Protein 1 ; Vesicular Glutamate Transport Protein 2

Mounting evidence supports an important role of chemokines, produced by spinal cord astrocytes, in promoting central sensitization and chronic pain. In particular, CCL2 (C-C motif chemokine ligand 2) has been shown to enhance N-methyl-D-aspartate (NMDA)-induced currents in spinal outer lamina II (IIo) neurons. However, the exact molecular, synaptic, and cellular mechanisms by which CCL2 modulates central sensitization are still unclear. We found that spinal injection of the CCR2 antagonist RS504393 attenuated CCL2- and inflammation-induced hyperalgesia. Single-cell RT-PCR revealed CCR2 expression in excitatory vesicular glutamate transporter subtype 2-positive (VGLUT2) neurons. CCL2 increased NMDA-induced currents in CCR2/VGLUT2 neurons in lamina IIo; it also enhanced the synaptic NMDA currents evoked by dorsal root stimulation; and furthermore, it increased the total and synaptic NMDA currents in somatostatin-expressing excitatory neurons. Finally, intrathecal RS504393 reversed the long-term potentiation evoked in the spinal cord by C-fiber stimulation. Our findings suggest that CCL2 directly modulates synaptic plasticity in CCR2-expressing excitatory neurons in spinal lamina IIo, and this underlies the generation of central sensitization in pathological pain.
Animals ; Benzoxazines ; pharmacology ; therapeutic use ; Chemokine CCL2 ; antagonists & inhibitors ; genetics ; metabolism ; pharmacology ; Excitatory Amino Acid Agents ; pharmacology ; Excitatory Amino Acid Agonists ; pharmacology ; Female ; Freund's Adjuvant ; toxicity ; Hyperalgesia ; chemically induced ; metabolism ; prevention & control ; Long-Term Potentiation ; drug effects ; physiology ; Luminescent Proteins ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Myelitis ; chemically induced ; drug therapy ; metabolism ; Neurons ; drug effects ; Pain Management ; Somatostatin ; genetics ; metabolism ; Spinal Cord ; cytology ; Spiro Compounds ; pharmacology ; therapeutic use ; Vesicular Glutamate Transport Protein 2 ; genetics ; metabolism ; Vesicular Inhibitory Amino Acid Transport Proteins ; genetics ; metabolism