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Ts65Dn mice (TS), the most commonly used model of Down syndrome (DS), exhibit phenotypic characteristics of this condition. Both TS mice and DS individuals present cognitive disturbances, age-related cholinergic degeneration, and increased brain expression of β-amyloid precursor protein (AβPP). These neurodegenerative processes may contribute to the progressive cognitive decline observed in DS. Melatonin is a pineal indoleamine that has been reported to reduce neurodegenerative processes and improve cognitive deficits in various animal models. In this study, we evaluated the potentially beneficial effects of long-term melatonin treatment on the cognitive deficits, cholinergic degeneration, and enhanced AβPP and β-amyloid levels of TS mice. Melatonin was administered for 5 months to 5- to 6-month-old TS and control (CO) mice. Melatonin treatment improved spatial learning and memory and increased the number of choline acetyltransferase (ChAT)-positive cells in the medial septum of both TS and CO mice. However, melatonin treatment did not significantly reduce AβPP or β-amyloid levels in the cortex or the hippocampus of TS mice. Melatonin administration did reduce anxiety in TS mice without inducing sensorimotor alterations, indicating that prolonged treatment with this indoleamine is devoid of noncognitive behavioral side effects (e.g., motor coordination, sensorimotor abilities, or spontaneous activity). Our results suggest that melatonin administration might improve the cognitive abilities of both TS and CO mice, at least partially, by reducing the age-related degeneration of basal forebrain cholinergic neurons. Thus, chronic melatonin supplementation may be an effective treatment for delaying the age-related progression of cognitive deterioration found in DS., (© 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.)

Background: Mitochondria-related mechanisms have been suggested to mediate methamphetamine (METH) toxicity. However, changes in brain energetics associated with high-energy phosphate metabolism have not been investigated in METH users. Phosphorus-31 ((31)P) magnetic resonance spectroscopy (MRS) was used to evaluate changes in mitochondrial high energy phosphates, including phosphocreatine (PCr) and β-nucleoside triphosphate (β-NTP, primarily ATP in brain) levels. We hypothesized that METH users would have decreased high-energy PCr levels in the frontal gray matter., Methods: Study participants consisted of 51 METH (age=32.8±6.7) and 23 healthy comparison (age=31.1±7.5) subjects. High-energy phosphate metabolite levels were compared between the groups and potential gender differences were explored., Results: METH users had lower ratios of PCr to total pool of exchangeable phosphate (PCr/TPP) in the frontal lobe as compared to the healthy subjects (p=.001). The lower PCr levels in METH subjects were significantly associated with lifetime amount of METH use (p=.003). A sub-analysis for gender differences revealed that female METH users, who had lower daily amounts (1.1±1.0g) of METH use than males (1.4±1.7g), had significantly lower PCr/TPP ratios than male METH users, controlling for the amount of METH use (p=.02)., Conclusions: The present findings suggest that METH compromises frontal lobe high-energy phosphate metabolism in a dose-responsive manner. Our findings also suggest that the abnormality in frontal lobe high-energy phosphate metabolism might be more prominent in female than in male METH users. This is significant as decreased PCr levels have been associated with depressive symptoms, and poor responses to antidepressant treatment have been reported in those with decreased PCr levels., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

An early but transient decrease in oxygen availability occurs during experimentally induced seizures. Using pimonidazole, which probes hypoxic insults, we found that by increasing the duration of pilocarpine-induced status epilepticus (SE) from 30 to 120 min, counts of pimonidazole-immunoreactive neurons also increased (P < 0.01, 120 vs 60 and 30 min). All the animals exposed to SE were immunopositive to pimonidazole, but a different scenario emerged during epileptogenesis when a decrease in pimonidazole-immunostained cells occurred from 7 to 14 days, so that only 1 out of 4 rats presented with pimonidazole-immunopositive cells. Pimonidazole-immunoreactive cells robustly reappeared at 21 days post-SE induction when all animals (7 out of 7) had developed spontaneous recurrent seizures. Specific neuronal markers revealed that immunopositivity to pimonidazole was present in cells identified by neuropeptide Y (NPY) or somatostatin antibodies. At variance, neurons immunopositive to parvalbumin or cholecystokinin were not immunopositive to pimonidazole. Pimonidazole-immunopositive neurons expressed remarkable immunoreactivity to hypoxia-inducible factor 1α (HIF-1α). Interestingly, surgical samples obtained from pharmacoresistant patients showed neurons co-labeled by HIF-1α and NPY antibodies. These interneurons, along with parvalbumin-positive interneurons that were negative to HIF-1α, showed immunopositivity to markers of cell damage, such as high-mobility group box 1 in the cytoplasm and cleaved caspase-3 in the nucleus. These findings suggest that interneurons are continuously endangered in rodent and human epileptogenic tissue. The presence of hypoxia and cell damage markers in NPY interneurons of rats and patients presenting with recurrent seizures indicates a mechanism of selective vulnerability in a specific neuronal subpopulation.

Epilepsy is characterised by the propensity of the brain to generate spontaneous recurrent bursts of excessive neuronal activity, seizures. GABA-mediated inhibition is critical for restraining neuronal excitation in the brain, and therefore potentiation of GABAergic neurotransmission is commonly used to prevent seizures. However, data obtained in animal models of epilepsy and from human epileptic tissue suggest that GABA-mediated signalling contributes to interictal and ictal activity. Prolonged activation of GABA(A) receptors during epileptiform bursts may even initiate a shift in GABAergic neurotransmission from inhibitory to excitatory and so have a proconvulsant action. Direct targeting of the membrane mechanisms that reduce spiking in glutamatergic neurons may better control neuronal excitability in epileptic tissue. Manipulation of brain pH may be a promising approach and recent advances in gene therapy and optogenetics seem likely to provide further routes to effective therapeutic intervention.

Postnatal maternal separation in rats causes a reduction of GABAergic parvalbumin-containing interneurons in the prefrontal cortex that first occurs in adolescence. This parvalbumin loss can be prevented by pre-adolescent treatment with a non-steroidal anti-inflammatory drug that also protects against excitotoxicity. Therefore, the neuropsychiatric disorders associated with early life adversity and interneuron dysfunction may involve neuroinflammatory processes and/or aberrant glutamatergic activity. Here, we aimed to determine whether delayed parvalbumin loss after maternal separation was due to inflammatory activity, and whether central administration of the anti-inflammatory cytokine interleukin (IL)-10 could protect against such loss. We also investigated the effects of maternal separation and IL-10 treatment on cortical NMDA receptor expression. Male rat pups were isolated for 4h/day between postnatal days 2-20. IL-10 was administered intracerebroventricularly through an indwelling cannula between P30 and 38. Adolescent prefrontal cortices were analyzed using Western blotting and immunohistochemistry for parvalbumin and NMDA NR2A subunit expression. We demonstrate that central IL-10 administration during pre-adolescence protects maternally separated animals from parvalbumin loss in adolescence. Linear regression analyses revealed that increased circulating levels of the pro-inflammatory cytokines IL-1β and IL-6 predicted lowered parvalbumin levels in maternally separated adolescents. Maternal separation also increases cortical expression of the NR2A NMDA receptor subunit in adolescence, which is prevented by IL-10 treatment. These data suggest that inflammatory damage to parvalbumin interneurons may occur via aberrant glutamatergic activity in the prefrontal cortex. Our findings provide a novel interactive mechanism between inflammation and neural dysfunction that helps explain deleterious effects of early life adversity on prefrontal cortex interneurons., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: To observe changes in expression of tumor necrosis factor (TNF)-alpha and permeability of blood brain barrier after salidroside pretreatment in rats with injury induced by focal cerebralischemia-reperfusion., Methods: Forty-five male SD rats were randomly divided into three groups (n=15): control group, ischemia-reperfusion (IR) model group, and salidroside pretreatment group. Before the IR model establishment, the rats in the salidroside pretreatment group were intraperitoneally administered with salidroside at a dose of 24 mg/(kg·d) for 7 d. After 30 min post the last administration, the IR model was induced by occlusion of middle cerebral artery with a filament. After 24 h post the operation, the water content and Evens blue content in the ischemia cerebral hemisphere were determined, and the level of TNF-alpha mRNA was detected by the semi-quantitative RT-PCR., Results: Compared with the IR model group, the salidroside pretreatment group had significantly lower (P<0.05) water content and Evens blue content in the ischemia cerebral hemisphere and also had significantly lower (P<0.05) level of TNF-alpha in the ischemic cerebral cortex tissue., Conclusions: The salidroside pretreatment alleviated the focal cerebralischemia-reperfusion injury in the rat model, possibly by decreasing the permeability of blood brain barrier, attenuating brain edema and reducing TNF-alpha expression., (Copyright © 2013 Hainan Medical College. Published by Elsevier B.V. All rights reserved.)

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc). The transcription of Arc is rapidly and highly induced by intensified neuronal activity and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes.

Direct determination of cerebral metal ions in small volume biological samples is still the bottleneck for evaluating the roles that metal ions play in the physiological and pathological processes. In this work, selected copper ion (Cu(2+)) as a model, a facile and direct electrochemical method for detection of Cu(2+) has been developed on the basis of two new designed strategies: one is specific recognition molecule for Cu(2+)-AE-TPEA (N-(2-aminoethyl)-N,N',N'-tris(pyridine-2-yl-methyl)ethane-1,2-diamine); another is carbon dots (C-Dots) with high electrocatalytic activity. Based on the high affinity between TPEA and Cu(2+), the electrode assembled with C-Dot-TPEA hybridized nanocomposites shows high selectivity toward Cu(2+) over other metal ions, amino acids, and biological coexisting species, such as uric acid (UA), ascorbic acid (AA), and so on, which makes it possible to be used for determination of Cu(2+) in the complex brain system. By taking advantage of C-Dots, a dynamic linear range from 1 μM to 60 μM is first achieved with a detection limit of ∼100 nM in aCSF solution. In addition, the developed method with theoretical simplicity and less instrumental demands exhibits long-term stability and good reproducibility. As a result, the present strategy has been successfully applied in detection of cerebral Cu(2+) in normal rat brain and that followed by global cerebral ischemia, combined with in vivo microdialysis. The determined concentrations of Cu(2+) in the rat brain microdialysates by the present method are found to be identical to those obtained by the conventional ICP-AES method.

Glycated proteins are important biomarkers for age-related disorders, however their analysis is challenging because of the complexity of the protein-carbohydrate adducts. Here we report a method that enables the detection and identification of individual glycated proteins in complex samples using fluorescent boronic acids in gel electrophoresis. Using this method we identified glycated proteins in human serum, insect hemolymph and mouse brain homogenates, confirming this technique as a powerful proteomics tool that can be used for the identification of potential disease biomarkers.

The purpose of this study was to determine whether hippocampus and cerebral cortex fatty acids (FA) composition can be altered as a result of n-3 enriched diet, or modifications in FA can affect the age-related histological changes in these brain tissues. The study was performed on eighteen rats which were fed control (CD) or fish oil supplemented diet (FOD) for 12 months. We investigated the n-3 and n-6 brain FA profile by gas-chromatography analysis. Histomorphometry included the measurement of the quantity of pyramidal cells in the cerebral cortex (layer II-III) and in the hippocampal formation (CA1, CA3 and DG). The number of apoptotic cells (neuron and glial cells) was also calculated separately in three hippocampal areas and in the pyramidal cerebral cortex layer. Rats fed supplemented diet showed a significantly poorer content of the arachidonic acid (AA, 20:4n6) in all brain regions when compared to the control group. Furthermore, the level of the docosahexaenoic acid (DHA, 22:6n3) was significantly higher in the cerebral cortex in fish oil fed rats vs. the control group. The neurons of the pyramidal cortex showed significant changes in cell density in control animals when compared to the group of fish oil fed rats. Furthermore, the number of positive apoptotic cells was significantly higher in the CA1 area and cerebral pyramidal layer in rats fed control diet. Following dietary n-3 fatty acids supplementation, the increase in DHA content in the cerebral cortex resulted in consequential changes in histology of this tissue. The results obtained indicate that dietary intake of fish n-3 fatty acids may reduce the risk of age-related brain impairments.

Angiogenesis is a hallmark of many conditions, including cancer, stroke, vascular disease, diabetes, and high-altitude exposure. We have previously shown that one can study angiogenesis in animal models by using total hemoglobin (tHb) as a marker of cerebral blood volume (CBV), measured using broadband near-infrared spectroscopy (bNIRS). However, the method was not suitable for patients as global anoxia was used for the calibration. Here we determine if angiogenesis could be detected using a calibration method that could be applied to patients. CBV, as a marker of angiogenesis, is quantified in a rat cortex before and after hypoxia acclimation. Rats are acclimated at 370-mmHg pressure for three weeks, while rats in the control group are housed under the same conditions, but under normal pressure. CBV increased in each animal in the acclimation group. The mean CBV (%volume/volume) is 3.49%± 0.43% (mean ± SD) before acclimation for the experimental group, and 4.76%± 0.29% after acclimation. The CBV for the control group is 3.28%± 0.75%, and 3.09%± 0.48% for the two measurements. This demonstrates that angiogenesis can be monitored noninvasively over time using a bNIRS system with a calibration method that is compatible with human use and less stressful for studies using animals.

Quantifying protein and RNA expression within specific cell populations in vivo is an essential step in unraveling the complex mechanisms of neurological disease. The challenges associated with studying human brain tissue are commonly compounded by variations in postmortem interval, formalin fixation time, and tissue processing methods among others. The result is a sample population that is inherently heterogeneous, implying the need for reliable protocols that are sensitive to low levels of antigen while minimizing background and nonspecific staining. Here, we describe a single immunohistochemistry protocol on formalin-fixed, paraffin-embedded human cortex which can be adapted to (1) quantify the relative protein expression of the chemokine receptor, CXCR4, using multispectral image or (2) isolate neuronal RNA through automated laser capture microdissection.

Objective: Although amyloid-beta (Aβ) peptide deposition into insoluble plaques is a pathological hallmark of Alzheimer disease; soluble oligomeric Aβ has been hypothesized to more directly underlie impaired learning and memory in dementia of the Alzheimer type. However, the lack of a sensitive, specific, and quantitative assay for Aβ oligomers has hampered rigorous tests of this hypothesis., Methods: We developed a plate-based single molecule counting fluorescence immunoassay for oligomeric Aβ sensitive to low pg/ml concentrations of synthetic Aβ dimers using the same Aβ-specific monoclonal antibody to both capture and detect Aβ. The Aβ oligomer assay does not recognize monomeric Aβ, amyloid precursor protein, or other non-Aβ peptide oligomers., Results: Aβ oligomers were detected in aqueous cortical lysates from patients with dementia of the Alzheimer type and nondemented patients with Aβ plaque pathology. However, Aβ oligomer concentrations in demented patients' lysates were tightly correlated with Aβ plaque coverage (r = 0.88), but this relationship was weaker in those from nondemented patients (r = 0.30) despite equivalent Aβ plaque pathology. The ratio of Aβ oligomer levels to plaque density fully distinguished demented from nondemented patients, with no overlap between groups in this derived variable. Other Aβ and plaque measures did not distinguish demented from nondemented patients. Aβ oligomers were not detected in cerebrospinal fluid with this assay., Interpretation: The results raise the intriguing hypothesis that the linkage between plaques and oligomers may be a key pathophysiological event underlying dementia of the Alzheimer type. This Aβ oligomer assay may be useful for many tests of the oligomer hypothesis., (Copyright © 2012 American Neurological Association.)

The purpose of this study was to elucidate the alteration of catecholamine metabolism and the contribution of catecholamines to the decline of learning and memory in the brain of the senescence-accelerated mouse prone 10 (SAMP10) with aging. Catecholamines and their metabolites in the cerebral cortex were measured by HPLC-ECD. The protein levels of tyrosine hydroxylase (TH) as well as TH phosphorylated at Ser19 or Ser40, dopamine-β-hydroxylase (DβH), and cAMP-dependent protein kinase (PKA) were determined by western blot analysis. Dopamine (DA) and norepinephrine (NE) levels in SAMP10 were significantly lower than those in control animals. However, no significant difference was observed in catecholamine metabolite levels between SAMP10 and control mice. The level of TH phosphorylation at Ser40 in SAMP10 was significantly lower than that in control mice, but no significant difference was observed in the levels of TH, TH phosphorylated at Ser19, or DβH. The amount of PKA, which regulates the phosphorylation of TH at Ser40, was significantly lower in SAMP10 than in control mice. The present study demonstrated that a decline in DA and NE concentrations was observed in the cerebral cortex of SAMP10 with aging, and this decrease of catecholamine levels was caused by impairment of their synthetic pathway. These impairments are considered to be caused by downregulation of TH phosphorylation at Ser40 as a result of PKA deficiency. The present study suggests that the decline of learning and memory abilities of SAMP10 is caused by a decrease in catecholamine synthesis in the cerebral cortex with aging., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Objective: We examined regional benzodiazepine receptors (rBZR) using single photon emission CT (SPECT) in patients with Alzheimer disease (AD), vascular dementia (VaD), and mixed AD/VaD dementia (MD) and compared the changes in the availability of rBZR with those of regional cerebral blood flow (rCBF)., Methods: A total of 7 patients with AD, 6 with MD, and 9 with VaD underwent SPECT studies with N-isopropyl-p-[(123)I] iodoamphetamine and (123)I-iomazenil to measure rCBF and rBZR. The ratios of rCBF and rBZR uptake in brain subregions to the average global activity were compared among these diseases. In addition, we acquired z-score maps using 3-dimensional stereotactic surface projections of SPECT data., Results: Compared with AD, VaD and MD showed rCBF and rBZR reduction predominantly in the frontal lobe, but rBZR images revealed more extensive and severe defects than rCBF images. In contrast, AD showed rCBF and rBZR reduction predominantly in the parietotemporal lobe compared with VaD and MD, but rCBF images revealed more extensive defects than rBZR images., Conclusion: rCBF imaging can detect parietotemporal abnormalities in AD, while rBZR imaging may enable the demonstration of underlying pathophysiological differences in the frontal lobe between VaD, MD and AD, reflecting neuronal integrity in the cerebral cortex., (Copyright © 2012 Elsevier B.V. All rights reserved.)

MS-based proteomics has been the method of choice for biomarker discovery in the field of traumatic brain injury (TBI). Due to its high sensitivity and specificity, MS is now being explored for biomarker quantitative validation in tissue and biofluids. In this study, we demonstrate the use of MS in both qualitative protein identification and targeted detection of acute TBI biomarkers released from degenerating cultured rat cortical mixed neuronal cells, mimicking intracellular fluid in the central nervous system after TBI. Calpain activation was induced by cell treatment with maitotoxin (MTX), a known calcium channel opener. Separate plates of mixed neuronal-glial culture were subjected to excitotoxin N-methyl-D-aspartate (NMDA) and apoptotic inducer staurosporine. Acute TBI biomarkers, GFAP and UCH-L1, were first detected and assessed in the culture media by Western blot. The cell-conditioned media were then trypsinized and subjected to bottom up proteomic analysis. GFAP was readily detected by data-dependent scanning but not UCH-L1. As a proof-of-principle study, rat glia-enriched cell cultures treated with MTX were used to investigate the time-dependent release of GFAP breakdown product by Western blot and for isotope dilution MS absolute quantitation method development. Absolute quantitation of the GFAP release was conducted using the three cortical mixed neuronal cell cultures treated with different agents. Other differentially expressed proteins identified in the glial-enriched and cortical mixed neuronal cell culture models were further analyzed by bioinformatic tools. In summary, this study demonstrates the use of MS in both protein identification and targeted quantitation of acute TBI biomarkers and is the preliminary step toward development of TBI biomarker validation by targeted MS., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The central nervous system (CNS)-based symptoms of Gulf War Illness (GWI) include motor dysfunction, anxiety, and cognitive impairment. Gulf War (GW) agents, such as pyridostigmine bromide (PB), permethrin (PER), N,N-diethyl-meta-toluamide (DEET), and stress, are among the contributory factors to the pathobiology of GWI. This study characterizes disturbances in phosphocholine-containing lipids that accompany neurobehavioral and neuropathological features associated with GW agent exposure. Exposed mice received PB orally, dermal application of PER and DEET and restraint stress daily for 28 days, while controls received vehicle during this period. Neurobehavioral studies included the rotarod, open field, and Morris water maze tests. Histopathological assessments included glial fibrillary acid protein, CD45, and Nissl staining. Liquid chromatography/mass spectrometry with source collision-induced dissociation in negative and positive ionization scanning modes was performed to characterize brain phosphatidylcholine (PC) and sphingomyelin (SM). A significant increase in ether containing PC (ePC34:0, ePC36:2, and ePC36:1) or long-chain fatty acid-containing PC (38:1, 40:4, 40:2) was observed in exposed mice compared with controls. Among differentially expressed PCs, levels of those with monounsaturated fatty acids were more affected than those with saturated and polyunsaturated fatty acids. Sensorimotor deficits and anxiety, together with an increase in astrocytosis, were observed in exposed mice compared with controls. These lipid changes suggest that alterations in peroxisomal pathways and stearoyl-CoA desaturase activity accompany neurobehavioral and neuropathological changes after GW agent exposure and represent possible treatment targets for the CNS symptoms of GWI.

Somatostatin (SST)-positive medium-sized aspiny interneurons are selectively spared in excitotoxicity. The biological effects of SST are mediated via five different receptors, namely somatostatin receptor (SSTR)1-5; however, SSTR subtype spared in excitotoxicity and involved in neuroprotection is not known. Dopamine- and cAMP-regulated phosphoprotein (DARPP-32) is predominantly expressed in medium-sized projection neurons that are most vulnerable in excitotoxicity. In the present study, we determined the colocalization of SST and SSTRs with DARPP-32 in rat brain cortical and striatal regions using immunofluorescence immunohistochemistry. We also determined the expression of DARPP-32 in SSTR1-5 immunoprecipitate prepared from cortex and striatum. SST-positive neurons in cortex and striatum are devoid of colocalization with DARPP-32. However, in cortical and striatal brain regions, three different neuronal populations either expressing SSTRs and DARPP-32 alone or displaying colocalization were identified. Quantitative analysis reveals that in cortex and striatum, SSTR1 and 5 are most predominant receptor subtypes colocalized with DARPP-32 followed by SSTR4, 2, and 3 in cortex whereas SSTR2, 4, and 3 in striatum. Importantly, DARPP-32 is expressed in SSTR1-5 immunoprecipitate prepared from cortex and striatum. Taken together, these results provide the first evidence that the SSTR-positive neurons lacking colocalization with DARPP-32 might be spared in excitotoxicity.

Like amyloid beta (Aβ) oligomers, tau aggregates are increasingly recognized as potential key toxic intermediates in Alzheimer's disease (AD) and as therapeutic targets. P-tau co-localizes with Aβ in cortical AD synapses and may contribute to synapse dysfunction and loss. Flow cytometry analysis of synaptosomes from AD compared with aged cognitively normal cortex demonstrates increased immunolabeling for three p-tau antibodies (AT8, PHF-1 and pS422), indicating phosphorylation at multiple tau epitopes. Sequential extraction experiments show increased soluble p-tau in AD synapses, but a sizable pool of p-tau requires detergent solubilization, suggesting endosomal/lysosomal localization. P-tau is co-localized with Aβ in individual synaptosomes in dual labeling experiments, and flow cytometry sorting of Aβ-positive synaptosomes from an AD case reveals a marked enrichment of p-tau aggregates. The p-tau enrichment, a 76-fold increase over the initial homogenate, is consistent with sequestration of p-tau in internal synaptic compartments. Western analysis of a series of AD and normal cases shows SDS-stable tau oligomers in the dimer/trimer size range in AD samples. These results indicate that widespread synaptic p-tau pathology accompanies Aβ accumulations in surviving synaptic terminals, particularly in late-stage AD., (© 2012 The Authors. Brain Pathology © 2012 International Society of Neuropathology.)

Microtubules are nearly uniformly oriented in the axons of vertebrate neurons but are non-uniformly oriented in their dendrites. Studies to date suggest a scenario for establishing these microtubule patterns whereby microtubules are transported into the axon and nascent dendrites with plus-ends-leading, and then additional microtubules of the opposite orientation are transported into the developing dendrites. Here, we used contemporary tools to confirm that depletion of kinesin-6 (also called CHO1/MKLP1 or kif23) from rat sympathetic neurons causes a reduction in the appearance of minus-end-distal microtubules in developing dendrites, which in turn causes them to assume an axon-like morphology. Interestingly, we observed a similar phenomenon when we depleted kinesin-12 (also called kif15 or HKLP2). Both motors are best known for their participation in mitosis in other cell types, and both are enriched in the cell body and dendrites of neurons. Unlike kinesin-12, which is present throughout the neuron, kinesin-6 is barely detectable in the axon. Accordingly, depletion of kinesin-6, unlike depletion of kinesin-12, has no effect on axonal branching or navigation. Interestingly, depletion of either motor results in faster growing axons with greater numbers of mobile microtubules. Based on these observations, we posit a model whereby these two motors generate forces that attenuate the transport of microtubules with plus-ends-leading from the cell body into the axon. Some of these microtubules are not only prevented from moving into the axon but are driven with minus-ends-leading into developing dendrites. In this manner, these so-called "mitotic" motors coregulate the microtubule patterns of axons and dendrites.

Study Objectives: GABAergic and cholinergic transmission within the basal forebrain and cerebral cortex contribute to the regulation of sleep and wakefulness. In contrast to levels of acetylcholine (ACh), levels of endogenous GABA in basal forebrain and cortex during sleep and wakefulness have not previously been quantified. This study (1) tested the hypothesis that there are differential, state-specific changes in GABA levels within the substantia innominata (SI) region of the basal forebrain and somatosensory cortex; and (2) quantified the ratio of GABAergic to cholinergic transmission in the SI, cortex, and pontine reticular formation during rapid eye movement sleep (REM), non-REM sleep (NREM), and wakefulness., Design: Within/between subjects., Setting: University of Michigan., Patients or Participants: Adult, male, purpose bred cats (n = 5)., Interventions: In vivo microdialysis, high performance liquid chromatography, electrophysiological recordings., Measurements and Results: In the SI, GABA levels were significantly greater during NREM (17%) than during REM. In the cortex, GABA levels were significantly greater during NREM than during wakefulness (39%) and REM (63%). During prolonged wakefulness, there was a linear increase in cortical GABA levels, and the amount of time spent awake accounted for 87% of the variance in GABA. The GABA-to-ACh ratio was largest during NREM for all brain regions. REM was characterized by a 68% decrease in the GABA-to-ACh ratio across brain regions, always due to a decrease in GABA levels., Conclusion: Three of the brain regions that comprise the anatomically distributed, sleep-generating network have in common a GABA-mediated, sleep-dependent decrease in the GABA-to-ACh ratio.

Near infrared spectroscopy (NIRS) measures the regional tissue oxygen saturation (rSO2) of various organs and provides a reflection of the balance between tissue oxygen supply and demand. Oxymetry assessed via NIRS has been proposed as a 'standard of care' and today it is already widely used in the NICU. This approach allows detection of any acute change in cerebral haemodynamics and continuous monitoring of cerebral and somatic oxygenation. This work describes three clinical cases of preterm VLBW infants which showed special points of interest during both cerebral and somatic NIRS monitoring.

Although sleep is defined as a behavioral state, at the cortical level sleep has local and use-dependent features suggesting that it is a property of neuronal assemblies requiring sleep in function of the activation experienced during prior wakefulness. Here we show that mature cortical cultured neurons display a default state characterized by synchronized burst-pause firing activity reminiscent of sleep. This default sleep-like state can be changed to transient tonic firing reminiscent of wakefulness when cultures are stimulated with a mixture of waking neurotransmitters and spontaneously returns to sleep-like state. In addition to electrophysiological similarities, the transcriptome of stimulated cultures strikingly resembles the cortical transcriptome of sleep-deprived mice, and plastic changes as reflected by AMPA receptors phosphorylation are also similar. We used our in vitro model and sleep-deprived animals to map the metabolic pathways activated by waking. Only a few metabolic pathways were identified, including glycolysis, aminoacid, and lipids. Unexpectedly large increases in lysolipids were found both in vivo after sleep deprivation and in vitro after stimulation, strongly suggesting that sleep might play a major role in reestablishing the neuronal membrane homeostasis. With our in vitro model, the cellular and molecular consequences of sleep and wakefulness can now be investigated in a dish.

Perineuronal nets (PNNs) are lattice-like substructures of the neural extracellular matrix that enwrap particular populations of neurons throughout the central nervous system. Previous work suggests that this structure plays a major role in modulating developmental neural plasticity and brain maturation. Understanding the precise role of these structures has been hampered by incomplete comprehension of their molecular composition and cellular contributions to their formation, which is studied herein using primary cortical cell cultures. By defining culture conditions to reduce (cytosine-β-d-arabinofuranoside/AraC addition) or virtually eliminate (elevated potassium chloride (KCl) and AraC application) glia, PNN components impacted by this cell type were identified. Effects of depolarizing KCl concentrations alone were also assessed. Our work identified aggrecan as the primary neuronal component of the PNN and its expression was dramatically up-regulated by both depolarization and glial cell inhibition and additionally, the development of aggrecan-positive PNNs was accelerated. Surprisingly, most of the other PNN components tested were made in a glial-dependent manner in our culture system. Interestingly, in the absence of these glial-derived components, an aggrecan- and hyaluronan-reactive PNN developed, demonstrating that these two components are sufficient for base PNN assembly. Other components were expressed in a glial-dependent manner. Overall, this work provides deeper insight into the complex interplay between neurons and glia in the formation of the PNN and improves our understanding of the molecular composition of these structures., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system (1-5). To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex (6-8). Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions. Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method( 9). However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice (10-11) (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA (12). These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C). Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

This protocol describes a technique for high-resolution chloride imaging of living cells using a quinoline-based chloride (Cl(-)) indicator dye, MQAE (N-[6-methoxyquinolyl] acetoethyl ester). Bath-applied to acute brain slices, MQAE provides high-quality labeling of neuronal cells and their processes. In living anesthetized mice, cortical cells are labeled using the multicell bolus loading procedure. In combination with two-photon microscopy, this procedure enables in vivo visualization of cell bodies of neurons and astrocytes as well as some astrocytic processes and allows one to monitor changes in the intracellular chloride concentration in dozens of individual cells.

Study Objective: It is well established that brain metabolism is higher during wake and rapid eye movement (REM) sleep than in nonrapid eye movement (NREM) sleep. Most of the brain's energy is used to maintain neuronal firing and glutamatergic transmission. Recent evidence shows that cortical firing rates, extracellular glutamate levels, and markers of excitatory synaptic strength increase with time spent awake and decline throughout NREM sleep. These data imply that the metabolic cost of each behavioral state is not fixed but may reflect sleep-wake history, a possibility that is investigated in the current report., Design: Chronic (4d) electroencephalographic (EEG) recordings in the rat cerebral cortex were coupled with fixed-potential amperometry to monitor the extracellular concentration of oxygen ([oxy]) and lactate ([lac]) on a second-by-second basis across the spontaneous sleep-wake cycle and in response to sleep deprivation., Setting: Basic sleep research laboratory., Patients or Participants: Wistar Kyoto (WKY) adult male rats., Interventions: N/A., Measurements and Results: Within 30-60 sec [lac] and [oxy] progressively increased during wake and REM sleep and declined during NREM sleep (n = 10 rats/metabolite), but with several differences. [Oxy], but not [lac], increased more during wake with high motor activity and/or elevated EEG high-frequency power. Meanwhile, only the NREM decline of [lac] reflected sleep pressure as measured by slow-wave activity, mirroring previous results for cortical glutamate., Conclusions: The observed state-dependent changes in cortical [lac] and [oxy] are consistent with higher brain metabolism during waking and REM sleep in comparison with NREM sleep. Moreover, these data suggest that glycolytic activity, most likely through its link with glutamatergic transmission, reflects sleep homeostasis.

Objective: To investigate the effect of icariin (ICA) on learning and memory abilities and cholinergic system in senescence-accelerated mice SAMP10., Method: The 8-month-old senescence-accelerated mice were randomly divided into the model SAMP10 group and the positive Donepezil group (1 mg x kg(-1)) and ICA groups (50, 100, 200 mg x kg(-1)), with 12 mice in each group. Another 12 8-month-old mice SAMR1 were selected as the normal control group. After 30 days of oral administration, Morris water maze, SMG-2 water maze and experimental platform were used to test the effects of ICA on learning and memory abilities of SAMP10 groups. By colorimetric determination of AChE activity in the cortex, enzyme-linked immunosorbent assay detection of ACh, ChAT, MCBC of the cerebral cortex, the effect of ICA on the cholinergic system of SAMP10 was observed., Result: ICA could improve the abilities of space exploration and positioning navigation of SAMP10, shorten the latency in SMG-2 water maze, enhance their jumping ability in response to the passive test, and increase levels of ACh, ChAT, MCBC in the cerebral cortex of SAMP10. But its active effect on AChE in SAMP10 cortex was not obvious., Conclusion: Different doses of icariin can improve learning and memory abilities of SAMP10 to varying degrees, which may be related to its effect on the cholinergic system.

This study aimed to evaluate the adenine nucleotides and nucleoside concentration in serum and cerebral cortex of rats infected with Trypanosma evansi. Each rat was intraperitoneally infected with 1 × 10(6) trypomastigotes suspended in cryopreserved blood (Group A; n = 18). Twelve animals were used as controls (Group B). The infected animals were monitored daily by blood smears. At days 4 and 20 post-infection (PI) it was collected serum and cerebral cortex to measure the levels of ATP, ADP, AMP and adenosine by high performance liquid chromatography (HPLC). In serum there was a significant (P < 0.05) increase in the ATP, AMP and adenosine concentrations at days 4 and 20 PI in infected rats when compared to not-infected. Furthermore, in the cerebral cortex it was observed a significant (P < 0.05) increase in the concentrations of ATP, AMP and decreased adenosine levels at day 4 PI. At day 20 PI it was only observed an increase in the AMP and adenosine concentrations in cerebral cortex of infected rats when compared to not-infected. It was not observed any difference in ADP concentration in serum and brain at days 4 and 20 PI. No change was observed histologically in the cerebral cortex of infected animals. The results allow us to conclude that infection with T. evansi in rats causes an increase in the concentrations of ATP, AMP and adenosine in serum and cerebral cortex the time periods evaluated. These alterations occurred as a result of T. evansi infection which involves neurotransmission, neuromodulation and immune response impairment confirm the importance of the purinergic system in this pathology., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The plasticity of neural networks is a complex process determined by changes in physiological status, gene expression and phenotype of a cell. A detailed study of this process dynamics requires the simultaneous recording of electrical and genomic activities in networks of neurons. This sets up one of the tasks for modern neuroscience as development of integration of electrophysiology and molecular biology methods. In the paper we review the current approaches to such integration, as well as the choice of molecular markers for detection of genomic and synaptic plasticity of neurons by use of physiological micro-sensorial system based on neuronal cells cultured on the micro-electrode arrays.

Objectives: Alzheimer's disease (AD) is a neurodegenerative disorder marked by progressive loss of memory and impairment of cognitive ability. One current hypothesis for AD pathogenesis is that neuronal death is linked to aberrant cell-cycle re-entry. In AD, neurons have been shown to enter the cell cycle inappropriately without the ability to complete it fully and the aberrant re-entry leads to its death. Curcumin has been reported as having a neural protective effect on the AD model, and could modulate the proliferation of tumor cells through the regulation of cyclin D1 and c-myc cell signaling pathways. In this study, we first observed the protective action of curcumin on Abeta-induced neuron damage, and then investigated whether this protective effect was a result of the inhibition of cell cycle advance., Materials and Methods: We used MTT assay and TUNEL assay to observe the effect of curcumin on Abeta-induced neuron death, and then examined the activated caspase-3 protein level to further confirm the protective effect of curcumin against Abeta-induced neuron toxicity. Next, we further investigate whether the inhibition of cell cycle reentry was mediated by the therapeutic effect of curcumin on Abeta induced primary cultured neuron damage by Brdu label assay and western blot assay., Results: The results showed that administration of curcumin (1-10 microM) could inhibit Abeta25-35 (40 microg/ml) induced primary cultured rat cortical neuron death, down-regulating activated caspase-3 protein expression. Furthermore, treatment with curcumin could inhibit abnormal activated cyclin D1 protein level, and decrease the Brdu positive cells in proportion to the Abeta25-35 treatment neurons., Conclusions: All the results suggest that curcumin has a protective effect against Abeta-induced toxicity in cultured rat cortical neurons, the inhibition of cell cycle re-entry at least partly mediating the therapeutic effect of curcumin in the AD model in vitro.

Amyloid beta (Aβ) oligomers and phosphorylated tau (p-tau) aggregates are increasingly identified as potential toxic intermediates in Alzheimer's disease (AD). In cortical AD synapses, p-tau co-localizes with Aβ, but the Aβ and p-tau peptide species responsible for synaptic dysfunction and demise remains unclear. The present experiments were designed to use high-speed cell sorting techniques to purify synaptosome population based on size, and then extend the method to physically isolate Aβ-positive synaptosomes with the goal of understanding the nature of Aβ and tau pathology in AD synapses. To examine the purity of size-gated synaptosomes, samples were first gated on size; particles with sizes between 0.5 and 1.5 microns were collected. Electron microscopy documented a homogenous population of spherical particles with internal vesicles and synaptic densities. Next, size-gated synaptosomes positive for Aβ were collected by fluorescence activated sorting and then analyzed by immunoblotting techniques. Sorted Aβ-positive synaptosomes were enriched for amyloid precursor protein (APP) and for Aβ oligomers and aggregates; immunolabeling for p-tau showed a striking accumulation of p-tau aggregates compared to the original homogenate and purified synaptosomes. These results confirm co-localization of Aβ and p-tau within individual synaptic terminals and provide proof of concept for the utility of flow sorting synaptosomes., (Copyright © 2011 International Society for Advancement of Cytometry.)

We studied the subcellular levels of melatonin in cerebral cortex and liver of rats under several conditions. The results show that melatonin levels in the cell membrane, cytosol, nucleus, and mitochondrion vary over a 24-hr cycle, although these variations do not exhibit circadian rhythms. The cell membrane has the highest concentration of melatonin followed by mitochondria, nucleus, and cytosol. Pinealectomy significantly increased the content of melatonin in all subcellular compartments, whereas luzindole treatment had little effect on melatonin levels. Administration of 10 mg/kg bw melatonin to sham-pinealectomized, pinealectomized, or continuous light-exposed rats increased the content of melatonin in all subcellular compartments. Melatonin in doses ranging from 40 to 200 mg/kg bw increased in a dose-dependent manner the accumulation of melatonin on cell membrane and cytosol, although the accumulations were 10 times greater in the former than in the latter. Melatonin levels in the nucleus and mitochondria reached saturation with a dose of 40 mg/kg bw; higher doses of injected melatonin did not further cause additional accumulation of melatonin in these organelles. The results suggest some control of extrapineal accumulation or extrapineal production of melatonin and support the existence of regulatory mechanisms in cellular organelles, which prevent the intracellular equilibration of the indolamine. Seemingly, different concentrations of melatonin can be maintained in different subcellular compartments. The data also seem to support a requirement of high doses of melatonin to obtain therapeutic effects. Together, these results add information that assists in explaining the physiology and pharmacology of melatonin., (© 2011 John Wiley & Sons A/S.)

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg(1). We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 μm. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Nitric oxide synthases (NOSs) have been shown to modulate thermal hyperalgesia and mechanical hypersensitivity in inflammatory and neuropathic pain. However, little is known about the effect of NOSs on baseline function of sensory nerve fibers. Using genetic deficiency and pharmacologic inhibition of NOSs, we examined the impact of the three isoforms NOS1, NOS2, and NOS3 on baseline nocifensive behavior by measuring current vocalization threshold in response to electrical stimulation at 5, 250, 2000 Hz that preferentially stimulate C, Aδ, and Aβ fibers. In response to 5, 250 and 2000 Hz, NOS1-deficient animals had significantly higher current vocalization thresholds compared with wild-type. Genetic deficiency of NOS2 was associated with higher current vocalization thresholds in response to 5 Hz (C-fiber) stimulation. In contrast, NOS3-deficient animals had an overall weak trend toward lower current vocalization thresholds at 5 Hz and significantly lower current vocalization threshold compared with wild-type animals at 250 and 2000 Hz. Therefore, NOSs distinctively affect baseline mouse current vocalization threshold and appear to play a role on nocifensive response to electrical stimulation of sensory nerve fibers., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The perirhinal cortex (PRC) composed of areas 35 and 36 forms an important route for activity transfer between the hippocampus-entorhinal cortex and neocortex. Its function in memory formation and consolidation as well as in the initiation and spreading of epileptic activity was already partially elucidated. We studied the general pattern of calretinin (CR), parvalbumin (PV) and calbindin (CB) immunoreactivity and its corrected relative optical density (cROD) as well as morphological features and density of CR and PV immunoreactive (CR+, PV+) interneurons in the rat PRC. Neighboring neocortical association area Te3V was analyzed as well. The PRC differed from the Te3V in higher CR and lower PV overall immunoreactivity level. On CR immunostained sections, the difference between high cROD value in area 35 and low cROD value in area Te3V reached statistical significance (p<0.05). The pattern of CB immunoreactivity was similar to that of the neocortex. Vertically oriented bipolar neurons were the most common morphological type of CR+ neurons, multipolar neuronal morphology was typical among PV+ neurons and vertically oriented bipolar neurons and multipolar neurons were approximately equally frequent among CB+ neurons. The density of CR+ and PV+ neurons was stereologically measured. While the density of PV+ neurons was not significantly different in PRC when compared to Te3V, density of CR+ neurons in area 35 was significantly higher by comparison with Te3V (p<0.05). Further, the overall neuronal density was measured on Nissl stained sections and the proportion of CR+ and PV+ interneurons was expressed as a percentage of the total neurons counts. The percentage of CR+ interneurons was higher in area 35 by comparison with area Te3 (p<0.05), while the percentage of PV+ interneurons did not significantly differ among the examined areas. In conclusion, the PRC possesses specific interneuronal equipment with unusually high proportion of CR+ interneurons, what might be of importance for the presumed gating function of PRC in normal and diseased states., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We developed and validated silicon-based neural probes for neural stimulating and recording in long-term implantation in the brain. The probes combine the deep reactive ion etching process and mechanical shaping of their tip region, yielding a mechanically sturdy shank with a sharpened tip to reduce insertion force into the brain and spinal cord, particularly, with multiple shanks in the same array. The arrays' insertion forces have been quantified in vitro. Five consecutive chronically-implanted devices were fully functional from 3 to 18 months. The microelectrode sites were electroplated with iridium oxide, and the charge injection capacity measurements were performed both in vitro and after implantation in the adult feline brain. The functionality of the chronic array was validated by stimulating in the cochlear nucleus and recording the evoked neuronal activity in the central nucleus of the inferior colliculus. The arrays' recording quality has also been quantified in vivo with neuronal spike activity recorded up to 566 days after implantation. Histopathology evaluation of neurons and astrocytes using immunohistochemical stains indicated minimal alterations of tissue architecture after chronic implantation., (© 2011 IEEE)

Recent studies have shown that time-resolved optical measurements of the head can estimate changes in the absorption coefficient with depth discrimination. Thus, changes in tissue oxygenation, which are specific to intracranial tissues, can be assessed using this advanced technique, and this method allows us to avoid the influence of changes to extracerebral tissue oxygenation on the measured signals. We report the results of time-resolved optical imaging that was carried out during carotid endarterectomy. This surgery remains the "gold standard" treatment for carotid stenosis, and intraoperative brain oxygenation monitoring may improve the safety of this procedure. A time-resolved optical imager was utilized within the operating theater. This instrument allows for the simultaneous acquisition of 32 distributions of the time-of-flight of photons at two wavelengths on both hemispheres. Analysis of the statistical moments of the measured distributions of the time-of-flight of photons was applied for estimating changes in the absorption coefficient as a function of depth. Time courses of changes in oxy- and deoxyhemoglobin of the extra- and intracerebral compartments during cross-clamping of the carotid arteries were obtained. A decrease in the oxyhemoglobin concentration and an increase in the deoxyhemoglobin concentrations were observed in a large area of the head. Large changes were observed in the hemisphere ipsilateral to the site of clamped carotid arteries. Smaller amplitude changes were noted at the contralateral site. We also found that changes in the hemoglobin signals, as estimated from intracerebral tissue, are very sensitive to clamping of the internal carotid artery, whereas its sensitivity to clamping of the external carotid artery is limited. We concluded that intraoperative multichannel measurements allow for imaging of brain tissue hemodynamics. However, when monitoring the brain during carotid surgery, a single-channel measurement may be sufficient.

Intracerebral injection of brain extracts containing amyloid or tau aggregates in transgenic animals can induce cerebral amyloidosis and tau pathology. We extracted pure populations of tau oligomers directly from the cerebral cortex of Alzheimer disease (AD) brain. These oligomers are potent inhibitors of long term potentiation (LTP) in hippocampal brain slices and disrupt memory in wild type mice. We observed for the first time that these authentic brain-derived tau oligomers propagate abnormal tau conformation of endogenous murine tau after prolonged incubation. The conformation and hydrophobicity of tau oligomers play a critical role in the initiation and spread of tau pathology in the naïve host in a manner reminiscent of sporadic AD.

Neuronal LRRTM3 (leucine-rich repeat transmembrane 3) protein has been reported to promote amyloid-β protein precursor (AβPP) processing and LRRTM3 is a candidate gene in late-onset Alzheimer's disease. To address the role of LRRTM3 in AβPP processing and amyloid-β (Aβ) production in vivo, we analyzed amyloidogenic processing of AβPP in the brains of LRRTM3-deficient mice and transgenic AβPP/PS1 mice with or without LRRTM3. We did not find differences between the genotypes in the levels of Aβ or AβPP C-terminal fragments indicating that LRRTM3 is not an essential regulator of Aβ production in adult mice. Moreover, Aβ levels in primary cortical neurons were similar between the genotypes, indicating that LRRTM3 is not required for Aβ generation in developing mice.

Docosahexenoic acid (DHA, 22:6n-3) plays an important role in development of proper brain function in mammals. We have previously reported that DHA promotes synaptogenesis and synaptic function in hippocampal neurons while DHA-depletion in the brain due to n-3 fatty acid deficiency produces opposite effects. To gain insight into underlying molecular mechanisms, we investigated whether the brain DHA status affects the synaptic plasma membrane (SPM) proteome by using nanoLC-ESI-MS/MS and (16)O/(18)O labeling. The DHA level in mouse brains was lowered by dietary depletion of n-3 fatty acids, and SPM was prepared by differential centrifugation followed by osmotic shock. SPM proteins from DHA-adequate and depleted brains were analyzed by nanoLC-ESI-MS/MS after SDS-PAGE, in-gel digestion, and differential O(18)/O(16) labeling. This strategy allowed comparative quantitation of more than 200 distinct membrane or membrane-associated proteins from DHA-adequate or depleted brains. We found that 18 pre- and postsynaptic proteins that are relevant to synaptic physiology were significantly down-regulated in DHA-depleted mouse brains. The protein network analysis suggests involvement of CREB and caspase-3 pathways in the DHA-dependent modulation of synaptic proteome. Reduction of specific synaptic proteins due to brain DHA-depletion may be an important mechanism for the suboptimal brain function associated with n-3 fatty acid deficiency.

Reduced glutamatergic signaling may contribute to cognitive dysfunction in schizophrenia. Glutamatergic synapses might be the site of primary abnormalities in this disorder with the dopaminergic changes being secondary to altered glutamatergic transmission. Isolation rearing of rats from weaning has been used as an experimental model for affective disorders like schizophrenia. In this immunohistochemistry study we evaluate the changes in the expression of GluR1 and GluR2 AMPA receptors in the hippocampus, amygdala and entorhinal cortex induced by isolation rearing. Two groups of Wistar rats (grouped and isolated, n = 6/each) were used. Isolation rearing induced a significant decrease in GluR1- and GluR2-immunopositive cells in the hippocampus. For GluR1 the reduction was 31% in the hilus of dentate gyrus (p = 0.02) and 47% in CA3 (p = 0.002). For GluR2 the reduction was 52% in the hilus of dentate gyrus (p < 0.0001) and 29% in CA1 (p = 0.002). Isolation rearing induced a non-significant decrease in GluR1-immunopositive cells in the basolateral amygdala (p = 0.066) while no alteration was found in the lateral nucleus (p = 0.657). For GluR2 no changes were induced by isolation in both nuclei of the amygdala. In the entorhinal cortex no apparent difference was seen in GluR1- or GluR2-immunopositive cells when isolated reared rats were compared to grouped rats. The results suggest that isolation rearing from weaning induces changes on the expression of AMPA glutamate receptors in the hippocampus similar to those reported for postmortem human brains with schizophrenia. These findings also contribute to additional evidence for using isolation rearing of rats from weaning as an animal model for schizophrenia.

Myelin sheath disruption is responsible for multiple neuropathies in the central and peripheral nervous system. Myelin imaging has thus become an important diagnosis tool. However, in vivo imaging has been limited to either low-resolution techniques unable to resolve individual fibers or to low-penetration imaging of single fibers, which cannot provide quantitative information about large volumes of tissue, as required for diagnostic purposes. Here, we perform myelin imaging without labeling and at micron-scale resolution with >300-μm penetration depth on living rodents. This was achieved with a prototype [termed deep optical coherence microscopy (deep-OCM)] of a high-numerical aperture infrared full-field optical coherence microscope, which includes aberration correction for the compensation of refractive index mismatch and high-frame-rate interferometric measurements. We were able to measure the density of individual myelinated fibers in the rat cortex over a large volume of gray matter. In the peripheral nervous system, deep-OCM allows, after minor surgery, in situ imaging of single myelinated fibers over a large fraction of the sciatic nerve. This allows quantitative comparison of normal and Krox20 mutant mice, in which myelination in the peripheral nervous system is impaired. This opens promising perspectives for myelin chronic imaging in demyelinating diseases and for minimally invasive medical diagnosis.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a valuable tool for the analysis of molecules directly from tissue. Imaging of phospholipids is gaining widespread interest, particularly as these lipids have been implicated in a variety of pathologic processes. Formalin fixation (FF) is the standard protocol used in histology laboratories worldwide to preserve tissue for analysis, in order to aid in the diagnosis and prognosis of diseases. This study assesses MALDI imaging of phospholipids directly in formalin fixed tissue, with a view to future analysis of archival tissue. This investigation proves the viability of MALDI-MSI for studying the distribution of lipids directly in formalin fixed tissue, without any pretreatment protocols. High quality molecular images for several phosphatidylcholine (PC) and sphingomyelin (SM) species are presented. Images correspond well with previously published data for the analysis of lipids directly from freshly prepared tissue. Different ionization pathways are observed when analyzing fixed tissue compared with fresh, and this change was found to be associated with formalin buffers employed in fixation protocols. The ability to analyze lipids directly from formalin fixed tissue opens up new doors in the investigation of disease profiles. Pathologic specimens taken for histologic investigation can be analyzed by MALDI-MS to provide greater information on the involvement of lipids in diseased tissue.

Although disrupted in schizophrenia 1 (DISC1) has been implicated in many psychiatric disorders, including schizophrenia, bipolar disorder, schizoaffective disorder and major depression, its biological role in these disorders is unclear. To better understand this gene and its role in psychiatric disease, we conducted transcriptional profiling and genome-wide association analysis in 1232 pedigreed Mexican-American individuals for whom we have neuroanatomic images, neurocognitive assessments and neuropsychiatric diagnoses. SOLAR was used to determine heritability, identify gene expression patterns and perform association analyses on 188 quantitative brain-related phenotypes. We found that the DISC1 transcript is highly heritable (h(2)=0.50; P=1.97 × 10(-22)), and that gene expression is strongly cis-regulated (cis-LOD=3.89) but is also influenced by trans-effects. We identified several DISC1 polymorphisms that were associated with cortical gray matter thickness within the parietal, temporal and frontal lobes. Associated regions affiliated with memory included the entorhinal cortex (rs821639, P=4.11 × 10(-5); rs2356606, P=4.71 × 10(-4)), cingulate cortex (rs16856322, P=2.88 × 10(-4)) and parahippocampal gyrus (rs821639, P=4.95 × 10(-4)); those affiliated with executive and other cognitive processing included the transverse temporal gyrus (rs9661837, P=5.21 × 10(-4); rs17773946, P=6.23 × 10(-4)), anterior cingulate cortex (rs2487453, P=4.79 × 10(-4); rs3738401, P=5.43 × 10(-4)) and medial orbitofrontal cortex (rs9661837; P=7.40 × 10(-4)). Cognitive measures of working memory (rs2793094, P=3.38 × 10(-4)), as well as lifetime history of depression (rs4658966, P=4.33 × 10(-4); rs12137417, P=4.93 × 10(-4)) and panic (rs12137417, P=7.41 × 10(-4)) were associated with DISC1 sequence variation. DISC1 has well-defined genetic regulation and clearly influences important phenotypes related to psychiatric disease.

Purpose: Statins are selective inhibitors of 3-hydroxyl-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of the mevalonate pathway for cholesterol biosynthesis. Increasing evidence indicates that statins, particularly atorvastatin, are neuroprotective in several conditions, including stroke, cerebral ischemia, traumatic brain injury, and excitotoxic amino acid exposure. However, only a few studies have investigated whether statins modulate seizure activity. In the current study we investigated whether atorvastatin or simvastatin alters the seizures induced by pentylenetetrazol (PTZ), a classical convulsant., Methods: Adult male Wistar rats were treated with atorvastatin or simvastatin for 7 days (10 mg/kg/day). Seizure activity was induced by PTZ (60 mg/kg, i.p.), and evaluated by behavioral and electrographic methods. Cholesterol levels were determined by a standard spectrophotometric method. Blood-brain barrier (BBB) permeability was assessed by the fluorescein method. Atorvastatin levels in the plasma and cerebral cortex were determined by high-performance liquid chromatography tandem mass spectrometry., Key Findings: We found that oral atorvastatin treatment increased the latency to PTZ-induced generalized seizures. In contrast, when the 7-day atorvastatin treatment was withheld for 1 day (i.e., atorvastatin withdrawal), PTZ-induced seizures were facilitated, as evidenced by a decrease in the latency to clonic and generalized tonic-clonic seizures induced by PTZ. In contrast, simvastatin treatment for 7 days (10 mg/kg/day, p.o.), with or without withdrawal, did not alter PTZ-induced seizures. Interestingly, the effects of atorvastatin treatment and withdrawal were not accompanied by changes in plasma or cerebral cortex cholesterol levels or in the BBB permeability. Atorvastatin levels in the plasma and cerebral cortex after 7 days of treatment were above the half maximal inhibitory concentration for inhibition of HMG-CoA reductase, whereas atorvastatin was not detectable in the plasma or cerebral cortex following a 24 h washout period (atorvastatin withdrawal)., Significance: We conclude that atorvastatin treatment and withdrawal have differential effects on pentylenetetrazol-induced seizures, which are not related to changes in plasma or cerebral cortex cholesterol levels or in BBB permeability. Additional studies are necessary to evaluate the molecular mechanisms underlying our findings as well as its clinical implications., (Wiley Periodicals, Inc. © 2011 International League Against Epilepsy.)

The FoxO3-dependent increase in type II deiodinase (D2), which converts the prohormone thyroxine (T(4)) to 3,5,3'-triiodothyronine (T(3)), is required for normal mouse skeletal muscle differentiation and regeneration. This implies a requirement for an increase in D2-generated intracellular T(3) under these conditions, which has not been directly demonstrated despite the presence of D2 activity in skeletal muscle. We directly show that D2-mediated T(4)-to-T(3) conversion increases during differentiation in C(2)C(12) myoblast and primary cultures of mouse neonatal skeletal muscle precursor cells, and that blockade of D2 eliminates this. In adult mice given (125)I-T(4) and (131)I-T(3), the intracellular (125)I-T(3)/(131)I-T(3) ratio is significantly higher than in serum in both the D2-expressing cerebral cortex and the skeletal muscle of wild-type, but not D2KO, mice. In D1-expressing liver and kidney, the (125)I-T(3)/(131)I-T(3) ratio does not differ from that in serum. Hypothyroidism increases D2 activity, and in agreement with this, the difference in (125)I-T(3)/(131)I-T(3) ratio is increased further in hypothyroid wild-type mice but not altered in the D2KO. Notably, in wild-type but not in D2KO mice, the muscle production of (125)I-T(3) is doubled after skeletal muscle injury. Thus, D2-mediated T(4)-to-T(3) conversion generates significant intracellular T(3) in normal mouse skeletal muscle, with the increased T(3) required for muscle regeneration being provided by increased D2 synthesis, not by T(3) from the circulation.

1,3,5,7-Tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-s-indacene (TMBB-Su), a new BODIPY-based fluorescent probe, was designed and synthesized for the labeling of amino compounds. It was used as a pre-column derivatizing reagent for determination of amino acid neurotransmitters by high-performance liquid chromatography (HPLC). The fluorescence quantum yield in acetonitrile increased from 0.84 to 0.95 when it reacted with amino acid neurotransmitters. Derivatization of TMBB-Su with seven amino acid neurotransmitters was completed within 30 min at 25 °C in 24.0 mmol L(-1) pH 7.8 boric acid buffer. The separation was performed on a C(18) column with methanol-water-buffer 55:35:10 (v/v) as mobile phase (buffer: 0.10 mol L(-1) H(3)Cit-0.10 mol L(-1) NaOH). Interference from the other concomitant amino acids was eliminated successfully by means of pH gradient elution. With fluorescence detection at 494 and 504 nm for excitation and emission, respectively, the limits of detection (signal-to-noise ratio = 3) were from 2.1 to 12.0 nmol L(-1). The proposed method has been used to determine amino acid neurotransmitters in the cerebral cortex of mice with cerebral ischemia at the convalescence stage with satisfactory recoveries varying from 94.9 to 105.2%.