Ciprofol alleviates remifentanil-induced hyperalgesia by regulating α2GABAARs, NR2B, and P-CaMKIIα levels in the spinal cord and hippocampus.

OBJECTIVES: Several studies have shown that propofol administration during surgery effectively attenuates remifentanil-induced hyperalgesia (RIH). Ciprofol, a novel intravenous sedative agent analogous to propofol, has not yet been proven efficacious in alleviating RIH. The present study aimed to investigate the effect of ciprofol on RIH and the possible mechanisms involved. METHODS: The RIH model was established by an infusion of remifentanil (1 µg·kg-1·min-1) 60 min in rats with incisional pain. Ciprofol (0.1, 0.25, and 0.4 mg·kg-1·min-1) was simultaneously infused to evaluate its effect on RIH. The antinociception of ciprofol was verified by measured paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL). γ-aminobutyric acid type A receptor α2 subunit (α2GABAAR), N-methyl-d-aspartate receptor NR2B subunit (NR2B), calcium/calmodulin-dependent protein kinase II α (CaMKIIα), and phosphorylated CaMKIIα (P-CaMKIIα) in the spinal cord and hippocampus of rats were assessed by western blotting and immunohistochemistry. KEY FINDINGS: The results showed that ciprofol dose-dependently increased PWMT and PWTL values in RIH rats. Moreover, ciprofol upregulated α2GABAAR and downregulated NR2B and P-CaMKIIα in the rat spinal cord and hippocampus. CONCLUSIONS: Ciprofol alleviates RIH effectively, and the anti-hyperalgesic mechanisms may involve increasing α2GABAAR levels and decreasing NR2B and P-CaMKIIα levels in the spinal cord and hippocampus.

Repeated Morphine Exposure Alters Temporoamonic-CA1 Synaptic Plasticity in Male Rat Hippocampus.

In this study, the electrophysiological and biochemical consequences of repeated exposure to morphine in male rats on glutamatergic synaptic transmission, synaptic plasticity, the expression of GABA receptors and glutamate receptors at the temporoammonic-CA1 synapse along the longitudinal axis of the hippocampus (dorsal, intermediate, ventral, DH, IH, VH, respectively) were investigated. Slice electrophysiological methods, qRT-PCR, and western blotting techniques were used to characterize synaptic plasticity properties. We showed that repeated morphine exposure (RME) reduced excitatory synaptic transmission and ability for long-term potentiation (LTP) in the VH as well as eliminated the dorsoventral difference in paired-pulse responses. A decreased expression of NR2B subunit in the VH and an increased expression GABAA receptor of α1 and α5 subunits in the DH were observed following RME. Furthermore, RME did not affect the expression of NR2A, AMPA receptor subunits, and γ2GABAA and GABAB receptors in either segment of the hippocampus. In sum, the impact of morphine may differ depending on the region of the hippocampus studied. A distinct change in the short- and long-term synaptic plasticity along the hippocampus long axis due to repeated morphine exposure, partially mediated by a change in the expression profile of glutamatergic receptor subunits. These findings can be useful in further understanding the cellular mechanism underlying deficits in information storage and, more generally, cognitive processes resulting from chronic opioid abuse.

Tat-NR2B9c attenuates oxidative stress via inhibition of PSD95-NR2B-nNOS complex after subarachnoid hemorrhage in rats.

Oxidative stress plays important roles in the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Tat-NR2B9c has shown efficacy as a neuroprotective agent in several studies. Here, we identified the neuroprotective role of Tat-NR2B9c after SAH and its related mechanisms. The results showed that Tat-NR2B9c treatment attenuated oxidative stress, therefore alleviated neuronal apoptosis and neurological deficits after SAH. Tat-NR2B9c treatment could alleviate mitochondrial vacuolization induced by SAH. Compared to SAH + vehicle group, Tat-NR2B9c resulted in the decrease of Acetylated superoxide dismutase2 (Ac-SOD2), Bcl-2-associated X protein (Bax) and cleaved-caspase3 (CC3) protein expression, and the up-regulation of Sirtunin 3 (Sirt3) and Bcl-2 protein level. Moreover, Tat-NR2B9c attenuated excitotoxicity by inhibiting the interaction of PSD95-NR2B-nNOS. Our results demonstrated that Tat-NR2B9c inhibited oxidative stress via inhibition of PSD95-NR2B-nNOS complex formation after SAH. Tat-NR2B9c may serve as a potential treatment for SAH induced brain injury.

Floralozone regulates MiR-7a-5p expression through AMPKα2 activation to improve cognitive dysfunction in vascular dementia.

BACKGROUND: The pathogenesis of vascular dementia (VD) is complex, and currently, no effective treatments have been recommended. Floralozone is a colorless liquid first discovered in Lagotis Gaertn. Recently, its medicinal value has been increasingly recognized. Our previous study has demonstrated that Floralozone can improve cognitive dysfunction in rats with VD by regulating the transient receptor potential melastatin 2 (TRPM2) and N-methyl-D-aspartate receptor (NMDAR) signaling pathways. However, the mechanism by which Floralozone regulates TRPM2 and NMDAR to improve VD remains unclear. AMP-activated protein kinase (AMPK) is an energy regulator in vivo; however, its role of AMPK activation in stroke remains controversial. MiR-7a-5p has been identified to be closely related to neuronal function. PURPOSE: To explore whether Floralozone can regulate the miR-7a-5p level in vivo through AMPKα2 activation, affect the TRPM2 and NR2B expression levels, and improve VD symptoms. METHODS: The VD model was established by a modified bilateral occlusion of the common carotid arteries (2-VO) of Sprague-Dawley (SD) rats and AMPKα2 KO transgenic (AMPKα2-/-) mice. Primary hippocampal neurons were modeled using oxygen and glucose deprivation (OGD). Morris water maze (MWM) test, hematoxylin-eosin staining (HE staining), and TUNEL staining were used to investigate the effects of Floralozone on behavior and hippocampal morphology in rats. Minichromosome maintenance complex component 2(MCM2) positive cells were used to investigate the effect of Floralozone on neurogenesis. Immunofluorescence staining, qRT-PCR, and western blot analysis were used to investigate the effect of Floralozone on the expression levels of AMPKα2, miR-7a-5p, TRPM2, and NR2B. RESULTS: The SD rat experiment revealed that Floralozone improved spatial learning and memory, improved the morphology and structure of hippocampal neurons, reduced apoptosis of hippocampal neurons and promoted neurogenesis in VD rats. Floralozone could increase the miR-7a-5p expression level, activate AMPKα2 and NR2B expressions, and inhibit TRPM2 expression in hippocampal neurons of VD rats. The AMPKα2 KO transgenic (AMPKα2-/-) mice experiment demonstrated that Floralozone could regulate miR-7a-5p, TRPM2, and NR2B expression levels through AMPKα2 activation. The cell experiment revealed that the TRPM2 and NR2B expression levels were regulated by miR-7a-5p, whereas the AMPKα2 expression level was not. CONCLUSION: Floralozone could regulate miR-7a-5p expression level by activating the protein expression of AMPKα2, control the protein expression of TRPM2 and NR2B, improve the morphology and structure of hippocampus neurons, reduce the apoptosis of hippocampus neurons, promote neurogenesis and improve the cognitive dysfunction.

Suppression of presynaptic corticostriatal glutamate activity attenuates L-dopa-induced dyskinesia in 6-OHDA-lesioned Parkinson's disease mice.

A common adverse effect of Parkinson's disease (PD) treatment is L-dopa-induced dyskinesia (LID). This condition results from both dopamine (DA)-dependent and DA-independent mechanisms, as glutamate inputs from corticostriatal projection neurons impact DA-responsive medium spiny neurons in the striatum to cause the dyskinetic behaviors. In this study, we explored whether suppression of presynaptic corticostriatal glutamate inputs might affect the behavioral and biochemical outcomes associated with LID. We first established an animal model in which 6-hydroxydopamine (6-OHDA)-lesioned mice were treated daily with L-dopa (10 mg/kg, i.p.) for 2 weeks; these mice developed stereotypical abnormal involuntary movements (AIMs). When the mice were pretreated with the NMDA antagonist, amantadine, we observed suppression of AIMs and reductions of phosphorylated ERK1/2 and NR2B in the striatum. We then took an optogenetic approach to manipulate glutamatergic activity. Slc17a6 (vGluT2)-Cre mice were injected with pAAV5-Ef1a-DIO-eNpHR3.0-mCherry and received optic fiber implants in either the M1 motor cortex or dorsolateral striatum. Optogenetic inactivation at either optic fiber implant location could successfully reduce the intensity of AIMs after 6-OHDA lesioning and L-dopa treatment. Both optical manipulation strategies also suppressed phospho-ERK1/2 and phospho-NR2B signals in the striatum. Finally, we performed intrastriatal injections of LDN 212320 in the dyskenesic mice to enhance expression of glutamate uptake transporter GLT-1. Sixteen hours after the LDN 212320 treatment, L-dopa-induced AIMs were reduced along with the levels of striatal phospho-ERK1/2 and phospho-NR2B. Together, our results affirm a critical role of corticostriatal glutamate neurons in LID and strongly suggest that diminishing synaptic glutamate, either by suppression of neuronal activity or by upregulation of GLT-1, could be an effective approach for managing LID.

Single exposure to anesthesia/surgery in neonatal mice induces cognitive impairment in young adult mice.

BACKGROUND: The effects of a solitary neonatal exposure to anesthesia plus surgery (anesthesia/surgery) on cognitive function and the underlying mechanism in developing brains remains largely undetermined. We, therefore, set out to investigate the impact of single exposure to anesthesia/surgery in neonatal mice. METHODS: Six-day-old male and female mice received abdominal surgery under 3% sevoflurane plus 50% oxygen for 2 h. The new object recognition (NOR) and Morris water maze (MWM) were used to evaluate cognitive function in young adult mice. Western blot, ELISA and RT-PCR were used to measure levels of NR2B and IL-6 in medial prefrontal cortex and IL-6 in blood of the mice. We employed NR2B siRNA and IL-6 antibody in the interaction studies. RESULTS: The anesthesia/surgery decreased the ratio of novel time to novel plus familiar time in NOR and the number of platform crossings, but not escape latency, in MWM compared to sham condition. The mice in anesthesia/surgery group had increased NR2B expression in medial prefrontal cortex, and IL-6 amounts in blood and medial prefrontal cortex. Local injection of NR2B siRNA in medial prefrontal cortex alleviated the anesthesia/surgery-induced cognitive impairment. IL-6 antibody mitigated the anesthesia/surgery-induced upregulation of NR2B and cognitive impairment in young adult mice. CONCLUSIONS: These results suggest that a single neonatal exposure to anesthesia/surgery causes impairment of memory, but not learning, in young adult mice through IL-6-regulated increases in NR2B concentrations in medial prefrontal cortex, highlighting the need for further research on the underlying mechanisms of anesthesia/surgery's impact on cognitive function in developing brains.

Ketamine ameliorates activity-based anorexia of adolescent female mice through changes in GluN2B-containing NMDA receptors at postsynaptic cytoplasmic locations of pyramidal neurons and interneurons of medial prefrontal cortex.

Anorexia nervosa (AN) is a mental illness with high rates of mortality and relapse, and no approved pharmacotherapy. Using the activity-based anorexia (ABA) model of AN, we previously showed that a single sub-anesthetic intraperitoneal injection of ketamine (30 mg/kg-KET, but not 3 mg/kg-KET), has an immediate and long-lasting effect of reducing anorexia-like behavior among adolescent female mice. We also showed previously that excitatory outflow from medial prefrontal cortex (mPFC) engages hunger-evoked hyperactivity, leading to the ABA condition of severe weight loss. Ketamine is known to target GluN2B-containing NMDARs (NR2B). Might synaptic plasticity involving NR2B in mPFC contribute to ketamine's ameliorative effects? We addressed this question through electron microscopic immunocytochemical quantification of GluN2B at excitatory synapses of pyramidal neurons (PN) and GABAergic interneurons (IN) in mPFC layer 1 of animals that underwent recovery from a second ABA induction (ABA2), 22 days after ketamine injection during the first ABA induction. The 30 mg/kg-KET evoked synaptic plasticity that differed for PN and IN, with changes revolving the cytoplasmic reserve pool of NR2B more than the postsynaptic membrane pool. Those individuals that suppressed hunger-evoked wheel running the most and increased food consumption during recovery from ABA2 the most showed the greatest increase of NR2B at PN and IN excitatory synapses. We hypothesize that 30 mg/kg-KET promotes long-lasting changes in the reserve cytoplasmic pool of NR2B that enables activity-dependent rapid strengthening of mPFC circuits underlying the more adaptive behavior of suppressed running and enhanced food consumption, in turn supporting better weight restoration.

PSD-95 inhibitor Tat-NR2B9c (NA-1) protects the integrity of the blood-brain barrier after transient middle artery occlusion in rats by downregulating matrix metalloprotease-9 and upregulating endothelial nitric oxide synthase.

BACKGROUND: Protection against ischemic stroke may be most effective when multiple components of the neurovascular unit are protected, yet current treatments target mainly neurons. Here we explored whether the PSD-95 inhibitor Tat-NR2B9c (NA-1) can protect not only neurons but also the blood-brain barrier. METHODS: Adult male Sprague-Dawley rats were randomly divided into three groups, which were subjected to either sham surgery or transient cerebral ischemia-reperfusion, after which some animals were treated with Tat-NR2B9c. The therapeutic efficacy of Tat-NR2B9c was assessed in terms of the degree of neurological deficit and cerebral infarction, integrity of the blood-brain barrier, cerebral water content, as well as expression of PSD-95, nitric oxide synthase, and matrix metalloprotease-9. RESULTS: Tat-NR2B9c (NA-1) ameliorated neurofunctional deficit, reduced cerebral infarction, mitigated blood-brain barrier injury and improved its integrity following ischemia-reperfusion, leading to less cerebral edema. These improvements were associated with upregulation of tight junction proteins in the blood-brain barrier. At the same time, Tat-NR2B9c (NA-1) downregulated neuronal nitric oxide synthase and matrix metalloprotease-9, while reversing the ischemia-induced downregulation of endothelial nitric oxide synthase in brain. We report here the first evidence that PSD-95 is expressed in vascular endothelial cells in the brain. CONCLUSION: Our experiments in a rat model of transient occlusion of the middle cerebral artery suggest that Tat-NR2B9c (NA-1) can mitigate ischemic injury to the blood-brain barrier, and that it may do so by downregulating matrix metalloprotease-9 and upregulating endothelial nitric oxide synthase.

Engineered exosomes mediated targeted delivery of neuroprotective peptide NR2B9c for the treatment of traumatic brain injury.

Neuroprotection is one of the core treatment strategies for brain injuries including traumatic brain injury (TBI). NR2B9c is a promising neuroprotective peptide but its clinical translation is limited because of poor brain penetrability. Exosomes are naturally occurring nanovesicles having therapeutic potential for TBI as well as an efficient drug delivery carrier to the brain. Here, we engineered exosomes with neuron targeting peptide rabies virus glycoprotein (RVG29) via bio-orthogonal click chemistry technique and loaded it with NR2B9c, developing RVG-ExoNR2B9c. RVG29 conjugated exosome had higher neuron targeting efficiency compared to naïve exosomes both in vivo and in vitro. RVG-ExoNR2B9c had great cytoprotective effect against oxygen glucose deprived Neuro2a cells. Intravenous administration of RVG-ExoNR2B9c significantly improved behavioral outcomes and reduced the lesion volume after TBI injury in a mice controlled cortical impact model. Due to their multifunctionality and significant efficacy, we anticipate that RVG-ExoNR2B9c have the potential to be translated both as therapeutic agent as well as cargo delivery system to the brain for the treatment of TBI.

Bergenin ameliorates chemotherapy-induced neuropathic pain in rats by modulating TRPA1/TRPV1/NR2B signalling.

Chemotherapy-induced neuropathic pain (CINP) is one of the most prominent and incapacitating complication associated with chemotherapeutic regimens. The exact mechanisms underlying CINP are not fully understood yet, which hampers the development of effective therapeutics. The current study has been designed to investigate the effect of bergenin on CINP and dissect the underlying cellular and molecular mechanisms. Behavioural responsiveness assays were conducted in rats before and after CINP induction and at different time points post-bergenin treatment. We also measured alterations in tight junction proteins, pro-inflammatory cytokines, microglia activity, transient receptor potential (TRP) channels (TRPV1, TRPA1 and TRPM8) and N-methyl-D-aspartate receptor subtype 2 (NR2B) in dorsal root ganglion (DRG) and spinal tissues of neuropathic rats. Bergenin treatment leads to a significant and dose-dependent reduction in evoked and spontaneous ongoing pain without causing central side effects in neuropathic rats. Furthermore, treatment with bergenin and gabapentin did not affect the baseline pain threshold in healthy, non-chemotherapy-treated rats, as evaluated through tail-flick and tail-clip assays. Chemotherapy administration leads to a significant activation of TRP channels, concurrent with microglial activation, disruption of spinal cord tight junction proteins, and subsequent infiltration of pro-inflammatory cytokines, as well as NR2B activation. Notably, bergenin treatment effectively reversed all of these alterations, with the exception of TRPM8, in both the DRG and spinal cord of neuropathic rats. Findings from the present study suggests that bergenin mitigates neuropathic pain by modulating the TRPA1/TRPV1/NR2B signalling and presents a promising therapeutic avenue for the treatment of chemotherapy-induced neuropathic pain.

The NR2B-targeted intervention alleviates the neuronal injuries at the sub-acute stage of cerebral ischemia: an exploration of stage-dependent strategy against ischemic insults.

Stroke is reported to be the second leading cause of death worldwide, among which ischemic stroke has fourfold greater incidence than intracerebral hemorrhage. Excitotoxicity induced by NMDAR plays a central role in ischemic stroke-induced neuronal death. However, intervention targeted NMDARs against ischemic stroke has failed, which may result from the complex composition of NMDARs and the dynamic changes of their subunits. In this current study, the levels of NR1, NR2A and NR2B subunits of NMDARs were observed upon different time points during the reperfusion after 1 h ischemia with the western blot assay. It was found that the changes of NR1 subunit were only detected after ischemia 1 h/reperfusion 1 day (1 d). While, the changes of NR2A and NR2B subunits may last to ischemia 1 h/reperfusion 7 day(7 d), indicating that NR2subunits may be a potential target for ischemia-reperfusion injuries at the sub-acute stage of ischemic stroke. Simultaneously, mitochondrial injuries in neurons were investigated with transmission electron microscopy (TEM), and mitochondrial dysfunction was evaluated with mitochondrial membrane proteins oxidative respiratory chain complex and OCR. When the antagonist of NMDARs was used before ischemic exposure, the neuronal mitochondrial dysfunction was alleviated, suggesting that these aberrant deviations of NMDARs from basal levels led to mitochondrial dysfunction. Furthermore, when the antagonist of NR2B was administrated intracerebroventricularly at the sub-acute cerebral ischemia, the volume of cerebral infarct region was decreased and the neural functions were improved. To sum up, the ratio of NR2B-containing NMDARs is vital for mitochondrial homeostasis and then neuronal survival. NR2B-targeted intervention should be chosen at the sub-acute stage of cerebral ischemia.

Bio-clickable, small extracellular vesicles-COCKTAIL therapy for ischemic stroke.

Delivering large therapeutic molecules via the blood-brain barrier to treat ischemic stroke remains challenging. NR2B9c is a potent neuroprotective peptide but it's safe and targeted delivery to the brain requires an efficient, natural, and non-immunogenic delivery technique. Small extracellular vesicles (sEVs) have shown great potential as a non-immunogenic, natural cargo delivery system; however, tailoring of its inefficient brain targeting is desired. Here, we coupled rabies virus glycoprotein 29 with sEVs surface via bio-orthogonal click chemistry reactions, followed by loading of NR2B9c, ultimately generating stroke-specific therapeutic COCKTAIL (sEVs-COCKTAIL). Primary neurons and Neuro-2a cells were cultured for in vitro and transient middle cerebral artery occlusion model was used for in vivo studies to evaluate neuron targeting and anti-ischemic stroke potential of the sEVs-COCKTAIL. Bio-clickable sEVs were selectively taken up by neurons but not glial cells. In the in vitro ischemic stroke model of oxygen-glucose deprivation, the sEVs-COCKTAIL exhibited remarkable potential against reactive oxygen species and cellular apoptosis. In vivo studies further demonstrated the brain targeting and increased half-life of bio-clickable sEVs, delivering NR2B9c to the ischemic brain and reducing stroke injury. Treatment with the sEVs-COCKTAIL significantly increased behavioral recovery and reduced neuronal apoptosis after transient middle cerebral artery occlusion. NR2B9c was delivered to neurons binding to post-synaptic density protein-95, inhibiting N-methyl-d-Aspartate receptor-mediated over production of oxidative stress and mitigating protein B-cell lymphoma 2 and P38 proteins expression. Our results provide an efficient and biocompatible approach to a targeted delivery system, which is a promising modality for stroke therapy.

The beneficial effects of vortioxetine on BDNF, CREB, S100B, ß amyloid, and glutamate NR2b receptors in chronic unpredictable mild stress model of depression.

BACKGROUND: Depression, one of the most significant mental disorders, is still poorly understood in terms of its pathogenetic mechanisms despite its well-recognized association with stress. OBJECTIVES: The current study's goal was to ascertain how the novel antidepressant drug vortioxetine (VOR) affected the BDNF (brain-derived neurotrophic factor), S100, amyloid ß (Aß), CREB (cAMP response element-binding protein), and NR2B, as well as its impact on depression-like behaviors, and tissue damage in an experimental rodent model of depression caused by chronic unpredictable stress. METHODS: We employed twenty-eight Wistar albino male rats, and we randomly divided them into four groups, each consisting of 7 rats: control, CUMS (chronic unpredictable mild stress), CUMS+vortioxetine (CUMS+VOR), and CUMS+fluoxetine (CUMS+FLU). Sucrose preference and forced swimming tests (SPT and FST, respectively), PCR, ELISA, and histopathological and immunohistochemical evaluation were made on brains. RESULTS: The behaviors of reduced immobility in the FST and increased sucrose preference were observed in the CUMS group and they improved in the groups treated with VOR and FLU. Compared with the control group, the group exposed to CUMS showed increased Aß and decreased BDNF, CREB, and S-100 expressions, as well as neuronal degeneration (p<0.001). VOR and FLU treatment ameliorate the findings. CONCLUSIONS: This study demonstrated significant ameliorative effects of VOR in an experimental model of chronic unpredictable depression to reduce brain tissue damage and depression-like behaviors in rats. Effects of CUMS on the brain and possible effects of VOR.

Activation of the N-methyl-D-aspartate receptor and calcium/calmodulin-dependent protein kinase IIα signal in the rostral anterior cingulate cortex is involved in pain-related aversion in rats with peripheral nerve injury.

The rostral anterior cingulate cortex (rACC) of rat brain is associated with pain-related emotions. However, the underlying molecular mechanism remains unclear. Here, we investigated the effects of the N-methyl-D-aspartate (NMDA) receptor and Ca2+/Calmodulin-dependent protein kinase type II (CaMKII)α signal on pain-related aversion in the rACC of a rat model of neuropathic pain (NP). Mechanical and thermal hyperalgesia were examined using von Frey and hot plate tests in a rat model of NP induced by spared nerve injury (SNI) of the unilateral sciatic nerve. Bilateral rACC pretreatment with the CaMKII inhibitor tat-CN21 (derived from the cell-penetrating tat sequence and CaM-KIIN amino acids 43-63) or tat-Ctrl (the tat sequence and the scrambled sequence of CN21) was performed on postoperative days 29-35 in Sham rats or rats with SNI. Spatial memory performance was tested using an eight-arm radial maze on postoperative days 34-35. Pain-related negative emotions (aversions) were evaluated using the place escape/avoidance paradigm on postoperative day 35 following the spatial memory performance test. The percentage of time spent in the light area was used to assess pain-related negative emotions (i.e., aversion). The expression levels of the NMDA receptor GluN2B subunit, CaMKIIα, and CaMKII-Threonine at position 286 (Thr286) phosphorylation in contralateral rACC specimens were detected by Western blot or real time PCR following the aversion test. Our data showed that pretreatment of the rACC with tat-CN21 increased determinate behavior but did not alter hyperalgesia or spatial memory performance in rats with SNI. In addition, tat-CN21 reversed the enhanced CaMKII-Thr286 phosphorylation and had no effect on the upregulated expression of GluN2B, CaMKIIα protein, and mRNA. Our data suggested that activation of the NMDA receptor-CaMKIIα signal in rACC is associated with pain-related aversion in rats with NP. These data may provide a new approach for the development of drugs that modulate cognitive and emotional pain aspects.

Dysfunction of inhibitory interneurons contributes to synaptic plasticity via GABABR-pNR2B signaling in a chronic migraine rat model.

Background: According to our previous study, the loss of inhibitory interneuron function contributes to central sensitization in chronic migraine (CM). Synaptic plasticity is a vital basis for the occurrence of central sensitization. However, whether the decline in interneuron-mediated inhibition promotes central sensitization by regulating synaptic plasticity in CM remains unclear. Therefore, this study aims to explore the role of interneuron-mediated inhibition in the development of synaptic plasticity in CM. Methods: A CM model was established in rats by repeated dural infusion of inflammatory soup (IS) for 7 days, and the function of inhibitory interneurons was then evaluated. After intraventricular injection of baclofen [a gamma-aminobutyric acid type B receptor (GABABR) agonist] or H89 [a protein kinase A (PKA) inhibitor), behavioral tests were performed. The changes in synaptic plasticity were investigated by determining the levels of the synapse-associated proteins postsynaptic density protein 95 (PSD95), synaptophysin (Syp) and synaptophysin-1(Syt-1)]; evaluating the synaptic ultrastructure by transmission electron microscopy (TEM); and determining the density of synaptic spines via Golgi-Cox staining. Central sensitization was evaluated by measuring calcitonin gene-related peptide (CGRP), brain-derived neurotrophic factor (BDNF), c-Fos and substance P (SP) levels. Finally, the PKA/Fyn kinase (Fyn)/tyrosine-phosphorylated NR2B (pNR2B) pathway and downstream calcium-calmodulin-dependent kinase II (CaMKII)/c-AMP-responsive element binding protein (pCREB) signaling were assessed. Results: We observed dysfunction of inhibitory interneurons, and found that activation of GABABR ameliorated CM-induced hyperalgesia, repressed the CM-evoked elevation of synapse-associated protein levels and enhancement of synaptic transmission, alleviated the CM-triggered increases in the levels of central sensitization-related proteins, and inhibited CaMKII/pCREB signaling via the PKA/Fyn/pNR2B pathway. The inhibition of PKA suppressed the CM-induced activation of Fyn/pNR2B signaling. Conclusion: These data reveal that the dysfunction of inhibitory interneurons contributes to central sensitization by regulating synaptic plasticity through the GABABR/PKA/Fyn/pNR2B pathway in the periaqueductal gray (PAG) of CM rats. Blockade of GABABR-pNR2B signaling might have a positive influence on the effects of CM therapy by modulating synaptic plasticity in central sensitization.

GluN2B-containing NMDARs in the mammalian brain: pharmacology, physiology, and pathology.

Glutamate N-methyl-D-aspartate receptor (NMDAR) is critical for promoting physiological synaptic plasticity and neuronal viability. As a major subpopulation of the NMDAR, the GluN2B subunit-containing NMDARs have distinct pharmacological properties, physiological functions, and pathological relevance to neurological diseases compared with other NMDAR subtypes. In mature neurons, GluN2B-containing NMDARs are likely expressed as both diheteromeric and triheteromeric receptors, though the functional importance of each subpopulation has yet to be disentangled. Moreover, the C-terminal region of the GluN2B subunit forms structural complexes with multiple intracellular signaling proteins. These protein complexes play critical roles in both activity-dependent synaptic plasticity and neuronal survival and death signaling, thus serving as the molecular substrates underlying multiple physiological functions. Accordingly, dysregulation of GluN2B-containing NMDARs and/or their downstream signaling pathways has been implicated in neurological diseases, and various strategies to reverse these deficits have been investigated. In this article, we provide an overview of GluN2B-containing NMDAR pharmacology and its key physiological functions, highlighting the importance of this receptor subtype during both health and disease states.

Cerebrolysin reduces excitotoxicity by modulation of cell-death proteins in delayed hours of ischemic reperfusion injury.

Recent preclinical and clinical reports suggest that cerebrolysin shows neuroprotective properties similar to endogenous neurotrophic factors in neurodegenerative disorders including ischemic stroke. However, little is known about its underlying antiexcitotoxic action. Adult male Wistar rats were intraperitoneally treated with cerebrolysin (0.15 or 0.30 mg/kg) or vehicle at 3, 6 and 12 h after ischemic reperfusion and were assessed 24 h after reperfusion in ischemic rats. We added cerebrolysin (2.5 or 5 mg/ml) or vehicle in primary cortical culture cells at 3, 6 and 12 h of post-glutamate exposure and performed cell viability assays at 24 h. Our in-vivo and in-vitro findings showed that cerebrolysin substantially reduced neuronal cell death in delayed hours of post ischemic- and glutamate-insult conditions respectively. Further, we have assessed the influence of NR-2 A/-2B receptor antagonism on neuroprotective action of cerebrolysin at 6 h in in-vivo as well as in-vitro conditions. Neuroprotective effect of cerebrolysin at 6 h of reperfusion was enhanced by pretreatment of NR2B antagonist RO25-6981.We found that cerebrolysin restrained upregulation of extrasynaptic NR2B responsible for triggering apoptotic pathways. Cerebrolysin reduced expression of important cell death proteins such as, JNK, PTEN, Calpain and Caspase-3 components. Importantly, we also found that cerebrolysin reduced SREBP1 expression, which gets activated only after 6 h of ischemia. These results demonstrate that cerebrolysin reduces excitotoxicity and protect neuronal cells in delayed hours of ischemic reperfusion injuries by decreasing cell death proteins.

The L-arginine-NO-cGMP pathway mediates the locomotor activity alterations during the expression of sensitization to mephedrone in mice.

Mephedrone is a representative of synthetic cathinones that is known from its rewarding and psychostimulant effects. It exerts behavioural sensitization after repeated and then interrupted administration. In our study, we investigated a role of the L-arginine-NO-cGMP-dependent signalling in the expression of sensitization to hyperlocomotion evoked by mephedrone. The study was carried out in male albino Swiss mice. The tested mice received mephedrone (2.5 mg/kg) for 5 consecutive days and on the 20th day of the experiment (the 'challenge' day) animals received both mephedrone (2.5 mg/kg) and a given substance that affects the L-arginine-NO-cGMP signalling, that is, L-arginine hydrochloride (125 or 250 mg/kg), 7-nitroindazole (10 or 20 mg/kg), L-NAME (25 or 50 mg/kg) or methylene blue (5 or 10 mg/kg). We observed that 7-nitroindazole, L-NAME and methylene blue inhibited the expression of sensitization to the mephedrone-induced hyperlocomotion. Moreover, we demonstrated that the mephedrone-induced sensitization is accompanied by lowered levels of D1 receptors and NR2B subunits in the hippocampus, whereas a concurrent administration of L-arginine hydrochloride, 7-nitroindazole and L-NAME with the mephedrone challenge dose reversed these effects. Methylene blue only reversed the mephedrone-induced effects on hippocampal levels of the NR2B subunit. Our study confirms that the L-arginine-NO-cGMP pathway contributes to mechanisms underlying the expression of sensitization to the mephedrone-evoked hyperlocomotion.

Activation of CB1R alleviates central sensitization by regulating HCN2-pNR2B signaling in a chronic migraine rat model.

BACKGROUND: Central sensitization has been widely accepted as an underlying pathophysiological mechanism of chronic migraine (CM), activation of cannabinoid type-1 receptor (CB1R) exerts antinociceptive effects by relieving central sensitization in many pain models. However, the role of CB1R in the central sensitization of CM is still unclear. METHODS: A CM model was established by infusing inflammatory soup (IS) into the dura of male Wistar rats for 7 days, and hyperalgesia was assessed by the mechanical and thermal thresholds. In the periaqueductal gray (PAG), the mRNA and protein levels of CB1R and hyperpolarization-activated cyclic nucleotide-gated cation channel 2 (HCN2) were measured by qRT-PCR and western blotting. After intraventricular injection of Noladin ether (NE) (a CB1R agonist), ZD 7288 (an HCN2 blocker), and AM 251 (a CB1R antagonist), the expression of tyrosine phosphorylation of N-methyl-D-aspartate receptor subtype 2B (pNR2B), calcium-calmodulin-dependent kinase II (CaMKII), and phosphorylated cAMP-responsive element binding protein (pCREB) was detected, and central sensitization was evaluated by the expression of calcitonin gene-related peptide (CGRP), c-Fos, and substance P (SP). Synaptic-associated protein (postsynaptic density protein 95 (PSD95) and synaptophysin (Syp)) and synaptic ultrastructure were detected to explore synaptic plasticity in central sensitization. RESULTS: We observed that the mRNA and protein levels of CB1R and HCN2 were both significantly increased in the PAG of CM rats. The application of NE or ZD 7288 ameliorated IS-induced hyperalgesia; repressed the pNR2B/CaMKII/pCREB pathway; reduced CGRP, c-Fos, SP, PSD95, and Syp expression; and inhibited synaptic transmission. Strikingly, the application of ZD 7288 relieved AM 251-evoked elevation of pNR2B, CGRP, and c-Fos expression. CONCLUSIONS: These data reveal that activation of CB1R alleviates central sensitization by regulating HCN2-pNR2B signaling in CM rats. The activation of CB1R might have a positive influence on the prevention of CM by mitigating central sensitization.

Candidate 3-benzazepine-1-ol type GluN2B receptor radioligands (11C-NR2B-Me enantiomers) have high binding in cerebellum but not to σ1 receptors.

INTRODUCTION: We recently reported 11C-NR2B-SMe ([S-methyl-11C](R,S)-7-thiomethoxy-3-(4-(4-methyl-phenyl)butyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ol) and its enantiomers as candidate radioligands for imaging the GluN2B subunit within rat N-methyl-D-aspartate receptors. However, these radioligands gave unexpectedly high and displaceable binding in rat cerebellum, possibly due to cross-reactivity with sigma-1 (σ1) receptors. This study investigated 11C-labeled enantiomers of a close analogue (7-methoxy-3-(4-(p-tolyl)butyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ol; NR2B-Me) of 11C-NR2B-SMe as new candidate GluN2B radioligands. PET was used to evaluate these radioligands in rats and to assess potential cross-reactivity to σ1 receptors. METHODS: NR2B-Me was assayed for binding affinity and selectivity to GluN2B in vitro. 11C-NR2B-Me and its enantiomers were prepared by Pd-mediated treatment of boronic ester precursors with 11C-iodomethane. Brain PET scans were conducted after radioligand intravenous injection into rats. Various ligands for GluN2B receptors or σ1 receptors were administered at set doses in pre-blocking or displacement experiments to assess their impact on imaging data. 18F-FTC146 and enantiomers of 11C-NR2B-SMe were used for comparison. Radiometabolites from brain and plasma were measured ex vivo and in vitro. RESULTS: NR2B-Me enantiomers showed high GluN2B affinity and selectivity in vitro. 11C-NR2B-Me enantiomers gave high early whole rat brain uptake of radioactivity, including high uptake in cerebellum, followed by slower decline. Radioactivity in brain at 30 min ex vivo was virtually all unchanged radioligand. Only less lipophilic radiometabolites appeared in plasma. When 11C-(R)-NR2B-Me was used, three high-affinity GluN2B ligands-NR2B-SMe, Ro25-6981, and CO101,244-showed increasing pre-block of whole brain radioactivity retention with increasing dose. Two σ1 receptor antagonists, FTC146 and BD1407, were ineffective pre-blocking agents. Together, these results strongly resemble those obtained with 11C-NR2B-SMe enantiomers, except that 11C-NR2B-Me enantiomers showed faster reversibility of binding. When 18F-FTC146 was used as a radioligand, FTC146 and BD1407 showed strong pre-blocking effects whereas GluN2B ligands showed only weak blocking effects. CONCLUSION: 11C-NR2B-Me enantiomers showed specific binding to GluN2B receptors in rat brain in vivo. High unexpected specific binding in cerebellum was not due to σ1 receptors. Additional investigation is needed to identify the source of the high specific binding.