Paired associative stimulation improves synaptic plasticity and functional outcomes after cerebral ischemia.
ABSTRACT: Paired associative stimulation is a relatively new non-invasive brain stimulation technique that combines transcranial magnetic stimulation and peripheral nerve stimulation. The effects of paired associative stimulation on the excitability of the cerebral cortex can vary according to the time interval between the transcranial magnetic stimulation and peripheral nerve stimulation. We established a model of cerebral ischemia in rats via transient middle cerebral artery occlusion. We administered paired associative stimulation with a frequency of 0.05 Hz 90 times over 4 weeks. We then evaluated spatial learning and memory using the Morris water maze. Changes in the cerebral ultra-structure and synaptic plasticity were assessed via transmission electron microscopy and a 64-channel multi-electrode array. We measured mRNA and protein expression levels of brain-derived neurotrophic factor and N-methyl-D-aspartate receptor 1 in the hippocampus using a real-time polymerase chain reaction and western blot assay. Paired associative stimulation treatment significantly improved learning and memory in rats subjected to cerebral ischemia. The ultra-structures of synapses in the CA1 area of the hippocampus in rats subjected to cerebral ischemia were restored by paired associative stimulation. Long-term potentiation at synapses in the CA3 and CA1 regions of the hippocampus was enhanced as well. The protein and mRNA expression of brain-derived neurotrophic factor and N-methyl-D-aspartate receptor 1 increased after paired associative stimulation treatment. These data indicate that paired associative stimulation can protect cognition after cerebral ischemia. The observed effect may be mediated by increases in the mRNA and protein expression of brain-derived neurotrophic factor and N-methyl-D-aspartate receptor 1, and by enhanced synaptic plasticity in the CA1 area of the hippocampus. The animal experiments were approved by the Animal Ethics Committee of Tongji Medical College, Huazhong University of Science & Technology, China (approval No. TJ-A20151102) on July 11, 2015.
Project description:Background and Purpose- Glutamate NMDARs (N-methyl-D-aspartate receptors) play a major role in the initiation of ischemic brain damage. However, NMDAR antagonists have no protective effects in stroke patients, possibly because they impair physiological functions of NMDARs. ?2?-1 (encoded by Cacna2d1) is strongly expressed in many brain regions. We determined the contribution of ?2?-1 to NMDAR hyperactivity and brain injury induced by ischemia and reperfusion. Methods- Mice were subjected to 90 minutes of middle cerebral artery occlusion followed by 24 hours of reperfusion. Neurological deficits, brain infarct volumes, and calpain/caspase-3 activity in brain tissues were measured. NMDAR activity of hippocampal CA1 neurons was measured in an in vitro ischemic model. Results- Middle cerebral artery occlusion increased ?2?-1 protein glycosylation in the cerebral cortex, hippocampus, and striatum. Coimmunoprecipitation showed that ischemia rapidly enhanced the ?2?-1-NMDAR physical interaction in the mouse brain tissue. Inhibiting ?2?-1 with gabapentin, uncoupling the ?2?-1-NMDAR interaction with an ?2?-1 C terminus-interfering peptide, or genetically ablating Cacna2d1 had no effect on basal NMDAR currents but strikingly abolished oxygen-glucose deprivation-induced NMDAR hyperactivity in hippocampal CA1 neurons. Systemic treatment with gabapentin or ?2?-1 C-terminus-interfering peptide or Cacna2d1 genetic knock-out reduced middle cerebral artery occlusion-induced infarct volumes, neurological deficit scores, and calpain/caspase-3 activation in brain tissues. Conclusions- ?2?-1 is essential for brain ischemia-induced neuronal NMDAR hyperactivity, and ?2?-1-bound NMDARs mediate brain damage caused by cerebral ischemia. Targeting ?2?-1-bound NMDARs, without impairing physiological ?2?-1-free NMDARs, may be a promising strategy for treating ischemic stroke.
Project description:In mouse hippocampal CA1 pyramidal neurons, the activity of synaptic small-conductance Ca(2+)-activated K(+) channels type 2 (SK2 channels) provides a negative feedback on N-methyl-D-aspartate receptors (NMDARs), reestablishing Mg(2+) block that reduces Ca(2+) influx. The well-established role of NMDARs in ischemia-induced excitotoxicity led us to test the neuroprotective effect of modulating SK2 channel activity following cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Administration of the SK channel positive modulator, 1-ethyl-benzimidazolinone (1-EBIO), significantly reduced CA1 neuron cell death and improved CA/CPR-induced cognitive outcome. Electrophysiological recordings showed that CA/CPR-induced ischemia caused delayed and sustained reduction of synaptic SK channel activity, and immunoelectron microscopy showed that this is associated with internalization of synaptic SK2 channels, which was prevented by 1-EBIO treatment. These results suggest that increasing SK2 channel activity, or preventing ischemia-induced loss of synaptic SK2 channels, are promising and novel approaches to neuroprotection following cerebral ischemia.
Project description:Introduction:Nucleobinding-2 (NUCB2) or nesfatin-1, a newly identified anorexigenic peptide, has antioxidant, anti-inflammatory, and anti-apoptotic properties. Brain ischemiareperfusion induces irreversible damages, especially in the hippocampus area. However, the therapeutic effects of NUCB2 have not been well investigated in cerebral ischemia. This study was designed for the first time to investigate the protective effects of NUCB2/Nesfatin-1 on the expression of apoptosis-related proteins and reactive astrogliosis level in the CA1 area of hippocampus in an experimental model of transient global cerebral ischemia. Methods:The male Wistar rats were randomly allocated into 4 groups (sham, NUCB2, ischemia-reperfusion, and ischemia-reperfusion+NUCB21) (n =7). The model of cerebral ischemia was prepared by common carotid arteries occlusion for 20 minutes. Nesfatin-1 (20 ?g/kg) and saline (as a vehicle) were injected (intraperitoneally) at the beginning of the reperfusion period. The assessment of the protein expression levels was performed by immunofluorescence and immunohistochemical staining. Results:NUCB2 significantly reduced the Bax and GFAP protein levels in the CA1 area after ischemia (P<0.05). Also, NUCB2 increased Bcl-2 protein level (P<0.05). NUCB2 exerted protective effects against ischemic injury by the inhibition of astrocytes activation as an inflammatory response and decreased neuronal cell apoptosis. Conclusion:The present study provides the possible neuroprotective view of nesfatin-1 in the treatment of ischemia injury model in rat hippocampus.
Project description:Abstract Excitotoxicity is thought to play key roles in brain neurodegeneration and stroke. Here we show that neuroprotection against excitotoxicity by trophic factors EFNB1 and brain-derived neurotrophic factor (called here factors) requires de novo formation of ‘survival complexes’ which are factor-stimulated complexes of N-methyl-d-aspartate receptor with factor receptor and presenilin 1. Absence of presenilin 1 reduces the formation of survival complexes and abolishes neuroprotection. EPH receptor B2- and N-methyl-d-aspartate receptor-derived peptides designed to disrupt formation of survival complexes also decrease the factor-stimulated neuroprotection. Strikingly, factor-dependent neuroprotection and levels of the de novo factor-stimulated survival complexes decrease dramatically in neurons expressing presenilin 1 familial Alzheimer disease mutants. Mouse neurons and brains expressing presenilin 1 familial Alzheimer disease mutants contain increased amounts of constitutive presenilin 1–N-methyl-d-aspartate receptor complexes unresponsive to factors. Interestingly, the stability of the familial Alzheimer disease presenilin 1–N-methyl-d-aspartate receptor complexes differs from that of wild type complexes and neurons of mutant-expressing brains are more vulnerable to cerebral ischaemia than neurons of wild type brains. Furthermore, N-methyl-d-aspartate receptor-mediated excitatory post-synaptic currents at CA1 synapses are altered by presenilin 1 familial Alzheimer disease mutants. Importantly, high levels of presenilin 1–N-methyl-d-aspartate receptor complexes are also found in post-mortem brains of Alzheimer disease patients expressing presenilin 1 familial Alzheimer disease mutants. Together, our data identify a novel presenilin 1-dependent neuroprotective mechanism against excitotoxicity and indicate a pathway by which presenilin 1 familial Alzheimer disease mutants decrease factor-depended neuroprotection against excitotoxicity and ischaemia in the absence of Alzheimer disease neuropathological hallmarks which may form downstream of neuronal damage. These findings have implications for the pathogenic effects of familial Alzheimer disease mutants and therapeutic strategies. We describe a presenilin 1-dependent mechanism by which neurotrophic factors protect neurons rom excitotoxicity, a condition with crucial roles in neurodegeneration and stroke. The mechanism includes presenilin1-dependent N-methyl-d-aspartate receptor complexes with factor receptors. Familial Alzheimer disease mutants alter N-methyl-d-aspartate receptor-presenilin1 associations and block factor-dependent neuroprotection in absence of Alzheimer disease hallmarks. Graphical Abstract Graphical Abstract
Project description:Glehnia littoralis, as a traditional herbal medicine to heal various health ailments in East Asia, displays various therapeutic properties including antioxidant effects. However, neuroprotective effects of G. littoralis against cerebral ischemic insults have not yet been addressed. Therefore, in this study, we first examined its neuroprotective effects in the hippocampus using a gerbil model of transient global cerebral ischemia (TGCI).Gerbils were subjected to TGCI for 5 min. G. littoralis extract (GLE; 100 and 200 mg/kg) was administrated orally once daily for 7 days before ischemic surgery. Neuroprotection was examined by neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. Gliosis was observed by immunohistochemistry for glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1. For neuroprotective mechanisms, immunohistochemistry for superoxide dismutase (SOD) 1 and brain-derived neurotrophic factor (BDNF) was done.Pretreatment with 200 mg/kg of GLE protected pyramidal neurons in the cornu ammonis 1 (CA1) area from ischemic insult area (F = 29.770, P < 0.05) and significantly inhibited activations of astrocytes (F = 22.959, P < 0.05) and microglia (F = 44.135, P < 0.05) in the ischemic CA1 area. In addition, pretreatment with GLE significantly increased expressions of SOD1 (F = 28.561, P < 0.05) and BDNF (F = 55.298, P < 0.05) in CA1 pyramidal neurons of the sham- and ischemia-operated groups.Our findings indicate that pretreatment with GLE can protect neurons from ischemic insults, and we suggest that its neuroprotective mechanism may be closely associated with increases of SOD1 and BDNF expressions as well as attenuation of glial activation.
Project description:AIM AND METHODS:Changes in cerebrospinal fluid osmotic pressure modulate brain excitability. Transient receptor potential vanilloid 4 (TRPV4), which is sensitive to hypotonic stimulation, is expressed in hippocampus. The present study investigated the effect of hypotonic stimulation on hippocampal synaptic transmission and the role of TRPV4 in hypotonicity-action using electrophysiological recording and pharmacological technique. RESULTS:Accompanied with the decrease in paired pulse facilitation, field excitatory postsynaptic potential (fEPSP) was enhanced by hypotonicity and TRPV4 agonist 4α-PDD in hippocampal slices, which was sensitive to TRPV4 antagonist HC-067047. Hypotonicity-induced increase in fEPSP was blocked by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, but not N-methyl-d-aspartate receptor or N- or P/Q-type voltage-gated calcium channel antagonist. High voltage-gated calcium current (ICa ) in hippocampal CA3 pyramidal neurons was not affected by hypotonicity. AMPA-activated current (IAMPA ) in hippocampal CA1 pyramidal neurons was increased by hypotonicity and 4α-PDD, which was attenuated by HC-067047. Inhibition of protein kinase C or protein kinase A markedly attenuated hypotonicity-increased IAMPA , whereas antagonism of calcium/calmodulin-dependent protein kinase II had no such effect. CONCLUSION:TRPV4 is involved in hypotonicity-induced enhancement of hippocampal synaptic transmission, which may be mediated through promoting presynaptic glutamate release and increasing postsynaptic AMPA receptor function.
Project description:Previous work has demonstrated that ischemic preconditioning neuroprotection is associated with inhibition of JNK pathway activation. The present study was designed to examine the hypothesis that the suppression of JNK3 activation by preconditioning is mediated by NMDA receptors and crosstalk between ERK1/2 and JNK3. Preconditioning (3 min ischemia) 2 days before global cerebral ischemia (8-min) markedly decreased neuronal degeneration in hippocampus CA1, an effect abolished by pretreatment with the NMDA receptor antagonist, MK-801. Furthermore, preconditioning abolished cerebral ischemia-induced JNK3 activation and enhanced ERK1/2 activation, an effect reversed by MK-801. Due to the inverse relationship between ERK1/2 and JNK3 activation following preconditioning, we hypothesized that ERK1/2 may regulate JNK3 activation following preconditioning. In support of this contention, pretreatment with the MEK inhibitor, PD98059 significantly attenuated preconditioning-induced ERK1/2 phosphorylation, and strongly reversed preconditioning down-regulation of JNK3 phosphorylation. This finding suggests that ERK1/2 signaling is responsible for preconditioning-induced down-regulation of JNK3 activation. Western blot analysis and immunohistochemistry further demonstrated that preconditioning, in an NMDA-dependent manner, enhanced activation of the pro-survival factors, p-CREB and Bcl-2, while attenuating activation of putative pro-death factors, p-c-Jun and Fas-L in the hippocampus CA1. As a whole, the study demonstrates that preconditioning attenuation of pro-death JNK3 in the hippocampus CA1 following global cerebral ischemia is mediated by NMDA receptor-induced crosstalk between ERK1/2 and JNK3. The ERK1/2-mediated reduction of JNK3 activation leads to enhanced pro-survival signaling (P-CREB and Bcl-2 induction) and attenuation of pro-death signaling (p-c-Jun and Fas-L), with subsequent induction of ischemic tolerance.
Project description:Mechanisms for electroacupuncture (EA) in disease treatments are still enigmatic. Here, we studied whether hippocampus was involved in the protection of EA stimulation on myocardial ischemia injury. Acute myocardial ischemia (AMI) model was produced. EA stimulation at heart meridian from Shenmen (HT7) to Tongli (HT5) was applied to rats 3 times a day for continuous three days. Coronary occlusion related tachycardia and hypotension, indicated by heart rate, mean arterial pressure, and rate pressure product, were apparently impaired after AMI injury. By contrast, EA stimulating could ameliorate the impairments of heart function (P < 0.05). Interestingly, lesion of CA1 region of hippocampus abolished the protection of EA. Neuronal activity in CA1 area was affected by AMI. As evidenced, cell counts, cell types, and frequency of the discharged neurons were facilitated after AMI, while EA stimulation attenuated the abnormalities. Furthermore, c-Fos expression was significantly facilitated in CA1 area after AMI, which was reduced by EA stimulation. Correlations were established between c-Fos expression and cell counts of discharged neurons, as well as between heart function and cell counts of discharged neurons. Taken together, EA stimulation at heart meridian protects against heart dysfunction induced by AMI possibly through suppressing the neuronal activity in CA1 region.
Project description:Transient global or forebrain ischemia induced experimentally in animals can cause selective, delayed neuronal death of hippocampal CA1 pyramidal neurons. A striking feature is a delayed rise in intracellular free Zn(2+) in CA1 neurons just before the onset of histologically detectable cell death. Here we show that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) at Schaffer collateral to CA1 synapses in postischemic hippocampus exhibit properties of Ca(2+)/Zn(2+)-permeable, Glu receptor 2 (GluR2)-lacking AMPARs before the rise in Zn(2+) and cell death. At 42 h after ischemia, AMPA excitatory postsynaptic currents exhibited pronounced inward rectification and marked sensitivity to 1-naphthyl acetyl spermine (Naspm), a selective channel blocker of GluR2-lacking AMPARs. In control hippocampus, AMPA excitatory postsynaptic currents were electrically linear and relatively insensitive to Naspm. Naspm injected intrahippocampally at 9-40 h after insult greatly reduced the late rise in intracellular free Zn(2+) in postischemic CA1 neurons and afforded partial protection against ischemia-induced cell death. These results implicate GluR2-lacking AMPA receptors in the ischemia-induced rise in free Zn(2+) and death of CA1 neurons, although a direct action at the time of the rise in Zn(2+) is unproven. This receptor subtype appears to be an important therapeutic target for intervention in ischemia-induced neuronal death in humans.
Project description:Multiple studies have demonstrated that brain-derived neurotrophic factor (BDNF) is a potent modulator of neuronal structure and function in the hippocampus. However, the majority of studies to date have relied on the application of recombinant BDNF. We herein report that endogenous BDNF, released via theta burst stimulation of mossy fibers (MF), elicits a slowly developing cationic current and intracellular Ca(2+) elevations in CA3 pyramidal neurons with the same pharmacological profile of the transient receptor potential canonical 3 (TRPC3)-mediated I(BDNF) activated in CA1 neurons by brief localized applications of recombinant BDNF. Indeed, sensitivity to both the extracellular BDNF scavenger tropomyosin-related kinase B (TrkB)-IgG and small hairpin interference RNA-mediated TRPC3 channel knockdown confirms the identity of this conductance as such, henceforth-denoted MF-I(BDNF). Consistent with such activity-dependent release of BDNF, these MF-I(BDNF) responses were insensitive to manipulations of extracellular Zn(2+) concentration. Brief theta burst stimulation of MFs induced a long-lasting depression in the amplitude of excitatory postsynaptic currents (EPSCs) mediated by both AMPA and N-methyl-d-aspartate (NMDA) receptors without changes in the NMDA receptor/AMPA receptor ratio, suggesting a reduction in neurotransmitter release. This depression of NMDAR-mediated EPSCs required activity-dependent release of endogenous BDNF from MFs and activation of Trk receptors, as it was sensitive to the extracellular BDNF scavenger TrkB-IgG and the tyrosine kinase inhibitor k-252b. These results uncovered the most immediate response to endogenously released--native--BDNF in hippocampal neurons and lend further credence to the relevance of BDNF signaling for synaptic function in the hippocampus.