Mitochondrial dysfunction and dysregulated calcium homeostasis contribute to Alzheimer’s disease (AD) pathogenesis. The extrasynaptic N-methyl-D-aspartic acid (NMDA) receptor (eNMDAR) plays a crucial role in calcium influx and subsequent signaling cascades. In individuals with AD, the reduced expression and function of glutamate transporter-1 (GLT-1) result in glutamate spillover from the synaptic clefts to the extrasynaptic region, thereby activating eNMDAR and inducing mitochondrial damage. Ceftriaxone (Cef) has been reported to ameliorate cognitive deficits in APPswe/PS1dE9 (APP/PS1) mice by upregulating GLT-1. This study aimed to explore whether Cef alleviates mitochondrial dysfunction to improve cognitive impairment and the roles of GLT-1 and eNMDAR, particularly the participation of eNMDAR-induced intracellular calcium signaling in this process. C57BL/6J, APP/PS1, and GLT-1-knockdown APP/PS1 mice were used. NMDA (1 mM, 2 µL per ventricle) was injected cerebroventricularly into APP/PS1 mice once to activate eNMDAR. Cef (200 mg/kg) was intraperitoneally administered for 14 days. Cognitive function was evaluated by novel object recognition, novel location recognition and Morris water maze tests. Hippocampal mitochondrial ultrastructure was observed using transmission electron microscopy. Hippocampal mitochondrial membrane potential (MMP) was detected using JC-1 staining. The expression of eNMDAR and proteins related to mitochondrial biogenesis and dynamics was evaluated by western blot. A neuron‒astrocyte coculture derived from the cerebral cortex of embryonic mice was used to evaluate the effects of Cef on eNMDAR-induced neuronal calcium influx, mitochondrial calcium accumulation and MMP loss using live-cell imaging. Cef treatment attenuated hippocampal mitochondrial dysfunction, including ultrastructural damage, reduced aspect ratio, dysregulation of MMP, impaired biogenesis and dynamics, and cognitive deficits, and prevented the upregulation of eNMDAR expression in APP/PS1 mice in a GLT-1-dependent manner. These protective effects on hippocampal mitochondrial dysfunction and cognitive deficits were counteracted by eNMDAR activation. Furthermore, Cef incubation inhibited eNMDAR-mediated calcium influx in a GLT-1-dependent way and reduced MMP in primary cortical neurons. Notably, Cef incubation significantly suppressed mitochondrial calcium overload, which was mechanistically linked to the observed decline in MMP. Cef treatment prevented the upregulation of eNMDAR expression and the subsequent extracellular calcium influx in a GLT-1-dependent manner, thereby reducing mitochondrial calcium loading and ultimately mitigating mitochondrial damage and cognitive deficits in APP/PS1 mice.

Tau phosphorylation and mislocalization are hallmark pathological features of Alzheimer's disease (AD), with endoplasmic reticulum stress (ERS) contributing to tauopathy. We previously showed that ceftriaxone (Cef) improves cognition in APP/PS1 AD mice through regulating GLT-1-mediated glutamate homeostasis. Here, we examined Cef's neuroprotection against ERS-related tauopathy. C57BL/6J and APP/PS1 AD mice were used. Cognitive functions were assessed by new object recognition (NOR), new location recognition (NLR) and Morris water maze (MWM) tests. Hippocampal synaptosomes were isolated using the Syn-PER™ Synaptic Protein Extraction Kit. Western blot analysis evaluates the protein levels of ERS markers, total and phosphorylated tau (Ser396/Ser262/Thr181), and Gsk3β. Transmission electron microscopy examined the endoplasmic reticulum ultrastructural changes of the hippocampus. Confocal 3D-reconstructed imaging assessed the phosphorylated tau (Ser396) distribution on the dendrites in the hippocampal region. The results showed that Cef treatment effectively reduced protein levels of ERS markers and restored endoplasmic reticulum ultrastructural integrity of hippocampus. Simultaneously, Cef treatment significantly alleviated tau phosphorylation levels, decreased accumulation of total and phosphorylated tau in synaptosomes, reduced phosphorylated tau (Ser396) distribution in dendritic compartments and inhibited Gsk3β activity in the hippocampus of APP/PS1 AD mice. Tunicamycin, a promoter of ERS, exacerbated cognitive impairments, tau phosphorylation levels and mislocalization, and Gsk3β activity, and notably, this exacerbation was inhibited by Cef treatment. Simultaneously, the ERS activation significantly inhibited Cef's above benefits on APP/PS1 AD mice. In conclusion, Cef improves cognitive impairment by alleviating ERS, decreasing Gsk3β activity, and reducing tau phosphorylation and mislocalization in the hippocampus of APP/PS1 AD mice.

Cognitive dysfunction is an important comorbidity of type 2 diabetes mellitus (T2DM). Sodium butyrate (NaB) is a short-chain fatty acid and has an effect improving T2DM-associated cognitive dysfunction. Using a high-fat diet (HFD)/streptozotocin (STZ)-induced T2DM mouse model, the present study investigated the mechanism involved in the beneficial effect of butyrate on diabetic cognitive dysfunction, with a focus on ameliorating mitochondrial damage through regulating the adenosine monophosphate-activated protein kinase/peroxisome proliferator- activated receptor gamma coactivator 1α (AMPK/PGC-1α) pathway considering the important role of mitochondrial impairments in the occurrence of T2DM-associated cognitive dysfunction. We found, based on reconfirmation of the improvement of NaB on cognitive impairment, that NaB treatment improved damaged synaptic structural plasticity including the decrease in dendritic spine density and downregulation in the expression of postsynaptic density protein 95 and synaptophysin in the hippocampus in the model mice. NaB treatment also ameliorated mitochondrial ultrastructural damage, increased mitochondrial membrane potential and adenosine 5'-triphosphate content, and improved mitochondrial biogenesis and dynamics in the model mice. Furthermore, the expression of phosphorylated AMPK and PGC-1α was upregulated after NaB treatment in the model mice. In particular, the above beneficial effects of NaB were blocked by the inhibition of either AMPK or PGC-1α. In conclusion, NaB treatment improved cognitive impairment and damaged synaptic structural plasticity in the hippocampus by ameliorating damage to mitochondrial morphology and function through regulating the AMPK/PGC-1α pathway in HFD/STZ-induced T2DM mice.

Group II metabotropic glutamate receptors (Group II mGluRs) are the peri-synaptic receptor of glutamatergic neurons and negatively regulate glutamate release from presynaptic neurons. Glutamate in the synaptic cleft is mainly taken into astrocytes by glutamate transporter-1 (GLT-1), which is primarily expressed in astrocytes. Increasing evidence showed that inhibiting or suppressing the activation of Group II mGluRs would contribute to the improvement of learning and memory deficits in Alzheimer's disease (AD) animal models. Ceftriaxone (Cef) has been reported to alleviate the spatial memory deficits in AD model mice by improving GLT-1-related clearance and metabolism of glutamate. Therefore, the present study further investigates the improving effect of Cef on recognition memory deficits and the involvement of Group II mGluRs in the process using the APP/PS1 AD mouse model. Novel object recognition tests showed that the Cef treatment significantly improved the recognition memory deficits of the AD mice. The Western blot and immunohistochemistry analysis showed that the Cef treatment significantly suppressed the upregulation of Group II mGluRs expression in APP/PS1 AD mice. The above suppression effect of Cef was blocked by dihydrokainic acid, an inhibitor of GLT-1 uptake activity. Furthermore, the Cef treatment significantly restored the downregulation in the downstream molecules of Group II mGluRs activation, including the expression of PKA and phosphorylated SNAP-25 in the APP/PS1 AD mice. The Cef treatment had no effect on the content of Aβ40 and Aβ42 in the hippocampus of APP/PS1 AD mice. The above results suggested that the suppression of Group II mGluRs contributed to the Cef-induced reversal of the recognition memory deficits in APP/PS1 AD mice.

Dopaminergic neurons in the ventral tegmental area (VTA) encode behavioral patterns important in reward and drug addiction as well as in emotional disorders. These functions of dopamine neurons are directly related to the release of dopamine in the targeted regions of the brain which are, thus, controlled by the excitability of dopamine neurons. One mechanism for modulation of dopamine neuronal excitability is mediated by the auto dopamine type 2 (D2) receptors, through activation of a Kir3/GIRK K+ channel which inhibits the firing of dopamine neurons. In this study, we provide evidence that Kv7.4, in addition to Kir3.2 channels, contributes to dopamine (DA)-mediated auto-inhibition of DA activity projecting to NAc and to basolateral amygdale (BLA). Furthermore, we demonstrate that D2 receptors enhance Kv7.4 currents through Gi/o protein and redox-dependent cellular pathway. Finally, we show this D2 mediated auto-inhibition is blunted in a social defeat mice model of depression, a phenomenon that may contribute to the altered excitability of VTA DA neurons in depressed animals. These results provide a new perspective for understanding the molecular mechanism of the excitability of VTA DA neurons and for potential new strategies against mental disorders involving altered excitability of DA neurons, such as major depression and drug addictions.

Aim To investigate the effect of p38 MAPK AS-ODNs on the expression of GLT-1 during the induction of brain ischemic tolerance induced by cerebral ischemic preconditioning (CIP).Methods Eighty healthy male Wistar rats with permanent occlusion of the bilateral vertebral arteries were randomly divided into 6 groups: ①Sham group (n=10);②CIP group (n=10);③ischemic insult (Ⅱ) group (n=10);④CIP+Ⅱ group (n=10);⑤p38 MAPK AS-ODNs+CIP+Ⅱ group (n=30);⑥p38 MAPK S-ODNs+CIP+Ⅱ group (n=10).Group ⑤ was divided into 5 nmol, 10 nmol and 15 nmol subgroups according to the dose of p38 MAPK AS-ODNs (n=10).The dose of p38 MAPK S-ODNs was 15 nmol.All the rats were sacrificed 6 h and 2 d after the sham operation or the last time of global brain ischemia reperfusion.Western blot and immunohistochemistry analysis were used for detecting the expression of p-p38 MAPK and GLT-1 protein.Results CIP moderately up-regulated the expression of p-p38 MAPK and significantly up-regulated the expression of GLT-1 protein, inhibited the excessively up-regulation of p-p38 MAPK and the down-regulation of GLT-1 induced by ischemic insult.p38 MAPK AS-ODNs significantly inhibited the up-regulation of p-p38 MAPK and GLT-1 protein in a dose-dependent manner during the induction of brain ischemic tolerance by CIP.Conclusion p38 MAPK AS-ODNs inhibit the up-regulation of GLT-1 during the induction of brain ischemic tolerance induced by CIP.

Peripheral sensory ganglia contain somata of afferent fibres conveying somatosensory inputs to the central nervous system. Growing evidence suggests that the somatic/perisomatic region of sensory neurons can influence peripheral sensory transmission. Control of resting membrane potential (Erest) is an important mechanism regulating excitability, but surprisingly little is known about how Erest is regulated in sensory neuron somata or how changes in somatic/perisomatic Erest affect peripheral sensory transmission. We first evaluated the influence of several major ion channels on Erest in cultured small-diameter, mostly capsaicin-sensitive (presumed nociceptive) dorsal root ganglion (DRG) neurons. The strongest and most prevalent effect on Erest was achieved by modulating M channels, K2P and 4-aminopiridine-sensitive KV channels, while hyperpolarization-activated cyclic nucleotide-gated, voltage-gated Na(+), and T-type Ca(2+) channels to a lesser extent also contributed to Erest. Second, we investigated how varying somatic/perisomatic membrane potential, by manipulating ion channels of sensory neurons within the DRG, affected peripheral nociceptive transmission in vivo. Acute focal application of M or KATP channel enhancers or a hyperpolarization-activated cyclic nucleotide-gated channel blocker to L5 DRG in vivo significantly alleviated pain induced by hind paw injection of bradykinin. Finally, we show with computational modelling how somatic/perisomatic hyperpolarization, in concert with the low-pass filtering properties of the t-junction within the DRG, can interfere with action potential propagation. Our study deciphers a complement of ion channels that sets the somatic Erest of nociceptive neurons and provides strong evidence for a robust filtering role of the somatic and perisomatic compartments of peripheral nociceptive neuron.

The aim of this study was to investigate the effect of the μ-opioid receptor gene (OPRM1) A118G polymorphism on the requirement for post-operative fentanyl analgesia in patients undergoing radical gastrectomy. One hundred and twenty-eight gastric cancer patients scheduled to undergo radical gastrectomy under general anesthesia were enrolled in the study. Post-operative, patient-controlled intravenous analgesia of fentanyl was provided for satisfactory analgesia until 48 h after surgery. OPRM1 A118G was screened by DNA sequence analysis of polymerase chain reaction (PCR)-amplified DNA. Differences in fentanyl consumption and adverse effects were compared among the different genotypes at 24 and 48 h after surgery. The ranges of fentanyl dose in the 128 patients at 24 and 48 h after surgery were 5.4-17.3 μg/kg and 12.4-29.9 μg/kg, respectively. Among these patients, there were 54 wild-type homozygotes (AA), 53 heterozygotes (AG) and 21 mutant homozygotes (GG). The frequency of the G allele was 0.371 in the OPRM1 polymorphism. There were no significant differences in fentanyl dose or adverse effects, including nausea, vomiting and dizziness, for the OPRM1 A118G polymorphism (P>0.05). The OPRM1 A118G polymorphism does not play a significant role in post-operative fentanyl analgesic dose or post-operative nausea, vomiting and dizziness in patients undergoing radical gastrectomy.

Objective To study the effects of rehabilitation training on the synaptic ultrastructure of the brain cortex after cerebral ischemia.Methods Sixty rats were randomly divided into a sham operation group,a cerebral ischemia group and a rehabilitation training group.A model of focal cerebral ischemia was created in the rats of the cerebral ischemia and rehabilitation training groups.The rehabilitation training group was given balancing,grasping,rotation and walking training every day.The sham operation group and cerebral ischemia group were kept in normal cages,taking food and moving freely.Electron microscopy was used to observe the density and ultrastructure of the cortical synapses at days 1,7,14 and 21 after the operation.Results In the sham operation group there were numerous synaptic terminals in the cortical neuropil.The majority of synapses were Gray type Ⅱ,containing spherical synaptic vesicles in presynaptic elements.After cerebral ischemia,the number of synaptic terminals had decreased and the synaptic structure was damaged.The synaptic vesicles lessened and dissolved.The presynaptic and postsynaptic membranes became unclear.At 21d after ischemia,the synaptic vesicles had disappeared and the presynaptic and postsynaptic membranes were damaged so seriously that the typical synaptic structure disappeared.After rehabilitation training,the damaged synaptic terminals recovered.Conclusions The synaptic structure was damaged after cerebral ischemia,and the damaged synaptic terminals recovered after rehabilitation training.Rehabilitation training can enhance synaptogenesis in the brain's cortex after cerebral ischemia and promote the recovery of motor function,at least in rats.

Objective To study the effects of rehabilitation training on the expression of synaptophysin in the cortex after cerebral ischemia. Methods A model of focal cerebral ischemia was created in rats. The rats were divided into 4 groups at random 24 h after the cerebral ischemia was induced. The rehabilitation group was given bal-ancing, grasping, rotation, walking and other training every day. An immobilization group was fixed in their cages. The model control group and cerebral ischemia group were kept in general cages, taking food and moving freely. Im-munohistochemisty method was used to detect synaptophysin expression at the 1st, 7th, 14th, 21st and 28th day after the cerebral ischemia was induced. Results In the model control group, synaptophysin immunoreactive positive products were observed. In the cerebral ischemia group, the expression of synaptophysin decreased gradually. After rehabilitation training, synaptophysin immunoreactive positive products decreased slowly, but remained more abun-dant than among the immobilized rats. Conclusion After cerebral ischemia, the expression of synaptophysin de-creases. This demonstrates that the synaptic terminals were less injured. Rehabilitation training can enhance synapto-genesis in the cortex after cerebral ischemia.

Abstract： The present study was undertaken to observe changes in concentrations of glutamate, aspartate and γ-aminobutyric acid (GABA) in the CA1 hippocampus during the acquirement of brain ischemic tolerance induced by cerebral ischemic preconditioning (CIP) using cerebral microdialysis and high performance liquid chromatography techniques in rats. An initial, moderate and synchronous increase in glutamate, aspartate and GABA was first evoked by the lethal ischemic insult for 8 min which was verified to induce delayed neuronal death (DND) in the CA1 hippocampus. GABA quickly recovered to and kept at control level after reperfusion, but both glutamate and aspartate levels increased secondly in more magnitudes within the early stage of reperfusion. When the animals were pretreated 2 days before the lethal ischemic insult with a CIP for 3 min which protected the pyramidal neurons against DND normally induced by the lethal brain ischemia, the second peaks of glutamate and aspartate was prevented completely. The results suggested first in vivo that the protective effect of the CIP against DND of pyramidal neurons in the CA1 hippocampus normally induced by lethal brain ischemia might be associated with suppressing the increase in extracellular concentrations of both glutamate and aspartate.