Increased Sestrin3 Contributes to Post-ischemic Seizures in the Diabetic Condition.

Seizures are among the most common neurological sequelae of stroke, and diabetes notably increases the incidence of post-ischemic seizures. Recent studies have indicated that Sestrin3 (SESN3) is a regulator of a proconvulsant gene network in human epileptic hippocampus. But the association of SESN3 and post-ischemic seizures in diabetes remains unclear. The present study aimed to reveal the involvement of SESN3 in seizures following transient cerebral ischemia in diabetes. Diabetes was induced in adult male mice and rats via intraperitoneal injection of streptozotocin (STZ). Forebrain ischemia (15 min) was induced by bilateral common carotid artery occlusion, the 2-vessel occlusion (2VO) in mice and 4-vessel occlusion (4VO) in rats. Our results showed that 59% of the diabetic wild-type mice developed seizures after ischemia while no seizures were observed in non-diabetic mice. Although no apparent cell death was detected in the hippocampus of seizure mice within 24 h after the ischemic insult, the expression of SESN3 was significantly increased in seizure diabetic mice after ischemia. The post-ischemic seizure incidence significantly decreased in SESN3 knockout mice. Furthermore, all diabetic rats suffered from post-ischemic seizures and non-diabetic rats have no seizures. Electrophysiological recording showed an increased excitatory synaptic transmission and intrinsic membrane excitability in dentate granule cells of the rat hippocampus, together with decreased I A currents and Kv4.2 expression levels. The above results suggest that SESN3 up-regulation may contribute to neuronal hyperexcitability and seizure generation in diabetic animals after ischemia. Further studies are needed to explore the molecular mechanism of SESN3 in seizure generation after ischemia in diabetic conditions.

Blood purification by nonselective hemoadsorption prevents death after traumatic brain injury and hemorrhagic shock in rats.

BACKGROUND: Patients who sustain traumatic brain injury (TBI) and concomitant hemorrhagic shock (HS) are at high risk of high-magnitude inflammation which can lead to poor outcomes and death. Blood purification by hemoadsorption (HA) offers an alternative intervention to reduce inflammation after injury. We tested the hypothesis that HA would reduce mortality in a rat model of TBI and HS. METHODS: Male Sprague Dawley rats were subjected to a combined injury of a controlled cortical impact to their brain and pressure-controlled HS. Animals were subsequently instrumented with an extracorporeal blood circuit that passed through a cartridge for sham or experimental treatment. In experimental animals, the treatment cartridge was filled with proprietary beads (Cytosorbents, Monmouth Junction, NJ) that removed circulating molecules between 5 kDa and 60 kDa. Sham rats had equivalent circulation but no blood purification. Serial blood samples were analyzed with multiplex technology to quantify changes in a trauma-relevant panel of immunologic mediators. The primary outcome was survival to 96 hours postinjury. RESULTS: Hemoadsorption improved survival from 47% in sham-treated rats to 86% in HA-treated rats. There were no treatment-related changes in histologic appearance. Hemoadsorption affected biomarker concentrations both during the treatment and over the ensuing 4 days after injury. Distinct changes in biomarker concentrations were also measured in survivor and nonsurvivor rats from the entire cohort of rats indicating biomarker patterns associated with survival and death after injury. CONCLUSION: Blood purification by nonselective HA is an effective intervention to prevent death in a combined TBI/HS rat model. Hemoadsorption changed circulating concentrations of multiple inmmunologically active mediators during the treatment time frame and after treatment. Hemoadsorption has been safely implemented in human patients with sepsis and may be a treatment option after injury.

Reduced expression of IA channels is associated with post-ischemic seizures.

PURPOSE: Post-stroke seizures are considered as a major cause of epilepsy in adults. The pathophysiologic mechanisms resulting in post-stroke seizures are not fully understood. The present study attempted to reveal a new mechanism underlying neuronal hyperexcitability responsible to the seizure development after ischemic stroke. METHODS: Transient global ischemia was produced in adult Wistar rats using the 4-vessel occlusion (4-VO) method. The spontaneous behavioral seizures were defined by the Racine scale III-V. The neuronal death in the brain was determined by hematoxylin-eosin staining. The expression levels of A-type potassium channels were analyzed by immunohistochemical staining and western blotting. RESULTS: We found that the incidence of spontaneous behavioral seizures increased according to the severity of ischemia with 0% after 15-min ischemia and ∼50% after 25-min ischemia. All behavioral seizures occurred with 48h after ischemia. Morphological analysis indicated that brain damage was not correlated with behavioral seizures. Immunohistochemical staining showed that the expression levels of the A-type potassium channel subunit Kv4.2 was significantly reduced in ischemic brains with behavioral seizures, but not in ischemic brains without seizures. In addition, rats failing to develop spontaneous behavioral seizures within 2days after ischemia were more sensitive to bicuculline-induced seizures at 2 months after ischemia than control rats. Meanwhile, Kv4.2 expression was decreased in brain at 2 months after ischemia. CONCLUSION: Our results demonstrated the reduction of Kv4.2 expression might contribute to the development of post-ischemic seizures and long-term increased seizure susceptibility after ischemia. The mechanisms underlying post-stroke seizures and epilepsy is unknown so far. The down-regulation of IA channels may explained the abnormal neuronal hyperexcitability responsible for the seizure development after ischemic stroke.

Enhanced autophagy signaling in diabetic rats with ischemia-induced seizures.

Seizures are among the most common neurological sequelae of stroke, and ischemic insult in diabetes notably increases the incidence of seizures. Recent studies indicated that autophagy influences the outcome of stroke and involved in epileptogenesis. However, the association of autophagy and post-ischemic seizures in diabetes remains unclear. The present study aimed to reveal the involvement of autophagy in the seizures following cerebral ischemia in diabetes. Diabetes was induced in adult male Wistar rats by intraperitoneal injection of streptozotocin (STZ). The diabetic rats were subjected to transient forebrain ischemia. The neuronal damage was assessed using hematoxylin-eosin staining. Western blotting and immunohistochemistry were performed to investigate the alteration of autophagy marker microtubule-associated protein light chain 1B (LC3B). The results showed that all diabetic animals developed seizures after ischemia. However, no apparent cell death was observed in the hippocampus of seizure rats 12h after the insult. The expression of LC3B was significantly enhanced in naïve animals after ischemia and was further increased in diabetic animals after ischemia. Immunofluorescence double-labeling study indicated that LC3B was mainly increased in neurons. Our study demonstrated, for the first time, that autophagy activity is significantly increased in diabetic animals with ischemia-induced seizures. Further studies are needed to explore the role of autophagy in seizure generation after ischemia in diabetic conditions.

Toll-like receptor 4 is associated with seizures following ischemia with hyperglycemia.

Seizures are a common sequel of cerebral ischemia, and hyperglycemia markedly increases the onset of seizures following an ischemic insult. However, the underlying mechanism of seizures is unclear. The toll-like receptor 4 (TLR4) pathway is known to be involved in temporal lobe epilepsy. The present study investigated the potential involvement of TLR4 in the pathogenesis of seizures following cerebral ischemia with hyperglycemia. Fifteen minutes of global ischemia was produced in adult Wistar rats using a 4-vessel occlusion method. Hyperglycemia was induced via an intraperitoneal injection of glucose 15 min prior to ischemia. We determined that 56.7% of the hyperglycemic rats, but none of the normoglycemic rats, developed tonic-clonic seizures within 12h after ischemia. TLR4 was mildly expressed in a few cells in the control hippocampus, primarily in interneurons, and was localized in the cytoplasm. The TLR4-positive cells were significantly increased 3-12h after ischemia. In the hyperglycemic ischemia group, TLR4-positive cells were further increased in quantity and intensity, with a peak at 3h after ischemia relative to the normoglycemic group. There was no difference in the expression of TLR4 between the hyperglycemic ischemia and LPS groups or between the hyperglycemic non-ischemia and control groups. Western blot analysis consistently exhibited an increase in TLR4 protein levels in the CA3 region 3h after hyperglycemic ischemia. High mobility group box 1 (HMGB1) (an endogenous ligand of TLR4) was localized in the nucleus of neuronal cells throughout the hippocampus in the control animals. We observed a dramatic decrease in HMGB1 immunostaining at 3h after hyperglycemic ischemia that gradually returned to control levels. These results suggest that the TLR4 pathway is associated with seizures following global ischemia with hyperglycemia, which provides a new direction for the study of the pathogenesis of seizures that result from hyperglycemic ischemia.

Reduced expression of IA channels is associated with postischemic seizures in hyperglycemic rats.

Poststroke seizures are considered to be the major cause of epilepsy in the elderly. The mechanisms of poststroke seizures remain unclear. A history of diabetes mellitus has been identified as an independent predictor of acute poststroke seizures in stroke patients. The present study sought to reveal the mechanisms for the development of postischemic seizures under hyperglycemic conditions. Transient forebrain ischemia was produced in adult Wistar rats by using the four-vessel occlusion method. At the normal blood glucose level, seizures occurred in ∼50% of rats after 25 min of ischemia. However, in rats with hyperglycemia, the incidence rate of postischemic seizures was significantly increased to 100%. The occurrence of postischemic seizures was not correlated with the severity of brain damage in hyperglycemic rats. Mannitol, an osmotic diuretic agent, could neither prevent postischemic seizures nor alleviate the exacerbated brain damage in the presence of hyperglycemia. K(+) channels play a critical role in controlling neuronal excitability. The expression of A-type K(+) channel subunit Kv4.2 in the hippocampus and the cortex was significantly reduced in hyperglycemic rats with seizures compared with those without seizures. These results suggest that the reduction of Kv4.2 expression could contribute to the development of postischemic seizures in hyperglycemia.

Alteration of neuronal activity after digit amputation in rat anterior cingulate cortex.

Phantom limb pain is experienced by nearly 50 - 80% of the patients following limb amputation. The anterior cingulate cortex (ACC) is a part of the limbic system that is an essential component in mediating the affective and emotional component of pain responses. To explore the role of ACC in the phantom limb pain, we recorded evoked excitatory postsynaptic potentials (EPSPs), cortical network activity and electrophysiological properties of pyramid neurons in adult rat ACC before and after a third hind paw digit amputation using in vivo intracellular or extracellular recording and staining techniques. The recorded neurons were morphologically identified as pyramidal neurons in the ACC region. The spontaneous activity of ACC neurons significantly reduced with a more percentage of down state after amputation, this is correlated with a decrease in spontaneous spikes in medial thalamus. However, the amplitude of the evoked EPSPs was increased significantly shortly after amputation and lasted for up to 7 days. This potentiation is associated with an increase of paired-pulse facilitation (PPF), suggesting the involvement of presynaptic component in this process. No significant difference in membrane properties was observed after amputation. On the other hand, administration of Complete Freund's Adjuvant (CFA) into the hind paw, a model of inflammatory pain, induced the potentiation of EPSPs in ACC neurons at 7 days after injection. These results demonstrate that digit amputation induced a long-lasting potentiation of synaptic transmission and decrease of cortical network activity in ACC in rats, which might contribute to the phantom limb pain.

Thrombospondin-4 contributes to spinal sensitization and neuropathic pain states.

Neuropathic pain is a common cause of pain after nerve injury, but its molecular basis is poorly understood. In a post-gene chip microarray effort to identify new target genes contributing to neuropathic pain development, we report here the characterization of a novel neuropathic pain contributor, thrombospondin-4 (TSP4), using a neuropathic pain model of spinal nerve ligation injury. TSP4 is mainly expressed in astrocytes and significantly upregulated in the injury side of dorsal spinal cord that correlates with the development of neuropathic pain states. TSP4 blockade by intrathecal antibodies, antisense oligodeoxynucleotides, or inactivation of the TSP4 gene reverses or prevents behavioral hypersensitivities. Intrathecal injection of TSP4 protein into naive rats is sufficient to enhance the frequency of EPSCs in spinal dorsal horn neurons, suggesting an increased excitatory presynaptic input, and to cause similar behavioral hypersensitivities. Together, these findings support that injury-induced spinal TSP4 may contribute to spinal presynaptic hypersensitivity and neuropathic pain states. Development of TSP4 antagonists has the therapeutic potential for target-specific neuropathic pain management.

Whole-cell patch-clamp recordings on spinal cord slices.

Whole-cell patch-clamp recording technique is a powerful tool to study intrinsic membrane properties and synaptic interactions in the spinal cord. Spinal cord slice is an idea preparation for electrophysiological studies under physiological and pharmacological manipulation that is difficult to perform in an in vivo preparation. Depending on experimental purposes, the extracellular and intracellular environment of neurons can be easily controlled during whole-cell recording to isolate membrane conductance of interest and to manipulate its modulation, which is important for addressing cellular mechanisms under particular physiological and pathological conditions. Several methods for preparing spinal cord slices have been developed for whole-cell patch-clamp recordings. Here we describe practical procedures for preparing spinal cord slices from adult rats and whole-cell recording from neurons in the spinal dorsal horn.

Alterations of A-type potassium channels in hippocampal neurons after traumatic brain injury.

Traumatic brain injury (TBI) is associated with cognitive deficits, memory impairment, and epilepsy. Previous studies have reported neuronal loss and neuronal hyperexcitability in the post-traumatic hippocampus. A-type K+ currents (I(A)) play a critical role in modulating the intrinsic membrane excitability of hippocampal neurons. The disruption of I(A) is reportedly linked to hippocampal dysfunction. The present study investigates the changes of I(A) in the hippocampus after TBI. TBI in rats was induced by controlled cortical impact. The impact induced a reproducible lesion in the cortex and an obvious neuronal death in the ipsilateral hippocampus CA3 region. At one week after TBI, immunohistochemical staining and Western blotting showed that the expression of I(A) channel subunit Kv4.2 was markedly decreased in the ipsilateral hippocampus, but remained unchanged in the contralateral hippocampus. Meanwhile, electrophysiological recording showed that I(A) currents in ipsilateral CA1 pyramidal neurons were significantly reduced, which was associated with an increased neuronal excitability. Furthermore, there was an increased sensitivity to bicuculline-induced seizures in TBI rats. At 8 weeks after TBI, immunohistochemical staining and electrophysiological recording indicated that I(A) returned to control levels. These findings suggest that TBI causes a transient downregulation of I(A) in hippocampal CA1 neurons, which might be associated with the hyperexcitability in the post-traumatic hippocampus, and in turn leads to seizures and epilepsy.

Moderate traumatic brain injury causes acute dendritic and synaptic degeneration in the hippocampal dentate gyrus.

Hippocampal injury-associated learning and memory deficits are frequent hallmarks of brain trauma and are the most enduring and devastating consequences following traumatic brain injury (TBI). Several reports, including our recent paper, showed that TBI brought on by a moderate level of controlled cortical impact (CCI) induces immature newborn neuron death in the hippocampal dentate gyrus. In contrast, the majority of mature neurons are spared. Less research has been focused on these spared neurons, which may also be injured or compromised by TBI. Here we examined the dendrite morphologies, dendritic spines, and synaptic structures using a genetic approach in combination with immunohistochemistry and Golgi staining. We found that although most of the mature granular neurons were spared following TBI at a moderate level of impact, they exhibited dramatic dendritic beading and fragmentation, decreased number of dendritic branches, and a lower density of dendritic spines, particularly the mushroom-shaped mature spines. Further studies showed that the density of synapses in the molecular layer of the hippocampal dentate gyrus was significantly reduced. The electrophysiological activity of neurons was impaired as well. These results indicate that TBI not only induces cell death in immature granular neurons, it also causes significant dendritic and synaptic degeneration in pathohistology. TBI also impairs the function of the spared mature granular neurons in the hippocampal dentate gyrus. These observations point to a potential anatomic substrate to explain, in part, the development of posttraumatic memory deficits. They also indicate that dendritic damage in the hippocampal dentate gyrus may serve as a therapeutic target following TBI.

Downregulation of hippocampal GABA after hypoxia-induced seizures in neonatal rats.

This study aims to determine the expression of Gamma-aminobutyric acid (GABA) following hypoxia in neonatal rats and explore how it may increase susceptibility to epilepsy later in life. A modified model of neonatal hypoxia-induced epileptic susceptibility was simulated by 17 min of hypoxia (5% O(2) and 95% N(2)) in postnatal day (P) 10 rats. Hippocampal glutamate decarboxylase (GAD) and parvalbumin (PV) during the development with or without hypoxia were examined using immunohistochemistry. No detectable neuronal loss was observed in the hippocampus either immediately or 14 days after hypoxia. During the development GAD- and PV-immunoreactivity increased substantially during P 11-13 and reached mature expression in the control rats, and decreased significantly at different time points except for a transient increase during P 11-13 in the hypoxic groups. Our study indicates that downregulation of hippocampal GABA after hypoxia-induced seizures in neonatal rats may contribute to higher epileptic susceptibility in later life.

Up-regulation of A-type potassium currents protects neurons against cerebral ischemia.

Excitotoxicity is the major cause of many neurologic disorders including stroke. Potassium currents modulate neuronal excitability and therefore influence the pathological process. A-type potassium current (I(A)) is one of the major voltage-dependent potassium currents, yet its roles in excitotoxic cell death are not well understood. We report that, following ischemic insults, the I(A) increases significantly in large aspiny (LA) neurons but not medium spiny (MS) neurons in the striatum, which correlates with the higher resistance of LA neurons to ischemia. Activation of protein kinase Cα increases I(A) in LA neurons after ischemia. Cultured neurons from transgenic mice lacking both Kv1.4 and Kv4.2 subunits exhibit an increased vulnerability to ischemic insults. Increase of I(A) by recombinant expression of Kv1.4 or Kv4.2 is sufficient in improving the survival of MS neurons against ischemic insults both in vitro and in vivo. These results, taken together, provide compelling evidence for a protective role of I(A) against ischemia.

Contribution of Ih to neuronal damage in the hippocampus after traumatic brain injury in rats.

Traumatic brain injury (TBI) causes selective neuronal damage in the hippocampus; however, the underlying mechanisms are still unclear. Post-traumatic alterations of ion channel activity, which actively regulate neuronal excitability and thus impact on excitotoxicity, may be involved in TBI-induced neuronal injury. Here we report that hyperpolarization-activated cation current (I(h)) contributes to the distinct vulnerability of hippocampal neurons in TBI. In a rat model of controlled cortical injury, moderate TBI produced neuronal death of both hippocampal CA3 neurons and mossy cells in the hilus, but not CA1 pyramidal cells. Treatment with lamotrigine, which enhances dendritic I(h), ameliorated TBI-induced neuronal damage to CA3 neurons and mossy cells. In contrast, intraventricular administration of I(h) channel blocker caused cell death in the CA1 region after TBI. Whole-cell recordings revealed that, differently from CA3 neurons, CA1 pyramidal cells expressed larger I(h) and exhibited a post-traumatic increase of I(h) amplitude. Moreover, blocking I(h) led to an increase of neuronal excitability, with greater effects seen in post-traumatic CA1 pyramidal cells than in CA3 neurons. In addition, the I(h) in mossy cells was dramatically inhibited early after TBI. Our findings indicate that differential changes of I(h) in hippocampal neurons may be one of the mechanisms of selective cell death, and that an enhancement of functional I(h) may protect hippocampal neurons against TBI.

Partial epilepsy with antecedent febrile seizures and seizure aggravation by antiepileptic drugs: associated with loss of function of Na(v) 1.1.

PURPOSE: Generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy in infancy (SMEI) are associated with sodium channel α-subunit type-1 gene (SCN1A) mutations. Febrile seizures and partial seizures occur in both GEFS+ and SMEI; sporadic onset and seizure aggravation by antiepileptic drugs (AEDs) are features of SMEI. We thus searched gene mutations in isolated cases of partial epilepsy with antecedent FS (PEFS+) that showed seizure aggravations by AEDs. METHODS: Genomic DNA from four patients was screened for mutations in SCN1A, SCN2A, SCN1B, and GABRG2 using denaturing high-performance liquid chromatography (dHPLC) and sequencing. Whole-cell patch clamp analysis was used to characterize biophysical properties of two newly defined mutants of Na(v) 1.1 in tsA201 cells. RESULTS: Two heterozygous de novo mutations of SCN1A (R946H and F1765L) were detected, which were proven to cause loss of function of Na(v) 1.1. When the functional defects of mutants reported previously are compared, it is found that all mutants from PEFS+ have features of loss of function, whereas GEFS+ shows mild dysfunction excluding loss of function, coincident with mild clinical manifestations. PEFS+ is similar to SMEI clinically with possible AED-induced seizure aggravation and biophysiologically with features of loss of function, and different from SMEI by missense mutation without changes in hydrophobicity or polarity of the residues. CONCLUSIONS: Isolated milder PEFS+ may associate with SCN1A mutations and loss of function of Na(v) 1.1, which may be the basis of seizure aggravation by sodium channel-blocking AEDs. This study characterized phenotypes biologically, which may be helpful in understanding the pathophysiologic basis, and further in management of the disease.

Activation of Akt/FoxO signaling pathway contributes to induction of neuroprotection against transient global cerebral ischemia by hypoxic pre-conditioning in adult rats.

It is well established that pre-conditioning protects neuronal injury against ischemia. However, the molecular mechanisms underlying ischemic tolerance are not completely understood. The purpose of the present study was to investigate the role of Akt/forkhead transcription factor, class O (FoxO) pathway in hypoxic pre-conditioning (HPC) using a newly developed HPC to transient global cerebral ischemia (tGCI) model in adult rats. HPC for 30-120 min significantly reduced cell death in the CA1 subregion after 10 min of tGCI. HPC was effective only when applied 1-4 days before ischemia. The maximum protection was observed with 30 min of hypoxia and 1 day interval between hypoxia and ischemia. The phosphorylated Akt and FoxOs measured by western blot and immunohistochemistry were significantly increased after hypoxia-ischemia except for a transient decrease in the HPC group. Lateral ventricular infusion of LY294002 before HPC blocked the increase in phosphorylated Akt and FoxOs and increased neuronal damage in HPC animals. These results suggest that pre-exposure to hypoxia induces protection against tGCI in adult rats. Activation of Akt results in the inactivation of FoxOs which may mediate ischemic tolerance after HPC.

Characterization of the highly regulated antigen BBA05 in the enzootic cycle of Borrelia burgdorferi.

Dramatic alteration of surface lipoprotein profiles is a key strategy that Borrelia burgdorferi, the Lyme disease pathogen, has evolved for adapting to the diverse environments of arthropod and mammalian hosts. Several of these differentially expressed lipoproteins have been shown to play important roles in the enzootic cycle of B. burgdorferi. The BBA05 protein is a previously identified putative lipoprotein (P55 or S1 antigen) that elicits antibody responses in mammals. Recent microarray analyses indicate that the BBA05 gene is differentially expressed by many environmental factors, including temperature. However, the role of the BBA05 protein in the life cycle of B. burgdorferi has not been elucidated. Here we show that expression of the BBA05 gene was exclusively induced in feeding nymphal ticks during the spirochetal transmission from ticks to mammals. Upon generating a BBA05 mutant in an infectious strain of B. burgdorferi, we showed that the BBA05 mutant remained capable of establishing infection in mice, being acquired by ticks, persisting through tick molting, and reinfecting new mammalian hosts. These results indicate that, despite being a highly conserved and regulated antigen, the BBA05 protein has a nonessential role in the transmission cycle of B. burgdorferi, at least in the animal model.

Excitatory roles of protein kinase C in striatal cholinergic interneurons.

Protein kinase C (PKC) plays critical roles in neuronal activity and is widely expressed in striatal neurons. However, it is not clear how PKC activation regulates the excitability of striatal cholinergic interneurons. In the present study, we found that PKC activation significantly inhibited A-type potassium current (I(A)), but had no effect on delayed rectifier potassium currents. Consistently, application of PKC activator caused an increase of firing in response to depolarizing currents in cholinergic interneurons, which was persistent in the presence of both excitatory and inhibitory neurotransmission blockers. These excitatory effects of PKC could be partially mimicked and occluded by blockade of I(A) with potassium channel blocker 4-aminopyridine. In addition, immunostaining demonstrated that PKCalpha, but not PKCgamma and PKCepsilon, was expressed in cholinergic interneurons. Furthermore, activation of group I metabotropic glutamate receptors (mGluRs) led to an inhibition of I(A) through a PKC-dependent pathway. These data indicate that activation of PKC, most likely PKCalpha, increases the neuronal excitability of striatal cholinergic interneurons by down-regulating I(A). Group I mGluR-mediated I(A) inhibition might be important for the glutamatergic regulation of cholinergic tone in the neostriatum.

Enhanced pre-synaptic glutamate release in deep-dorsal horn contributes to calcium channel alpha-2-delta-1 protein-mediated spinal sensitization and behavioral hypersensitivity.

Nerve injury-induced expression of the spinal calcium channel alpha-2-delta-1 subunit (Cavalpha2delta1) has been shown to mediate behavioral hypersensitivity through a yet identified mechanism. We examined if this neuroplasticity modulates behavioral hypersensitivity by regulating spinal glutamatergic neurotransmission in injury-free transgenic mice overexpressing the Cavalpha2delta1 proteins in neuronal tissues. The transgenic mice exhibited hypersensitivity to mechanical stimulation (allodynia) similar to the spinal nerve ligation injury model. Intrathecally delivered antagonists for N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptors, but not for the metabotropic glutamate receptors, caused a dose-dependent allodynia reversal in the transgenic mice without changing the behavioral sensitivity in wild-type mice. This suggests that elevated spinal Cavalpha2delta1 mediates allodynia through a pathway involving activation of selective glutamate receptors. To determine if this is mediated by enhanced spinal neuronal excitability or pre-synaptic glutamate release in deep-dorsal horn, we examined wide-dynamic-range (WDR) neuron excitability with extracellular recording and glutamate-mediated excitatory postsynaptic currents with whole-cell patch recording in deep-dorsal horn of the Cavalpha2delta1 transgenic mice. Our data indicated that overexpression of Cavalpha2delta1 in neuronal tissues led to increased frequency, but not amplitude, of miniature excitatory post synaptic currents mediated mainly by AMPA/kainate receptors at physiological membrane potentials, and also by NMDA receptors upon depolarization, without changing the excitability of WDR neurons to high intensity stimulation. Together, these findings support a mechanism of Cavalpha2delta1-mediated spinal sensitization in which elevated Cavalpha2delta1 causes increased pre-synaptic glutamate release that leads to reduced excitation thresholds of post-synaptic dorsal horn neurons to innocuous stimuli. This spinal sensitization mechanism may mediate at least partially the neuropathic pain states derived from increased pre-synaptic Cavalpha2delta1 expression.

Diversity and fluctuation of spine morphology in CA1 pyramidal neurons after transient global ischemia.

Dendritic spines form postsynaptic components of excitatory synapses in CA1 pyramidal neurons and play a key role in excitatory signal transmission. Transient global ischemia is thought to induce excitotoxicity that triggers delayed neuronal death in the CA1 region. However, the mechanism underlying structural changes of excitatory synapses after ischemia is not completely understood. Here, we demonstrate how dendritic spines change in their density and structure at an acute stage after transient global ischemia. Intracellular staining in vivo showed that the total spine density in basal, proximal, and distal apical dendrites increased at 12 hr and 24 hr after ischemia, but returned to control levels at 48 hr after ischemia. Consistent increase of spine density mainly appeared in non-late depolarizing postsynaptic potential neurons, although late depolarizing postsynaptic potential neurons also showed slight increases in spine density in these dendrites at the same intervals after ischemia. Golgi staining showed increased spine density occurred in less swollen dendrites but decreased spine density appeared in severely swollen dendrites at 12 and 24 hr after ischemia. In addition, the density and percentage of stubby spines reduced at 12 hr and 48 hr, whereas the density of thin spines increased at 12 hr after ischemia. The density and percentage of filopodia increased nearly fivefold at 24 hr after ischemia. Moreover, the density of mushroom spines doubled and its percentage increased by 150% at 48 hr after ischemia. These morphological changes of spines may be related to neuronal injury in CA1 pyramidal neurons after ischemia.