A novel restrainer device for acquistion of brain images in awake rats.

Functional neuroimaging methods like fMRI and PET are vital in neuroscience research, but require that subjects remain still throughout the scan. In animal research, anesthetic agents are typically applied to facilitate the acquisition of high-quality data with minimal motion artifact. However, anesthesia can have profound effects on brain metabolism, selectively altering dynamic neural networks and confounding the acquired data. To overcome the challenge, we have developed a novel head fixation device designed to support awake rat brain imaging. A validation experiment demonstrated that the device effectively minimizes animal motion throughout the scan, with mean absolute displacement and mean relative displacement of 0.0256 (SD: 0.001) and 0.009 (SD: 0.002), across eight evaluated subjects throughout fMRI image acquisition (total scanning time per subject: 31 min, 12 s). Furthermore, the awake scans did not induce discernable stress to the animals, with stable physiological parameters throughout the scan (Mean HR: 344, Mean RR: 56, Mean SpO2: 94 %) and unaltered serum corticosterone levels (p = 0.159). In conclusion, the device presented in this paper offers an effective and safe method of acquiring functional brain images in rats, allowing researchers to minimize the confounding effects of anesthetic use.

Carry-Over Effect of Deep Cerebellar Stimulation-Mediated Motor Recovery in a Rodent Model of Traumatic Brain Injury.

BACKGROUND: We previously demonstrated that deep brain stimulation (DBS) of lateral cerebellar nucleus (LCN) can enhance motor recovery and functional reorganization of perilesional cortex in rodent models of stroke or TBI. OBJECTIVE: Considering the treatment-related neuroplasticity observed at the perilesional cortex, we hypothesize that chronic LCN DBS-enhanced motor recovery observed will carry-over even after DBS has been deactivated. METHODS: Here, we directly tested the enduring effects of LCN DBS in male Long Evans rats that underwent controlled cortical impact (CCI) injury targeting sensorimotor cortex opposite their dominant forepaw followed by unilateral implantation of a macroelectrode into the LCN opposite the lesion. Animals were randomized to DBS or sham treatment for 4 weeks during which the motor performance were characterize by behavioral metrics. After 4 weeks, stimulation was turned off, with assessments continuing for an additional 2 weeks. Afterward, all animals were euthanized, and tissue was harvested for further analyses. RESULTS: Treated animals showed significantly greater motor improvement across all behavioral metrics relative to untreated animals during the 4-week treatment, with functional gains persisting across 2-week post-treatment. This motor recovery was associated with the increase in CaMKIIα and BDNF positive cell density across perilesional cortex in treated animals. CONCLUSIONS: LCN DBS enhanced post-TBI motor recovery, the effect of which was persisted up to 2 weeks beyond stimulation offset. Such evidence should be considered in relation to future translational efforts as, unlike typical DBS applications, treatment may only need to be provided until such time as a new function plateau is achieved.

The role of dorsolateral striatum in the effects of deep cerebellar stimulation-mediated motor recovery following ischemic stroke in rodents.

Despite great advances in acute care and rehabilitation, stroke remains the leading cause of motor impairment in the industrialized world. We have developed a deep brain stimulation (DBS)-based approach for post-stroke rehabilitation that has shown reproducible effects in rodent models and has been recently translated to humans. Mechanisms underlying the rehabilitative effects of this novel therapy have been largely focused on the ipsilesional cortex, including cortical reorganization, synaptogenesis, neurogenesis and greater expression of markers of long-term potentiation. The role of subcortical structures on its therapeutic benefits, particularly the striatum, remain unclear. In this study, we compared the motor rehabilitative effects of deep cerebellar stimulation in two rodent models of cerebral ischemia: a) cortical ischemia; and b) combined striatal and cortical ischemia. All animals underwent the same procedures, including implantation of the electrodes and tethered connections for stimulation. Both experimental groups received four weeks of continuous lateral cerebellar nucleus (LCN) DBS and each was paired with a no stimulation, sham, group. Fine motor function was indexed using the pasta matrix task. Brain tissue was harvested for histology and immunohistochemical analyses. In the cortical-only ischemia, the average pasta matrix performance of both sham and stimulated groups reduced from 19 to 24 pieces to 7-8 pieces following the stroke induction. At the end of the four-week treatment, the performance of stimulated group was significantly greater than that of sham group (14 pieces vs 7 pieces, p < 0.0001). Similarly, in the combined cortical and striatal ischemia, the performance of both sham and stimulated groups reduced from 29 to 30 pieces to 7-11 pieces following the stroke induction. However, at the end of the four-week treatment, the performance of stimulated group was not significantly greater than that of sham group (15 pieces vs 11 pieces, p = 0.452). In the post-mortem analysis, the number of cells expressing CaMKIIα at the perilesional cortical and striatum of the LCN DBS treated animals receiving cortical-only stroke elevated but not those receiving cortical+striatal stroke. The current findings suggested that the observed, LCN DBS-enhanced motor recovery and perilesional plasticity may involve striatal mechanisms.

Neurostimulation for Functional Recovery After Traumatic Brain Injury: Current Evidence and Future Directions for Invasive Surgical Approaches.

We aim to provide a comprehensive review of the current scientific evidence supporting the use of invasive neurostimulation in the treatment of deficits associated with traumatic brain injury (TBI), as well as to identify future directions for research and highlight important questions that remain unaddressed. Neurostimulation is a treatment modality with expanding applications in modern medical practice. Targeted electrical stimulation of specific brain regions has been shown to increase synaptogenesis and enhance structural reorganization of neuronal networks. This underlying therapeutic effect might be of high value for patients suffering from TBI because it could modulate neuronal connectivity and function of areas that are partially or completely spared after injury. The current published literature exploring the application of invasive neurostimulation for the treatment of functional deficits associated with TBI is scarce but promising. Rodent models have shown that targeted stimulation of the hippocampus or connecting structures can result in significant cognitive recovery, while stimulation of the motor cortex and deep cerebellar nuclei is associated with motor improvements. Data from clinical studies are extremely limited; single-patient reports and case series found neurostimulation to be effective in relieving motor symptoms, improving visuospatial memory, and supporting emotional adjustment. Looking forward, it will be important to identify stimulation targets and paradigms that can maximize improvement over multiple functional domains. It will also be important to corroborate the observed behavioral improvements with histological, electrophysiological, and radiological evidence. Finally, the impact of biological variables such as sex and age on the treatment outcomes needs to be explored.

Changes in somatosensory evoked potentials elicited by lateral cerebellar nucleus deep brain stimulation in the naïve rodent.

Deep brain stimulation (DBS) of the deep cerebellar nuclei has been shown to enhance perilesional cortical excitability and promote motor rehabilitation in preclinical models of cortical ischemia and is currently being evaluated in patients with chronic, post-stroke deficits. Understanding the effects of cerebellar DBS on contralateral sensorimotor cortex may be key to developing approaches to optimize stimulation delivery and treatment outcomes. Using the naïve rat model, we characterized the effects of DBS of the lateral cerebellar nucleus (LCN) on somatosensory evoked potentials (SSEPs) and evaluated their potential use as a surrogate index of cortical excitability. SSEPs were recorded concurrently with continuous 30 Hz or 100 Hz LCN DBS and compared to the DBS OFF condition. Ratios of SSEP peak to peak amplitude during 100 Hz LCN DBS to DBS OFF at longer latency peaks were significantly>1, suggesting that cortical excitability was enhanced as a result of LCN DBS. Although changes in SSEP peak to peak amplitudes were observed, they were modest in relation to previously reported effects on motor cortical excitability. Overall, our findings suggest that LCN output influences thalamocortical somatosensory pathways, however further work is need to better understand the potential role of SSEPs in optimizing therapy.

Deep cerebellar stimulation enhances cognitive recovery after prefrontal traumatic brain injury in rodent.

Functional outcome following traumatic brain injury (TBI) varies greatly, with approximately half of those who survive suffering long-term motor and cognitive deficits despite contemporary rehabilitation efforts. We have previously shown that deep brain stimulation (DBS) of the lateral cerebellar nucleus (LCN) enhances rehabilitation of motor deficits that result from brain injury. The objective of the present study was to evaluate the efficacy of LCN DBS on recovery from rodent TBI that uniquely models the injury location, chronicity and resultant cognitive symptoms observed in most human TBI patients. We used controlled cortical impact (CCI) to produce an injury that targeted the medial prefrontal cortex (mPFC-CCI) bilaterally, resulting in cognitive deficits. Unilateral LCN DBS electrode implantation was performed 6 weeks post-injury. Electrical stimulation started at week eight post-injury and continued for an additional 4 weeks. Cognition was evaluated using baited Y-maze, novel object recognition task and Barnes maze. Post-mortem analyses, including Western Blot and immunohistochemistry, were conducted to elucidate the cellular and molecular mechanisms of recovery. We found that mPFC-CCI produced significant cognitive deficits compared to pre-injury and naïve animals. Moreover, LCN DBS treatment significantly enhanced the long-term memory process and executive functions of applying strategy. Analyses of post-mortem tissues showed significantly greater expression of CaMKIIα, BDNF and p75NTR across perilesional cortex and higher expression of postsynaptic formations in LCN DBS-treated animals compared to untreated. Overall, these data suggest that LCN DBS is an effective treatment of cognitive deficits that result from TBI, possibly by activation of ascending, glutamatergic projections to thalamus and subsequent upregulation of thalamocortical activity that engages neuroplastic mechanisms for facilitation of functional re-organization. These results support a role for cerebellar output neuromodulation as a novel therapeutic approach to enhance rehabilitation for patients with chronic, post-TBI cognitive deficits that are unresponsive to traditional rehabilitative efforts.

Cellular-resolution monitoring of ischemic stroke pathologies in the rat cortex.

Stroke is a leading cause of disability in the Western world. Current post-stroke rehabilitation treatments are only effective in approximately half of the patients. Therefore, there is a pressing clinical need for developing new rehabilitation approaches for enhancing the recovery process, which requires the use of appropriate animal models. Here, we demonstrate the use of nonlinear microscopy of calcium sensors in the rat brain to study the effects of ischemic stroke injury on cortical activity patterns. We longitudinally recorded from thousands of neurons labeled with a genetically-encoded calcium indicator before and after an ischemic stroke injury in the primary motor cortex. We show that this injury has an effect on the activity patterns of neurons not only in the motor and somatosensory cortices, but also in the more distant visual cortex, and that these changes include modified firing rates and kinetics of neuronal activity patterns in response to a sensory stimulus. Changes in neuronal population activity provided animal-specific, circuit-level information on the post-stroke cortical reorganization process, which may be essential for evaluating the efficacy of new approaches for enhancing the recovery process.

BrdU-induced hyperlocomotion in the stroked rat.

5-bromo-2'-dexoyuridine (BrdU) is often used in neuroscience research as a marker of newly-divided cells. However, several studies suggest that BrdU can produce unwanted side effects, including changes in animal behavior and cellular function. In this study, we investigated the effect of BrdU injections on locomotor behavior in a rodent model of ischemic stroke. Ischemic strokes were induced in adult rats, and 50 mg/kg BrdU was intraperitoneally injected over 5 days beginning 2 weeks post-stroke, while control animals received vehicle. Locomotor activity was evaluated by videotaping the rats in their home cages for 30 min, beginning one hour after BrdU injection. BrdU-injected rats showed a nearly three-fold increase in locomotor activity compared to control animals. These findings suggest that BrdU induces a hyperlocomotor effect in rats following brain injury, pointing to the need for caution when interpreting behavioral results in such studies.

A novel selective MAO-B inhibitor with neuroprotective and anti-Parkinsonian properties.

We previously reported that 1,3-bisbenzylimidazolium (DBZIM) bromide was neuroprotective for the dopaminergic system in Parkinson's disease (PD) models of rodent, however the underlying mechanism was unclear. We currently further confirmed that DBZIM ameliorated the Parkinsonian motor deficit and protected the nigrostriatal tract from the neurotoxicity of 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine (2'-CH3-MPTP) in C57Bl/6 mice. The dopaminergic degeneration in the substantia nigra par compacta (SNc) and striatum was analyzed by immunohistochemistry while the monoamine oxidase B (MAO-B) inhibition effect of DBZIM was determined by enzyme kinetics. DBZIM was at least as effective as the clinically approved anti-PD drug, l-deprenyl (Selegiline), for both neuroprotection and correction of motor deficits. Mechanistically, DBZIM inhibited the specific activity of MAO-B in the striatum and C6 cells without affecting the protein expression. DBZIM directly inhibited the enzymatic activity of a purified MAO-B protein with an estimated Ki value from 780 to 940nM, in par with that of l-deprenyl (970nM). The physical interaction between DBZIM and MAO-B was proven by NMR analysis, with Kd around 21.5-46.8µM. Our binding and modelling data further illustrated that DBZIM is a mixed inhibitor with its binding to active site partially hindering the substrate binding. Therefore, inhibiting MAO-B is a major mechanism through which DBZIM confers neuroprotection for the dopaminergic neurons against 2'-CH3-MPTP toxicity. Remarkably, the post-lesion treatment with DBZIM provided greater anti-parkinsonian and neuroprotective effects than the l-deprenyl. The current study, together with our previous findings in a 6-OHDA PD model, demonstrated that DBZIM is a promising neuroprotectant for PD with anti-MAO-B property.

Lateral Cerebellar Nucleus Stimulation has Selective Effects on Glutamatergic and GABAergic Perilesional Neurogenesis After Cortical Ischemia in the Rodent Model.

BACKGROUND: Chronic deep brain stimulation of the rodent lateral cerebellar nucleus (LCN) has been demonstrated to enhance motor recovery following cortical ischemia. This effect is concurrent with synaptogenesis and expression of long-term potentiation markers in the perilesional cerebral cortex. OBJECTIVE: To further investigate the cellular changes associated with chronic LCN stimulation in the ischemic rodent by examining neurogenesis along the cerebellothalamocortical pathway. METHODS: Rats were trained on the pasta matrix task, followed by induction of cortical ischemia and electrode implantation in the contralesional LCN. Electrical stimulation was initiated 6 wk after stroke induction and continued for 4 wk prior to sacrifice. Neurogenesis was examined using immunohistochemistry. RESULTS: Treated animals showed enhanced performance on the pasta matrix task relative to sham controls. Increased cell proliferation colabeled with 5'-Bromo-2'-deoxyuridine and neurogenic markers (doublecortin) was observed in the perilesional cortex as well as bilateral mediodorsal and ventrolateral thalamic subnuclei in treated vs untreated animals. The neurogenic effect at the level of motor cortex was selective, with stimulation-treated animals showing greater glutamatergic neurogenesis but significantly less GABAergic neurogenesis. CONCLUSION: These findings suggest that LCN deep brain stimulation modulates postinjury neurogenesis, providing a possible mechanistic foundation for the associated enhancement in poststroke motor recovery.

Lateral cerebellar nucleus stimulation promotes motor recovery and suppresses neuroinflammation in a fluid percussion injury rodent model.

BACKGROUND: Many traumatic brain injury (TBI) survivors live with persistent disability from chronic motor deficits despite contemporary rehabilitation services, underscoring the need for novel treatment. OBJECTIVE/HYPOTHESIS: We have previously shown that deep brain stimulation (DBS) of the lateral cerebellar nucleus (LCN) can enhance post-stroke motor recovery and increase the expression of markers of long-term potentiation in perilesional cerebral cortex. We hypothesize that a similar beneficial effect will be for motor deficits induced by unilateral fluid percussion injury (FPI) in rodents through long-term potentiation- and anti-inflammatory based mechanisms. METHODS: Male Long Evans rats with a DBS macroelectrode in the LCN underwent FPI over contralateral primary motor cortex. After 4 weeks of spontaneous recovery, DBS treatment was applied for 4 weeks, with the pasta matrix, cylinder, and horizontal ladder tests used to evaluate motor performance. All animals were euthanized and tissue harvested for further analysis by histology, immunohistochemistry, RNA microarray assay and Western Blot. RESULTS: LCN DBS-treated animals experienced a significantly greater rate of motor recovery than untreated surgical controls, with treated animals showing enhanced expression of RNA and protein for excitability related genes, suppressed expression of pro-inflammatory genes, suppressed microglial and astrocytic activation, but proliferation of c-fos positive cells. Finally, our data suggest a possible role for anti-apoptotic effects with LCN DBS. CONCLUSION: LCN DBS enhanced the motor recovery following TBI, possibly by elevating the neuronal excitability at the perilesional area and mediating anti-apoptotic and anti-inflammatory effects.

Crossed Cerebellar Atrophy of the Lateral Cerebellar Nucleus in an Endothelin-1-Induced, Rodent Model of Ischemic Stroke.

Crossed cerebellar diaschisis (CCD) is a functional deficit of the cerebellar hemisphere resulting from loss of afferent input consequent to a lesion of the contralateral cerebral hemisphere. It is manifested as a reduction of metabolism and blood flow and, depending on severity and duration, it can result in atrophy, a phenomenon known as crossed cerebellar atrophy (CCA). While CCA has been well-demonstrated in humans, it remains poorly characterized in animal models of stroke. In this study we evaluated the effects of cerebral cortical ischemia on contralateral cerebellar anatomy using an established rodent model of chronic stroke. The effects of cortical ischemia on the cerebellar hemispheres, vermis and deep nuclei were characterized. Intracortical microinjections of endothelin-1 (ET-1) were delivered to the motor cortex of Long Evans rats to induce ischemic stroke, with animals sacrificed 6 weeks later. Naive animals served as controls. Cerebral sections and cerebellar sections including the deep nuclei were prepared for analysis with Nissl staining. Cortical ischemia was associated with significant thickness reduction of the molecular layer at the Crus 1 and parafloccular lobule (PFL), but not in fourth cerebellar lobule (4Cb), as compared to the ipsilesional cerebellar hemisphere. A significant reduction in volume and cell density of the lateral cerebellar nucleus (LCN), the rodent correlate of the dentate nucleus, was also noted. The results highlight the relevance of corticopontocerebellar (CPC) projections for cerebellar metabolism and function, including its direct projections to the LCN.

Differential frequency modulation of neural activity in the lateral cerebellar nucleus in failed and successful grasps.

The olivo-cerebellar system has an essential role in the detection and adaptive correction of movement errors. While there is evidence of an error signal in the cerebellar cortex and inferior olivary nucleus, the deep cerebellar nuclei have been less thoroughly investigated. Here, we recorded local field potential activity in the rodent lateral cerebellar nucleus during a skilled reaching task and compared event-related changes in neural activity between unsuccessful and successful attempts. Increased low gamma (40-50 Hz) band power was present throughout the reach and grasp behavior, with no difference between successful and unsuccessful trials. Beta band (12-30 Hz) power, however, was significantly increased in unsuccessful reaches, compared to successful, throughout the trial, including during the epoch preceding knowledge of the trial's outcome. This beta band activity was greater in unsuccessful trials of high-performing days, compared to unsuccessful trials of low-performing days, indicating that this activity may reflect an error prediction signal, developed over the course of motor learning. These findings suggest an error-related discriminatory oscillatory hallmark of movement in the deep cerebellar nuclei.

Neuroprotective and behavioural assessments of an imidazolium compound (DBZIM) in a rat model of Parkinson's disease induced by 6-OHDA.

The neuroprotective effect of DBZIM, a novel imidazolium compound, has previously been documented to slow down neurodegeneration in a mouse model of Parkinson's disease. In this study, we conducted behavioural studies and further investigated the neuroprotection in a rat Parkinsonian model induced by 6-hydroxydopamine (6-OHDA). DBZIM was found to significantly reduce the 6-OHDA-induced asymmetrical rotation and preferential usage of contralateral forelimbs. Furthermore, the degeneration of tyrosine hydroxylase immunopositive (TH+) dopaminergic neurones in the substantia nigra par compacta (SNc) was illustrated by immunohistochemistry. The significant loss of TH+ neurones by 6-OHDA administration was ameliorated by three different doses of DBZIM treatment in a bell-shape manner. Such neuroprotection was also observed in the 6-OHDA-lesioned striata. High-performance liquid chromatography (HPLC) analysis of the striatal tissues revealed that DBZIM beneficially maintained the dopamine level by slowing down its metabolism. In addition, DBZIM attenuated the activation of astrocytes and microglia. This suggests that anti-inflammation may be an additional mechanism underlying the DBZIM-mediated neuroprotection. These findings warrant further investigation of DBZIM as a promising and potent agent for the future treatment of Parkinson's disease.

Bis(12)-hupyridone, a novel acetylcholinesterase inhibitor, protects against glutamate-induced neuronal excitotoxicity via activating α7 nicotinic acetylcholine receptor/phosphoinositide 3-kinase/Akt cascade.

Bis(12)-hupyridone (B12H), derived from the Chinese medicinal component huperzine A, was originally designed as a novel acetylcholinesterase (AChE) inhibitor. In this paper, we report that B12H (24-h pretreatment) effectively blocked glutamate-induced neuronal excitotoxicity in cerebellar granule neurons (CGNs). However, the huge discrepancy between the EC50 value and IC50 value of B12H, to protect against neuronal toxicity (0.09 µM) and to block the NMDA receptor (21.8 µM) respectively, suggests that the neuroprotection of B12H might be not primarily due to the blockade of the NMDA receptor. Pretreatment by specific antagonists of alpha7-nicotinic acetylcholine receptor (α7nAChR), but not muscarinic acetylcholine receptor (mAChR) or α4ß2nAChR, decreased the neuroprotection of B12H. The neuroprotection of B12H could also be abolished by the pretreatment of specific PI3-K inhibitors. Furthermore, B12H restored the suppressed activation of the Akt pathway caused by glutamate as evidenced by the decreased expressions of pSer473-Akt and pSer9-GSK3ß. All these results suggest that B12H substantially protected CGNs against glutamate-induced neuronal excitotoxicity via activating α7nAChR/PI3-K/Akt cascade.

Neuroprotection against excitotoxic and ischemic insults by bis(12)-hupyridone, a novel anti-acetylcholinesterase dimer, possibly via acting on multiple targets.

The activation of N-methyl-d-aspartate (NMDA) receptors by excessive release of glutamate is involved in the pathogenesis of ischemic stroke. Thus the NMDA receptor has become an attractive therapeutic target for the development of neuroprotectants, especially from antagonists with moderate to low affinity. In the current study, NMDA receptor blockage and neuroprotective effects of bis(12)-hupyridone (B12H), a novel dimeric acetylcholinesterase inhibitor derived from a naturally occurring monomeric analog huperzine A, were investigated in vitro and in vivo. In primary rat cerebellar granule neurons, B12H (0.1 nM to 1 µM) prevented glutamate-induced apoptosis in a concentration- and time-dependent manner. Receptor-ligand binding analysis showed that B12H competed with [(3)H]MK801 with a K(i) value of 7.7 µM. In the 2-hour middle cerebral artery occlusion rat model, B12H (0.4 and 0.8 mg/kg, 30 min before-ischemia and 15 min post-ischemia, i.p.) significantly attenuated ischemia-induced apoptosis in the penumbra region, improved neurological behavior impairment, and decreased cerebral infarct volume, cerebral edema and neuronal apoptosis in the stroke model. Together, these results showed that B12H moderately blocks NMDA receptors at MK801 site and exerts neuroprotection against excitotoxic and ischemic insults in vitro and in vivo. Combined with our previous publications, we conjecture that B12H might exert neuroprotection via acting on multiple targets.

Synergistic neuroprotection by bis(7)-tacrine via concurrent blockade of N-methyl-D-aspartate receptors and neuronal nitric-oxide synthase.

The excessive activation of the N-methyl-D-aspartate receptor (NMDAR)/nitric oxide (NO) pathway has been proposed to be involved in the neuropathology of various neurodegenerative disorders. In this study, NO was found to mediate glutamate-induced excitotoxicity in primary cultured neurons. Compared with the NO synthase (NOS) inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), and the NMDAR antagonist memantine, bis(7)-tacrine was found to be more potent in reducing NO-mediated excitotoxicity and the release of NO caused by glutamate. Moreover, like L-NMMA but not like 5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) and memantine, bis(7)-tacrine showed greater neuroprotection and inhibition on NO release when neurons were pretreated for a prolonged time between 0 and 24 h and remained quite potent even when neurons were post-treated 1 h after the glutamate challenge. Bis(7)-tacrine was additionally found to be as moderately potent as memantine in competing with [(3)H]MK-801, inhibiting NMDA-evoked currents and reducing glutamate-triggered calcium influx, which eventually reduced neuronal NOS activity. More importantly, at neuroprotective concentrations, bis(7)-tacrine substantially reversed the overactivation of neuronal NOS caused by glutamate without interfering with the basal activity of NOS. Furthermore, in vitro pattern analysis demonstrated that bis(7)-tacrine competitively inhibited both purified neuronal and inducible NOS with IC(50) values at 2.9 and 9.3 microM but not endothelial NOS. This result was further supported by molecular docking simulations that showed hydrophobic interactions between bis(7)-tacrine and three NOS isozymes. Taken together, these results strongly suggest that the substantial neuroprotection against glutamate by bis(7)-tacrine might be mediated synergistically through the moderate blockade of NMDAR and selective inhibition of neuronal NOS.

Novel dimeric acetylcholinesterase inhibitor bis7-tacrine, but not donepezil, prevents glutamate-induced neuronal apoptosis by blocking N-methyl-D-aspartate receptors.

The neuroprotective properties of bis(7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate-induced excitotoxicity were investigated in primary cultured cerebellar granule neurons (CGNs). Exposure of CGNs to 75 mum glutamate resulted in neuronal apoptosis as demonstrated by Hoechst staining, TUNEL, and DNA fragmentation assays. The bis(7)-tacrine treatment (0.01-1 mum) on CGNs markedly reduced glutamate-induced apoptosis in dose- and time-dependent manners. However, donepezil and other AChE inhibitors, even at concentrations of inhibiting AChE to the similar extents as 1 mum bis(7)-tacrine, failed to prevent glutamate-induced excitotoxicity in CGNs; moreover, both atropine and dihydro-beta-erythroidine, the cholinoreceptor antagonists, did not affect the anti-apoptotic properties of bis(7)-tacrine, suggesting that the neuroprotection of bis(7)-tacrine appears to be independent of inhibiting AChE and cholinergic transmission. In addition, ERK1/2 and p38 pathways, downstream signals of N-methyl-d-aspartate (NMDA) receptors, were rapidly activated after the exposure of glutamate to CGNs. Bis(7)-tacrine inhibited the apoptosis and the activation of these two signals with the same efficacy as the coapplication of PD98059 and SB203580. Furthermore, using fluorescence Ca(2+) imaging, patch clamp, and receptor-ligand binding techniques, bis(7)-tacrine was found effectively to buffer the intracellular Ca(2+) increase triggered by glutamate, to reduce NMDA-activated currents and to compete with [(3)H]MK-801 with an IC(50) value of 0.763 mum in rat cerebellar cortex membranes. These findings strongly suggest that bis(7)-tacrine prevents glutamate-induced neuronal apoptosis through directly blocking NMDA receptors at the MK-801-binding site, which offers a new and clinically significant modality as to how the agent exerts neuroprotective effects.

Minocycline prevents glutamate-induced apoptosis of cerebellar granule neurons by differential regulation of p38 and Akt pathways.

Minocycline has been shown to have remarkably neuroprotective qualities, but underlying mechanisms remain elusive. We reported here the robust neuroprotection by minocycline against glutamate-induced apoptosis through regulations of p38 and Akt pathways. Pre-treatment of cerebellar granule neurons (CGNs) with minocycline (10-100 microm) elicited a dose-dependent reduction of glutamate excitotoxicity and blocked glutamate-induced nuclear condensation and DNA fragmentations. Using patch-clamping and fluorescence Ca2+ imaging techniques, it was found that minocycline neither blocked NMDA receptors, nor reduced glutamate-caused rises in intracellular Ca2+. Instead, confirmed by immunoblots, minocycline in vivo and in vitro was shown to directly inhibit the activation of p38 caused by glutamate. A p38-specific inhibitor, SB203580, also attenuated glutamate excitotoxicity. Furthermore, the neuroprotective effects of minocycline were blocked by phosphatidylinositol 3-kinase (PI3-K) inhibitors LY294002 and wortmannin, while pharmacologic inhibition of glycogen synthase kinase 3beta (GSK3beta) attenuated glutamate-induced apoptosis. In addition, immunoblots revealed that minocycline reversed the suppression of phosphorylated Akt and GSK3beta caused by glutamate, as were abolished by PI3-K inhibitors. These results demonstrate that minocycline prevents glutamate-induced apoptosis in CGNs by directly inhibiting p38 activity and maintaining the activation of PI3-K/Akt pathway, which offers a novel modality as to how the drug exerts protective effects.