Objective: To study the effect of cocaine- and amphetamine-regulated transcript (CART) protein vaccine on morphine analgesia and tolerance. Methods: The expression plasmid pGEX-4T3-CART was constructed by gene cloning. The CART protein was purified by glutathione s-transferase (GST)-affinity chromatography. The experiment included 6 groups: blank control, normal saline (NS) GST + Freund's adjuvant, and CART protein vaccine (5 μg, 10 μg and 20 μg) groups. After immunization for twice, all groups were tested in hot plate. Morphine analgesia effect was evaluated through s. c. injection with 6 mg/kg morphine solution, calculated by MPE%. Then morphine tolerance model was established, and the tolerance to morphine was tested by s. c. injection with 6 mg/kg morphine 12 h after the last injection. Results: CART vaccine itself had no pronounced effect on the pain threshold (P>0.05). CART vaccine at 10 μg significantly depressed the analgesic effect of morphine analgesia (P<0.05). Compared with NS group, vaccine groups showed a potential antagonizing tolerance effect, especially in the 10 μg group, with the MPE% significantly increased (P<0. 05). Conclusion: CART vaccine itself has no influence on the pain response; however, it can impair the analgesia effect of morphine and can antagonize the analgesia tolerance to morphine.

Objective: To study the interaction between RET (GDNF receptor) with sucl-bingding neurotrophic target (SNT1) and search for the possible downstream substrates or regulatory proteins of RET, so as to reveal the mechanism of downstream signal transduction of RET. Methods: The RETIC (intracellular domain of RET) was fused to LexA and the product was used as a DNA-binding domain protein. SNT1, SNT1PTB or SNT1ΔPTB (SNT1 without PTB) was separately fused to B42AD and their products were used as an activation domain protein. The cotransformants were tested by β-galactosidase activity analysis and leucine medium growth analysis was used to study the interaction between SNT1 and RET. Results: Interaction between RET and SNT1 or SNT1PTB was found in yeast, but that between SNT1ΔPTB and RET was not observed. Conclusion: It i s confirmed that RET can interact with SNT1 in yeast, and the interaction may be mediated by PTB domain of SNT1.

Objective: To study the interaction between TrkA and downstream of kinases 1(dok 1) and search for the possible intracellular substrates or regulatory proteins of TrkA, so as to better understand the mechanism of downstream signal transduction of TrkA. Methods: The TrkAIC (intracellular domain of TrkA) was fused with LexA and the product was used as a DNA-binding domain protein; dok1, dok1PTB and dok1ΔPTB(dok1 without PTB) were separately fused with B42AD and their products were used as activation domain protein. Cotransformants were subjected to β-galactosidase activity analysis and leucine medium growth analysis for understanding of their interaction. Results: Dok1 and dok1PTB interacted with TrkA in yeast, while dok1ΔPTB did not. Conclusion: It is confirmed that TrkA can interact with dok1 in yeast, and the interaction may be mediated by PTB domain of dok1.

Demyelination disables saltatory conduction and leads to neural dysfunction. Remyelination is mediated by oligodendrocyte precursor cells (OPCs), which are widely distributed in the central nervous system (CNS) of adults. A belter understanding of oligodendrocyte biology, mechanisms of myelination and maintenance of myelin sheaths, and the relationship of failed remyelination in the CNS with the number, migration, and myelinating ability of endogenous OPCs, will greatly improve the remyelination strategies in chronic inflammatory demyelinating diseases, including muitiple sclerosis.

Objective To investigate the expression and activation of MMP-2, MMP-9 in cerebral tissues of cerebral ischemia-reperfusion rats and the effects of doxycycline on the cerebral edema. Methods Cerebral ischemia-reperfusion model was established by middle cerebral artery occlusion (MCAO). The rats were randomly divided into a sham-operated and 6 reperfusion groups according to different reperfusion periods (3 h, 6 h, 12 h, 24 h, 72 h, and 120 h groups). Dry weight method was used to evaluate hemispheric water content of the ischemic side after treatment with doxycycline. Western blotting analysis and Gelatin zymography were used to determine the expression and activity of MMP-2 and MMP-9 in the ischemic cerebral tissues. Results The protein expression and activity of MMP-2 were significantly increased in the ischemia tissue at 3 h and 120 h after reperfusion(P<0.01) ; the protein expression and activity of MMP-9 began to increase at 6 h after reperfusion (P<0. 01) , reached the peak at 24 h, and then returned to the basal levels at 120 h after reperfusion (P>0.05). The hemispheric water content of the ischemic side gradually increased with reperfusion periods compared with the sham-operated group. Dexycycline significantly reduced the hemispheric water contents compared with the normal saline at the same time points(P<0.05 or P<0.01). Conclusion MMP-2, MMP-9 can induce vasogenic brain edema by degrading basal membrane of capillary vessel. The alteration of MMP-2, MMP-9 expression and activities are likely associated with brain edema during cerebral ischemia-reperfusion.

Medial pontine reticular formation (mPRF),an important part of brainstem reticular formation, has drawn great attention from anesthesiologists due to its close relation with not only consciousness, but also analgesia, muscle relaxation and autonomic reflex. Progress in related research may provide evidence for understanding the mechanism of general anesthesia; meanwhile,it may serve as an effective anesthesia target site, which may contribute to improvement in anesthetic effect and reduction of complications.

BACKGROUND: Previous studies have shown that neurotrophin 3 (NT3)-chitosan can induce endogenous neurogenesis and axon regeneration in rats with spinal cord injury, and promote recovery of motor and sensory functions in rats. OBJECTIVE: To observe the effect of rehabilitation training combined with NT3-chitosan biomaterial scaffold on skeletal muscle morphological changes and functional recovery in rats with complete spinal cord injury. METHODS: Fifty adult female Wistar rats were randomly divided into five groups, 10 in each group. The sham group was not modeled; the remaining four groups were prepared with T7-T8 complete 5-mm spinal cord injury model, and the lesion control was not performed any intervention after modeling. The other three groups were given rehabilitation training, NT3-chitosan active biomaterial scaffold, NT3-chitosan active biomaterial scaffold combined with rehabilitation training intervention. Rehabilitation training started 2 weeks after modeling. Before operation, 2, 4, 6, 8, 10, and 12 weeks after operation, all of rats were subjected to double-blind open-field BBB scores. After 12 weeks, the skeletal muscles of the hind limbs (tibialis anterior muscle, gastrocnemius muscle, and soleus muscle) were taken for hematoxylin-eosin staining and acetylcholinesterase staining. The changes in muscle atrophy and motor endplates were assessed in each group. The experimental plan was approved by the Animal Experiment Committee of Capital Medical University (approval No. AEEI-2018-105). RESULTS AND CONCLUSION: (1) The BBB score at each time point in the sham group was higher than that in the other four groups (P < 0.05); and the scores at 8, 10, and 12 weeks after the NT3-chitosan combined rehabilitation training group were higher than the lesion control group, the lesion control combined rehabilitation training group, and NT3-chitosan group (P < 0.05). (2) At 12 weeks after operation, hematoxylin-eosin staining showed that the cross-sectional area and diameter of muscle fibers of each skeletal muscle were smaller in the other four groups than that in the sham group (P < 0.05). The cross-sectional area and diameter of muscle fibers of each skeletal muscle in the NT3-chitosan combined rehabilitation training group were higher than the lesion control group, the lesion control combined rehabilitation training group, and NT3-chitosan group (P < 0.05). (3) At 12 weeks after operation, the acetylcholinesterase staining showed that the average optical density of the acetylcholinesterase on motor endplate of the muscle was lower in the other four groups than that in the sham group (P < 0.05); the average optical density of the acetylcholinesterase of the motor endplate in the NT3-chitosan combined rehabilitation training was significantly higher than that in the lesion control, lesion control combined rehabilitation training, and NT3-chitosan groups (P < 0.05). (4) The results show that NT3-chitosan combined with rehabilitation training can effectively prevent muscular atrophy of hind limb skeletal muscles in rats with complete spinal cord injury, improve the average optical density of the acetylcholinesterase of the motor endplate, reduce neuromuscular joint degeneration, and improve rat hindlimb motor function.

Myasthenia gravis (MG) is a prototypical antibody-mediated neurological autoimmune disease with the involvement of humoral immune responses in its pathogenesis. T follicular helper (Tfh) cells have been implicated in many autoimmune diseases. However, whether and how Tfh cells are involved in MG remain unclear. Here, we established and studied a widely-used and approved animal model of human MG, the rat model with acetylcholine receptor alpha (AChRα) subunit (R-AChR)-induced experimental autoimmune myasthenia gravis (EAMG). This model presented mild body-weight loss 10 days after the first immunization (representing the early stage of disease) and more obvious clinical manifestations and body-weight loss 7 days after the second immunization (representing the late stage of disease). AChR-specific pre-Tfh cells and mature Tfh cells were detected in these two stages, respectively. In co-cultures of Tfh cells and B cells, the number of IgG2b-secreting B cells and the level of anti-AChR antibodies in the supernatant were higher in the cultures containing EAMG-derived Tfh cells. In immunohistochemistry and immunofluorescence assays, a substantial number of CD4/Bcl-6 T cells and a greater number of larger germinal centers were observed in lymph node tissues resected from EAMG rats. Based on these results, we hypothesize that an AChR-specific Tfh cell-mediated humoral immune response contributes to the development of EAMG.
Animals ; B-Lymphocytes ; immunology ; Disease Models, Animal ; Female ; Immunity, Humoral ; Lymph Nodes ; immunology ; Myasthenia Gravis, Autoimmune, Experimental ; immunology ; Protein Subunits ; immunology ; Proto-Oncogene Proteins c-bcl-6 ; immunology ; Rats, Inbred Lew ; Receptor Cross-Talk ; Receptors, Cholinergic ; immunology ; T-Lymphocytes, Helper-Inducer ; immunology

The anterior auditory field (AAF) is a core region of the auditory cortex and plays a vital role in discrimination tasks. However, the role of the AAF corticostriatal neurons in frequency discrimination remains unclear. Here, we used c-Fos staining, fiber photometry recording, and pharmacogenetic manipulation to investigate the function of the AAF corticostriatal neurons in a frequency discrimination task. c-Fos staining and fiber photometry recording revealed that the activity of AAF pyramidal neurons was significantly elevated during the frequency discrimination task. Pharmacogenetic inhibition of AAF pyramidal neurons significantly impaired frequency discrimination. In addition, histological results revealed that AAF pyramidal neurons send strong projections to the striatum. Moreover, pharmacogenetic suppression of the striatal projections from pyramidal neurons in the AAF significantly disrupted the frequency discrimination. Collectively, our findings show that AAF pyramidal neurons, particularly the AAF-striatum projections, play a crucial role in frequency discrimination behavior.
Acoustic Stimulation/methods* ; Neurons/physiology* ; Auditory Cortex/physiology* ; Auditory Perception ; Pyramidal Cells

The GABAergic neurons in the parafacial zone (PZ) play an important role in sleep-wake regulation and have been identified as part of a sleep-promoting center in the brainstem, but the long-range connections mediating this function remain poorly characterized. Here, we performed whole-brain mapping of both the inputs and outputs of the GABAergic neurons in the PZ of the mouse brain. We used the modified rabies virus EnvA-ΔG-DsRed combined with a Cre/loxP gene-expression strategy to map the direct monosynaptic inputs to the GABAergic neurons in the PZ, and found that they receive inputs mainly from the hypothalamic area, zona incerta, and parasubthalamic nucleus in the hypothalamus; the substantia nigra, pars reticulata and deep mesencephalic nucleus in the midbrain; and the intermediate reticular nucleus and medial vestibular nucleus (parvocellular part) in the pons and medulla. We also mapped the axonal projections of the PZ GABAergic neurons with adeno-associated virus, and defined the reciprocal connections of the PZ GABAergic neurons with their input and output nuclei. The newly-found inputs and outputs of the PZ were also listed compared with the literature. This cell-type-specific neuronal whole-brain mapping of the PZ GABAergic neurons may reveal the circuits underlying various functions such as sleep-wake regulation.
Animals ; Axons ; physiology ; Brain ; anatomy & histology ; Brain Mapping ; Brain Stem ; cytology ; GABAergic Neurons ; physiology ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Neural Pathways ; physiology ; Peptide Elongation Factor 1 ; genetics ; metabolism ; Rabies virus ; genetics ; metabolism ; Transduction, Genetic ; Vesicular Inhibitory Amino Acid Transport Proteins ; genetics ; metabolism