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Several studies have demonstrated that mouse models of Alzheimer's disease (AD) can exhibit impaired peripheral glucose tolerance. Further, in the APP/PS1 mouse model, this is observed prior to the appearance of AD-related neuropathology (e.g., amyloid-β plaques; Aβ) or cognitive impairment. In the current study, we examined whether impaired glucose tolerance also preceded AD-like changes in the triple transgenic model of AD (3xTg-AD). Glucose tolerance testing (GTT), insulin ELISAs, and insulin tolerance testing (ITT) were performed at ages prior to (1-3 months and 6-8 months old) and post-pathology (16-18 months old). Additionally, we examined for altered insulin signaling in the hippocampus. Western blots were used to evaluate the two-primary insulin signaling pathways: PI3K/AKT and MAPK/ERK. Since the PI3K/AKT pathway affects several downstream targets associated with metabolism (e.g., GSK3, glucose transporters), western blots were used to examine possible alterations in the expression, translocation, or activation of these targets. We found that 3xTg-AD mice display impaired glucose tolerance as early as 1 month of age, concomitant with a decrease in plasma insulin levels well prior to the detection of plaques (∼14 months old), aggregates of hyperphosphorylated tau (∼18 months old), and cognitive decline (≥18 months old). These alterations in peripheral metabolism were seen at all time points examined. In comparison, PI3K/AKT, but not MAPK/ERK, signaling was altered in the hippocampus only in 18-20-month-old 3xTg-AD mice, a time point at which there was a reduction in GLUT3 translocation to the plasma membrane. Taken together, our results provide further evidence that disruptions in energy metabolism may represent a foundational step in the development of AD.

Epidemiological data have shown that metabolic disease can increase the propensity for developing cognitive decline and dementia, particularly Alzheimer's disease (AD). While this interaction is not completely understood, clinical studies suggest that both hyper- and hypoinsulinemia are associated with an increased risk for developing AD. Indeed, insulin signaling is altered in post-mortem brain tissue from AD patients and treatments known to enhance insulin signaling can improve cognitive function. Further, clinical evidence has shown that AD patients and mouse models of AD often display alterations in peripheral metabolism. Since insulin is primarily derived from the periphery, it is likely that changes in peripheral insulin levels lead to alterations in central nervous system (CNS) insulin signaling and could contribute to cognitive decline and pathogenesis. Developing a better understanding of the relationship between alterations in peripheral metabolism and cognitive function might provide a foundation for the development of better treatment options for patients with AD. In this article we will begin to piece together the present data defining this relationship by briefly discussing insulin signaling in the periphery and CNS, its role in cognitive function, insulin's relationship to AD, peripheral metabolic alterations in mouse models of AD and how information from these models helps understand the mechanisms through which these changes potentially lead to impairments in insulin signaling in the CNS, and potential ways to target insulin signaling that could improve cognitive function in AD. This article is part of the Special Issue entitled 'Metabolic Impairment as Risk Factors for Neurodegenerative Disorders.', (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Alzheimer's disease (AD) is a devastating neurodegenerative disorder bearing multiple pathological hallmarks suggestive of complex cellular/molecular interplay during pathogenesis. Transgenic mice and nonhuman primates are used as disease models for mechanistic and translational research into AD; the extent to which these animal models recapitulate AD-type neuropathology is an issue of importance. Putative C-terminal fragments from sortilin, a member of the vacuolar protein sorting 10 protein (Vps10p) family, have recently been shown to deposit in the neuritic β-amyloid (Aβ) plaques in the human brain., Methods: We set out to explore if extracellular sortilin neuropathology exists in AD-related transgenic mice and nonhuman primates. Brains from different transgenic strains and ages developed overt cerebral Aβ deposition, including the β-amyloid precursor protein and presenilin 1 double-transgenic (APP/PS1) mice at ~ 14 months of age, the five familial Alzheimer's disease mutations transgenic (5×FAD) mice at ~ 8 months, the triple-transgenic Alzheimer's disease (3×Tg-AD) mice at ~ 22 months, and aged monkeys (Macaca mulatta and Macaca fascicularis) were examined. Brain samples from young transgenic mice, middle-aged/aged monkeys, and AD humans were used as negative and positive pathological controls., Results: The C-terminal sortilin antibody, which labeled senile plaques in the AD human cerebral sections, did not display extracellular immunolabeling in the transgenic mouse or aged monkey brain sections with Aβ deposition. In Western blot analysis, sortilin fragments ~ 15 kDa were not detectable in transgenic mouse cortical lysates, but they occurred in control AD lysates., Conclusions: In reference to their human brain counterparts, neuritic plaques seen in transgenic AD model mouse brains represent an incomplete form of this AD pathological hallmark. The species difference in neuritic plaque constituents also indicates more complex secondary proteopathies in the human brain relative to rodents and nonhuman primates during aging and in AD.

Alzheimer's disease (AD) is the primary cause of dementia in the elderly. The cause of the disease is still unknown, but amyloid plaques and neurofibrillary tangles in the brain are thought to play a role. However, transgenic mouse models expressing these neuropathological features do not show severe or consistent cognitive impairments. There is accumulating evidence that diabetes increases the risk for developing AD. We tested the hypothesis that experimentally induced diabetes would exacerbate cognitive symptoms in a mouse model of AD. Diabetes was induced in 12-month old 3xTg mice using streptozotocin (STZ; 90mg/kg, i.p., on two successive days). Hyperglycemia was verified by sampling blood glucose levels. Three months after injection (at 15 months of age), the mice were behaviorally tested in the Morris water maze and contextual fear conditioning. Subsequently, the hippocampal region was examined using immunohistochemistry (6E10 antibody for amyloid) and immunoblotting (AT8 antibody for phosphorylated tau). No differences were found in learning or memory between the vehicle-treated control and STZ-treated groups. A significant increase in the number of amyloid-positive plaques was observed in the subiculum of STZ-treated mice; very few plaques were seen in other hippocampal regions in either group. No differences in AT8 load were observed. These results reinforce that amyloid plaques, per se, are not sufficient to cause memory impairments. Further, while diabetes can enhance this aspect of brain pathology, the combination of disrupted glucose metabolism and the transgenes is still not sufficient to cause the severe cognitive impairments associated with clinical AD., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by beta-amyloid (Aβ) deposition, neurofibrillary tangles and cognitive decline. Clinical data suggests that both type 1 and type 2 diabetes are risk factors for AD-related dementia and several clinical studies have demonstrated that AD patients show alterations in peripheral glucose regulation characterized by insulin resistance (hyperinsulinemia) or hypoinsulinemia. Whether animal models of AD exhibit a pre-diabetic phenotype without additional dietary or experimental manipulation is unclear however, with contradictory data available. Further, most studies have not examined the time course of potential pre-diabetic changes relative to AD pathogenesis and cognitive decline. Thus, in this study we tested the hypothesis that a pre-diabetic phenotype (peripheral metabolic dysregulation) exists in the APP/PS1 transgenic model of AD under normal conditions and precedes AD-related pathology. Specifically, we examined glucose tolerance in male APP/PS1 mice on a C57BL/6J congenic background at 2, 4-6 and 8-9months of age by assessing fasting glucose levels, glucose tolerance, plasma insulin levels and insulin sensitivity as well as the development of pathological characteristics of AD and verified that our APP/PS1 mice develop cognitive impairment. Here we show that APP/PS1 mice, compared to wild-type controls, exhibit a significant impairment in glucose tolerance during an intraperitoneal glucose tolerance test (ipGTT) and a trend for increased fasting plasma insulin concentrations as early as 2months of age, while extracellular Aβ 1-42 deposition occurs later and cognitive decline exists at 8-9months of age. Moreover, APP/PS1 mice did not respond as well to exogenous insulin as the wild-type controls during an intraperitoneal insulin tolerance test (ipITT). Taken together, these data reveal that male APP/PS1 mice on a C57BL/6J congenic background exhibit a pre-diabetic phenotype prior to the development of AD-like pathology and that this metabolic deficit persists when they exhibit neuropathology and cognitive decline. This raises the question of whether altered glucose regulation and insulin production/secretion could contribute to AD pathogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cognitive function declines with age and appears to correlate with decreased cerebral metabolic rate (CMR). Caloric restriction, an antiaging manipulation that extends life-span and can preserve cognitive function, is associated with decreased glucose uptake, decreased lactate levels, and increased ketone body (KB) levels in the brain. Since the majority of brain nutrients come from the periphery, this study examined whether the capacity to regulate peripheral glucose levels and KB production differs in animals with successful cognitive aging (growth hormone receptor knockouts, GHRKOs) versus unsuccessful cognitive aging (the 3xTg-AD mouse model of Alzheimer's disease). Animals were fasted for 5 hours with their plasma glucose and KB levels subsequently measured. Intriguingly, in GHRKO mice, compared to those in controls, fasting plasma glucose levels were significantly decreased while their KB levels were significantly increased. Conversely, 3xTg-AD mice, compared to controls, exhibited significantly elevated plasma glucose levels and significantly reduced plasma KB levels. Taken together, these results suggest that the capacity to provide the brain with KBs versus glucose throughout an animal's life could somehow help preserve cognitive function with age, potentially through minimizing overall brain exposure to reactive oxygen species and advanced glycation end products and improving mitochondrial function.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by beta-amyloid (Aβ) deposition, neurofibrillary tangles and cognitive decline. Recent pharmacologic studies have found that ATP-sensitive potassium (K ATP ) channels may play a role in AD and could be a potential therapeutic target. Interestingly, these channels are found in both neurons and astrocytes. One of the hallmarks associated with AD is reactive gliosis and a change in astrocytic function has been identified in several neuropathological conditions including AD. Thus the goal of this study was to examine whether the pore-forming subunits of K ATP channels, Kir6.1 and Kir6.2, are altered in the hippocampus in a cell type-specific manner of the 3xTg-AD mouse model of AD and in human AD tissue obtained from the Chinese brain bank. Specifically, in old 3xTg-AD mice, and age-matched controls, we examined glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), Kir6.1 and Kir6.2 in hippocampal region CA1 with a combination of immunoblotting and immunohistochemistry (IHC). A time point was selected when memory impairment and histopathological changes have been reported to occur in 3xTg-AD mice. In human AD and age-matched control tissue IHC experiments were performed using GFAP and Kir6.2. In the hippocampus of 3xTg-AD mice, compared to wild-type controls, Western blots showed a significant increase in GFAP indicating astrogliosis. Further, there was an increase in Kir6.2, but not Kir6.1 in the plasma membrane fraction. IHC examination of hippocampal region CA1 in 3xTg-AD sections revealed an increase in Kir6.2 immunoreactivity (IR) in astrocytes as identified by GFAP and GS. In human AD tissue similar data were obtained. There was an increase in GFAP-IR in the stratum oriens (SO) and alveus (ALV) of CA1 concomitant with an increase in Kir6.2-IR in cells with an astrocytic-like morphology. Dual immunofluorescence revealed a dramatic increase in co-localization of Kir6.2-IR and GFAP-IR. Taken together, these data demonstrate that increased Kir6.2 is seen in reactive astrocytes in old 3xTg-AD mice and human AD tissue. These changes could dramatically alter astrocytic function and subsequently contribute to AD phenotype in either a compensatory or pathophysiological manner., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neural stem/progenitor cells have been characterized at neurogenic sites in adult mammalian brain with various molecular markers. Here it has been demonstrated that Sp8, a transcription factor typically expressed among mature GABAergic interneurons, also labels putative neural precursors in adult guinea pig and human cerebrum. In guinea pigs, Sp8 immunoreactive (Sp8+) cells were localized largely in the superficial layers of the cortex including layer I, as well as the subventricular zone (SVZ) and subgranular zone (SGZ). Sp8+ cells at the SGZ showed little colocalization with mature and immature neuronal markers, but co-expressed neural stem cell markers including Sox2. Some layer I Sp8+ cells also co-expressed Sox2. The amount of Sp8+ cells in the dentate gyrus was maintained 2 weeks after X-ray irradiation, while that of doublecortin (DCX+) cells was greatly reduced. Mild ischemic insult caused a transient increase of Sp8+ cells in the SGZ and layer I, with the subgranular Sp8+ cells exhibited an increased colabeling for the mitotic marker Ki67 and pulse-chased bromodeoxyuridine (BrdU). Sp8+ cells in the dentate gyrus showed an age-related decline in guinea pigs, in parallel with the loss of DCX+ cells in the same region. In adult humans, Sp8+ cells exhibited comparable morphological features as seen in guinea pigs, with those at the SGZ and some in cortical layer I co-expressed Sox2. Together, these results suggested that Sp8 may label putative neural progenitors in guinea pig and human cerebrum, with the labeled cells in the SGZ appeared largely not mitotically active under normal conditions. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 939-955, 2016., (© 2015 Wiley Periodicals, Inc.)

Glutamatergic synaptic activity entails a high energetic cost. During aging, a variety of neural metabolic changes have been reported that could compromise the capacity of neural circuits to maintain synaptic transmission during periods of reduced extracellular glucose. Indeed, a preferential compromise in evoked synaptic activity has been observed in hippocampal CA1 with age during exposure to low-glucose solutions. Whether this aging-related compromise in synaptic activity is regionally specific is unclear, however. Data suggest that the dentate gyrus (DG) preferentially exhibits hypometabolism with age and this region plays a critical role in spatial pattern separation, which is compromised with age. Therefore, we assessed whether synaptic activity is also preferentially affected in the DG with age. In vitro extracellular field potential recordings were used to monitor orthodromic and antidromic evoked activity in the DG granule cell layer in hippocampal slices from adult (8-12 months) and aged (22-27 months) rats in aCSF containing 10mM glucose, followed by a reduced glucose aCSF containing 1mM glucose. In 10mM glucose-aCSF, orthodromic- and antidromic-evoked field potential activity was comparable between age groups. However, orthodromic-evoked population spike amplitude and field excitatory post-synaptic potential (EPSP) slope were preferentially decreased in slices from aged rats during exposure to 1mM glucose-aCSF. Antidromic population spike amplitude was not differentially affected in slices from aged versus adult rats, however. These data suggest that synaptic efficacy is preferentially compromised with age under reduced glucose availability and, combined with a decreased capacity of the periphery to provide glucose to the central nervous system (CNS) during metabolically challenging conditions, could contribute to aging-related hippocampal dysfunction and cognitive decline., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Cells expressing doublecortin (DCX+) occur at cortical layer II, predominantly over the paleocortex in mice/rats, but also across the neocortex among larger mammals. Here, we explored the time of origin of these cells in neonatal and 2-month-old guinea pigs following prenatal BrdU pulse-chasing. In the neocortex, BrdU+ cells birth-dated at embryonic day 21 (E21), E28, and E35 laminated over the cortical plate with an inside-out order. In the piriform cortex, cells generated at E21 and E28 occurred with a greater density in layer II than III. Many cells were generated at later time points until birth, occurring in the cortex without a laminar preference. DCX+ cells in the neocortex and piriform cortex partially co-colocalized with BrdU (up to 7.5%) in the newborns after pulse-chasing from E21 to E49 and in the 2 month-old animals after pulse-chasing from E28 to E60/61, with higher rates seen among the E21-E35 groups. Together, layer II DCX+ cells in neonatal and young adult guinea pigs may be produced over a wide prenatal time window, but mainly during the early phases of corticogenesis. Our data also show an earlier establishment of the basic lamination in the piriform relative to neocortical areas in guinea pigs.

Microbleeds are a common finding in aged human brains. In Alzheimer's disease (AD), neuritic plaques composed of β-amyloid (Aβ) deposits and dystrophic neurites occur frequently around cerebral vasculature, raising a compelling question as to whether, and if so, how, microvascular abnormality and amyloid/neuritic pathology might be causally related. Here we used a guinea pig model of cerebral microembolism to explore a potential inductive effect of vascular injury on neuritic and amyloid pathogenesis. Brains were examined 7-30 days after experimental microvascular embolization occupying ~0.5% of total cortical area. Compared to sham-operated controls, glial fibrillary acidic protein immunoreactivity was increased in the embolized cerebrum, evidently around intracortical vasculature. Swollen/sprouting neurites exhibiting increased reactivity of nicotinamide adenine dinucleotide phosphate diaphorase, parvalbumin, vesicular glutamate transporter 1 and choline acetyltransferase appeared locally in the embolized brains in proximity to intracortical vasculature. The embolization-induced swollen/sprouting neurites were also robustly immunoreactive for β-amyloid precursor protein and β-secretase-1, the substrate and initiating enzyme for Aβ genesis. These experimental data suggest that microvascular injury can induce multisystem neuritic pathology associated with an enhanced amyloidogenic potential in wild-type mammalian brain.

Chronic neuroinflammation is thought to play an etiological role in Alzheimer's disease (AD), which is characterized pathologically by amyloid and tau formation, as well as neuritic dystrophy and synaptic degeneration. The causal relationship between these pathological events is a topic of ongoing research and discussion. Recent data from transgenic AD models point to a tight spatiotemporal link between neuritic and amyloid pathology, with the obligatory enzyme for β -amyloid (A β ) production, namely β -secretase-1 (BACE1), is overexpressed in axon terminals undergoing dystrophic change. However, the axonal pathology inherent with BACE1 elevation seen in transgenic AD mice may be secondary to increased soluble A β in these genetically modified animals. Here we explored the occurrence of the AD-like axonal and dendritic pathology in adult rat brain affected by LPS-induced chronic neuroinflammation. Unilateral intracerebral LPS injection induced prominent inflammatory response in glial cells in the ipsilateral cortex and hippocampal formation. BACE1 protein levels were elevated the ipsilateral hippocampal lysates in the LPS treated animals relative to controls. BACE1 immunoreactive dystrophic axons appeared in the LPS-treated ipsilateral cortex and hippocampal formation, colocalizing with increased β -amyloid precursor protein and A β antibody (4G8) immunolabeling. Quantitative Golgi studies revealed reduction of dendritic branching points and spine density on cortical layer III and hippocampal CA3 pyramidal neurons in the LPS-treated ipsilateral cerebrum. These findings suggest that Alzheimer-like amyloidogenic axonal pathology and dendritic degeneration occur in wildtype mammalian brain in partnership with neuroinflammation following LPS injection.

The spinal cord is composed of distinct neuronal groups with well-defined anatomic connections. In some transgenic (Tg) models of Alzheimer's disease (AD), amyloid plaques develop in this structure, although the underlying cellular mechanism remains elusive. We attempted to explore the origin, evolution, and modulation of spinal β-amyloid (Aβ) deposition using Tg mice harboring five familiar AD-related mutations (5XFAD) as an experiential model. Dystrophic neuritic elements with enhanced β-secretase-1 (BACE1) immunoreactivity (IR) appeared as early as 2 months of age, and increased with age up to 12 months examined in this study, mostly over the ventral horn (VH). Extracellular Aβ IR emerged and developed during this same period, site-specifically co-existing with BACE1-labeled neurites often in the vicinity of large VH neurons that expressed the mutant human APP. The BACE1-labeled neurites almost invariably colocalized with β-amyloid precursor protein (APP) and synaptophysin, and frequently with the vesicular glutamate transporter-1 (VGLUT). Reduced IR for the neuronal-specific nuclear antigen (NeuN) occurred in the VH by 12 months of age. In 8-month-old animals surviving 6 months after a unilateral sciatic nerve transection, there were significant increases of Aβ, BACE1, and VGLUT IR in the VN of the ipsilateral relative to contralateral lumbar spinal segments. These results suggest that extracellular Aβ deposition in 5XFAD mouse spinal cord relates to a progressive and amyloidogenic synaptic pathology largely involving presynaptic axon terminals from projection neurons in the brain. Spinal neuritic plaque formation is enhanced after peripheral axotomy, suggesting a retrograde transneuronal modulation on pathogenesis.

Deposition of β -amyloid (Aβ) peptides, cleavage products of β-amyloid precursor protein (APP) by β-secretase-1 (BACE1) and γ-secretase, is a neuropathological hallmark of Alzheimer's disease (AD). γ-Secretase inhibition is a therapeutical anti-Aβ approach, although changes in the enzyme's activity in AD brain are unclear. Cerebrospinal fluid (CSF) Aβ peptides are thought to derive from brain parenchyma and thus may serve as biomarkers for assessing cerebral amyloidosis and anti-Aβ efficacy. The present study compared active γ-secretase binding sites with Aβ deposition in aged and AD human cerebrum, and explored the possibility of Aβ production and secretion by the choroid plexus (CP). The specific binding density of [(3) H]-L-685,458, a radiolabeled high-affinity γ-secretase inhibitor, in the temporal neocortex and hippocampal formation was similar for AD and control cases with similar ages and post-mortem delays. The CP in post-mortem samples exhibited exceptionally high [(3) H]-L-685,458 binding density, with the estimated maximal binding sites (Bmax) reduced in the AD relative to control groups. Surgically resected human CP exhibited APP, BACE1 and presenilin-1 immunoreactivity, and β-site APP cleavage enzymatic activity. In primary culture, human CP cells also expressed these amyloidogenic proteins and released Aβ40 and Aβ42 into the medium. Overall, our results suggest that γ-secretase activity appears unaltered in the cerebrum in AD and is not correlated with regional amyloid plaque pathology. The CP appears to be a previously unrecognised non-neuronal contributor to CSF Aβ, probably at reduced levels in AD., (© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Network patterns are believed to provide unique temporal contexts for coordinating neuronal activity within and across different regions of the brain. Some of the characteristics of network patterns modeled in vitro are altered in the CA3 or CA1 subregions of hippocampal slices from aged mice. CA3-CA1 network interactions have not been examined previously. We used slices from aged and adult mice to model spontaneous sharp wave ripples and carbachol-induced gamma oscillations, and compared measures of CA3-CA1 network timing between age groups. Coherent sharp wave ripples and gamma oscillations were evident in the CA3-CA1 circuit in both age groups, but the relative timing of activity in CA1 stratum pyramidale was delayed in the aged. In another sample of aged slices, evoked Schaffer collateral responses were attenuated in CA3 (antidromic spike amplitude) and CA1 (orthodromic field EPSP slope). However, the amplitude and timing of spontaneous sharp waves recorded in CA1 stratum radiatum were similar to adults. In both age groups unit activity recorded juxtacellularly from unidentified neurons in CA1 stratum pyramidale and stratum oriens was temporally modulated by CA3 ripples. However, aged neurons exhibited reduced spike probability during the early cycles of the CA3 ripple oscillation. These findings suggest that aging disrupts the coordination of patterned activity in the CA3-CA1 circuit.

The brain is capable of remarkable synaptic reorganization following stress and injury, often using the same molecular machinery that governs neurodevelopment. This form of plasticity is crucial for restoring and maintaining network function. However, neurodegeneration and subsequent reorganization can also play a role in disease pathogenesis, as is seen in temporal lobe epilepsy and Alzheimer's disease. β-Secretase-1 (BACE1) is a protease known for cleaving β-amyloid precursor protein into β-amyloid (Aβ), a major constituent in amyloid plaques. Emerging evidence suggests that BACE1 is also involved with synaptic plasticity and nerve regeneration. Here we examined whether BACE1 immunoreactivity (IR) was altered in pilocarpine-induced epileptic CD1 mice in a manner consistent with the synaptic reorganization seen during epileptogenesis. BACE1-IR increased in the CA3 mossy fiber field and dentate inner molecular layer in pilocarpine-induced epileptic mice, relative to controls (saline-treated mice and mice 24-48 h after pilocarpine-status), and paralleled aberrant expression of neuropeptide Y. Regionally increased BACE1-IR also occurred in neuropil in hippocampal area CA1 and in subregions of the amygdala and temporal cortex in epileptic mice, colocalizing with increased IR for growth associated protein 43 (GAP43) and polysialylated-neural cell adhesion molecule (PSA-NCAM), but reduced IR for microtubule-associated protein 2 (MAP2). These findings suggest that BACE1 is involved in aberrant limbic axonal sprouting in a model of temporal lobe epilepsy, warranting further investigation into the role of BACE1 in physiological vs. pathological neuronal plasticity., (Published by Elsevier Inc.)

β-amyloid precursor protein (APP) and presenilins mutations cause early-onset familial Alzheimer's disease (FAD). Some FAD-based mouse models produce amyloid plaques, others do not. β-Amyloid (Aβ) deposition can manifest as compact and diffuse plaques; it is unclear why the same Aβ molecules aggregate in different patterns. Is there a basic cellular process governing Aβ plaque pathogenesis? We showed in some FAD mouse models that compact plaque formation is associated with a progressive axonal pathology inherent with increased expression of β-secretase (BACE1), the enzyme initiating the amyloidogenic processing of APP. A monoclonal Aβ antibody, 3D6, visualized distinct axon terminal labeling before plaque onset. The present study was set to understand BACE1 and axonal changes relative to diffuse plaque development and to further characterize the novel axonal Aβ antibody immunoreactivity (IR), using triple transgenic AD (3xTg-AD) mice as experimental model. Diffuse-like plaques existed in the forebrain in aged transgenics and were regionally associated with increased BACE1 labeled swollen/sprouting axon terminals. Increased BACE1/3D6 IR at axon terminals occurred in young animals before plaque onset. These axonal elements were also co-labeled by other antibodies targeting the N-terminal and mid-region of Aβ domain and the C-terminal of APP, but not co-labeled by antibodies against the Aβ C-terminal and APP N-terminal. The results suggest that amyloidogenic axonal pathology precedes diffuse plaque formation in the 3xTg-AD mice, and that the early-onset axonal Aβ antibody IR in transgenic models of AD might relate to a cross-reactivity of putative APP β-carboxyl terminal fragments.

Spectrins line the intracellular surface of plasmalemma and play a critical role in supporting cytoskeletal stability and flexibility. Spectrins can be proteolytically degraded by calpains and caspases, yielding breakdown products (SBDPs) of various molecular sizes, with SBDP120 being largely derived from caspase-3 cleavage. SBDPs are putative biomarkers for traumatic brain injury. The levels of SBDPs also elevate in the brain during aging and perhaps in Alzheimer's disease (AD), although the cellular basis for this change is currently unclear. Here we examined age-related SBDP120 alteration in forebrain neurons in rats and in the triple transgenic model of AD (3×Tg-AD) relative to non-transgenic controls. SBDP120 immunoreactivity (IR) was found in cortical neuronal somata in aged rats, and was prominent in the proximal dendrites of the olfactory bulb mitral cells. Western blot and densitometric analyses in wild-type mice revealed an age-related elevation of intraneuronal SBDP120 in the forebrain which was more robust in their 3×Tg-AD counterparts. The intraneuronal SBDP120 occurrence was not spatiotemporally correlated with transgenic amyloid precursor protein (APP) expression, β-amyloid plaque development, or phosphorylated tau expression over various forebrain regions or lamina. No microscopically detectable in situ activated caspase-3 was found in the nuclei of SBDP120-containing neurons. The present study demonstrates the age-dependent intraneuronal presence of an αII-spectrin cleavage fragment in mammalian forebrain which is exacerbated in a transgenic model of AD. This novel neuronal alteration indicates that impairments in membrane protein metabolism, possibly due to neuronal calcium mishandling and/or enhancement of calcium sensitive proteolysis, occur during aging and in transgenic AD mice.

The comorbidity between epilepsy and Alzheimer's disease (AD) is a topic of growing interest. Senile plaques and tauopathy are found in epileptic human temporal lobe structures, and individuals with AD have an increased incidence of spontaneous seizures. However, why and how epilepsy is associated with enhanced AD-like pathology remains unknown. We have recently shown β-secretase-1 (BACE1) elevation associated with aberrant limbic axonal sprouting in epileptic CD1 mice. Here we sought to explore whether BACE1 upregulation affected the development of Alzheimer-type neuropathology in mice expressing mutant human APP, presenilin and tau proteins, the triple transgenic model of AD (3×Tg-AD). 3×Tg-AD mice were treated with pilocarpine or saline (i.p.) at 6-8 months of age. Immunoreactivity (IR) for BACE1, β-amyloid (Aβ) and phosphorylated tau (p-tau) was subsequently examined at 9, 11 or 14 months of age. Recurrent convulsive seizures, as well as mossy fiber sprouting and neuronal death in the hippocampus and limbic cortex, were observed in all epileptic mice. Neuritic plaques composed of BACE1-labeled swollen/sprouting axons and extracellular AβIR were seen in the hippocampal formation, amygdala and piriform cortices of 9 month-old epileptic, but not control, 3×Tg-AD mice. Densities of plaque-associated BACE1 and AβIR were elevated in epileptic versus control mice at 11 and 14 months of age. p-Tau IR was increased in dentate granule cells and mossy fibers in epileptic mice relative to controls at all time points examined. Thus, pilocarpine-induced chronic epilepsy was associated with accelerated and enhanced neuritic plaque formation and altered intraneuronal p-tau expression in temporal lobe structures in 3×Tg-AD mice, with these pathologies occurring in regions showing neuronal death and axonal dystrophy.

Clinical data and experimental studies in rats have shown that the aged CNS is more susceptible to the proconvulsive effects of the excitotoxic glutamate analogues kainate (KA) and domoate (DA), which bind high-affinity receptors localized at mossy fiber (MF) synapses in the CA3 subregion of the hippocampus. Although decreased renal clearance appears to play a role in the hypersensitivity of the aged hippocampus to systemically-administered DA, it is unclear if the excitability of the CA3 network is also altered with age. Therefore, this study monitored CA3 field potential activity in hippocampal slices from aged and adult male Fischer 344 rats in response to electrical and pharmacological network stimulation targeted to the MF-CA3 circuit. Network challenges with repetitive hilar stimulation or bath application of nanomolar concentrations of KA more readily elicited excitable network activity (e.g. population spike facilitation, multiple population spikes, and epileptiform bursts) in slices from aged vs. adult rats, although basal network excitability was comparable between age groups. Additionally, exposure to 200 nM KA often abolished epileptiform activity and revealed theta or gamma oscillations instead. However, slices from aged rats were less sensitive to the rhythmogenic effects of 200 nM KA. Taken together, these findings suggest that aging decreases the capacity of the CA3 network to constrain the spread of excitability during focal excitatory network challenges.

Caloric restriction enhances N-methyl-D-aspartate (NMDA) receptor binding and upregulates messenger RNA expression of the GluN1 subunit during aging. Old growth hormone receptor knockout mice resemble old calorically restricted rodents in enhanced life span and brain function, as compared with aged controls. This study examined whether aged growth hormone receptor knockout mice also show enhanced expression of NMDA receptors. Six or 23- to 24-month-old male normal-sized control or dwarf growth hormone receptor knockout mice were assayed for NMDA-displaceable [(3)H]glutamate binding (autoradiography) and GluN1 subunit messenger RNA (in situ hybridization). There was slight sparing of NMDA receptor binding densities within aged medial prefrontal and motor cortices, similar to caloric restriction, but there were greater age-related declines in GluN1 messenger RNA in growth hormone receptor knockout versus control mice. These results suggest that some of the functional improvements in aged mice with altered growth hormone signaling may be due to enhancement of NMDA receptors, but not through the upregulation of messenger RNA for the GluN1 subunit.

Alzheimer's disease (AD) is the most common dementia-causing disorder in the elderly; it may be related to multiple risk factors, and is characterized pathologically by cerebral hypometabolism, paravascular β-amyloid peptide (Aβ) plaques, neuritic dystrophy, and intra-neuronal aggregation of phosphorylated tau. To explore potential pathogenic links among some of these lesions, we examined β-secretase-1 (BACE1) alterations relative to Aβ deposition, neuritic pathology and vascular organization in aged monkey and AD human cerebral cortex. Western blot analyses detected increased levels of BACE1 protein and β-site-cleavage amyloid precursor protein C-terminal fragments in plaque-bearing human and monkey cortex relative to controls. In immunohistochemistry, locally elevated BACE1 immunoreactivity (IR) occurred in AD but not in control human cortex, with a trend for increased overall density among cases with greater plaque pathology. In double-labeling preparations, BACE1 IR colocalized with immunolabeling for Aβ but not for phosphorylated tau. In perfusion-fixed monkey cortex, locally increased BACE1 IR co-existed with intra-axonal and extracellular Aβ IR among virtually all neuritic plaques, ranging from primitive to typical cored forms. This BACE1 labeling localized to swollen/sprouting axon terminals that might co-express one or another neuronal phenotype markers (GABAergic, glutamatergic, cholinergic, or catecholaminergic). Importantly, these BACE1-labeled dystrophic axons resided near to or in direct contact with blood vessels. These findings suggest that plaque formation in AD or normal aged primates relates to a multisystem axonal pathogenesis that occurs in partnership with a potential vascular or metabolic deficit. The data provide a mechanistic explanation for why senile plaques are present preferentially near the cerebral vasculature., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

A considerable number of cells expressing typical immature neuronal markers including doublecortin (DCX+) are present around layer II in the cerebral cortex of young and adult guinea pigs and other larger mammals, and their origin and biological implication await further characterization. We show here in young adult guinea pigs that these DCX+ cells are accompanied by in situ cell division around the superficial cortical layers mostly in layer I, but they co-express proliferating cell nuclear antigen (PCNA) and an early neuronal fate determining factor, PAX6. A small number of these DCX+ cells also colocalize with BrdU following administration of this mitotic indicator. Cranial X-ray irradiation causes a decline of DCX+ cells around layer II, and novel environmental exploration induces c-Fos expression among these cells in several neocortical areas. Together, these data are compatible with a notion that DCX+ cortical neurons around layer II might derive from proliferable neuronal precursors around layer I in young adult guinea pig cerebrum, and that these cells might be modulated by experience under physiological conditions., (Copyright 2010 Elsevier Inc. All rights reserved.)

Cerebral hypometabolism and amyloid accumulation are principal neuropathological manifestations of Alzheimer's disease (AD). Whether and how brain/neuronal activity might modulate certain pathological processes of AD are interesting topics of recent clinical and basic research in the field, and may be of potential medical relevance in regard to both the disease etiology and intervention. Using the Tg2576 transgenic mouse model of AD, this study characterized a promotive effect of neuronal hypoactivity associated with functional deprivation on amyloid plaque pathogenesis in the olfactory pathway. Unilateral naris-occlusion caused beta-secretase-1 (BACE1) elevation in neuronal terminals in the deprived relative to the non-deprived bulb and piriform cortex in young adult mice. In parallel with the overall age-related plaque development in the forebrain, locally increased BACE1 immunoreactivity co-occurred with amyloid deposition first in the piriform cortex then within the bulb, more prominent on the deprived relative to the non-deprived side. Biochemical analyses confirmed elevated BACE1 protein levels, enzymatic activity and products in the deprived relative to non-deprived bulbs. Plaque-associated BACE1 immunoreactivity in the bulb and piriform cortex was localized preferentially to swollen/sprouting glutamatergic axonal terminals, with Abeta immunoreactivity occurring inside as well as around these terminals. Together, these findings suggest that functional deprivation or neuronal hypoactivity facilitates amyloid plaque formation in the forebrain in a transgenic model of AD, which operates synergistically with age effect. The data also implicate an intrinsic association of amyloid accumulation and plaque formation with progressive axonal pathology.

The amyloid cascade hypothesis has guided much of the research into Alzheimer's disease (AD) over the last 25 years. We argue that the hypothesis of amyloid-β (Aβ) as the primary cause of dementia may not be fully correct. Rather, we propose that decline in brain metabolic activity, which is tightly linked to synaptic activity, actually underlies both the cognitive decline in AD and the deposition of Aβ. Aβ may further exacerbate metabolic decline and result in a downward spiral of cognitive function, leading to dementia. This novel interpretation can tie the disparate risk factors for dementia to a unifying hypothesis and present a roadmap for interventions to decrease the prevalence of dementia in the elderly population.

The presence of neuritic plaques is a pathological hallmark of Alzheimer's disease (AD). However, the origin of extracellular beta-amyloid peptide (Abeta) deposits and the process of plaque development remain poorly understood. The present study attempted to explore plaque pathogenesis by localizing beta-secretase-1 (BACE1) elevation relative to Abeta accumulation and synaptic/neuritic alterations in the forebrain, using transgenic mice harboring familial AD (FAD) mutations (5XFAD and 2XFAD) as models. In animals with fully developed plaque pathology, locally elevated BACE1 immunoreactivity (IR) coexisted with compact-like Abeta deposition, with BACE1 IR occurring selectively in dystrophic axons of various neuronal phenotypes or origins (GABAergic, glutamatergic, cholinergic or catecholaminergic). Prior to plaque onset, localized BACE1/Abeta IR occurred at swollen presynaptic terminals and fine axonal processes. These BACE1/Abeta-containing axonal elements appeared to undergo a continuing process of sprouting/swelling and dystrophy, during which extracellular Abeta IR emerged and accumulated in surrounding extracellular space. These data suggest that BACE1 elevation and associated Abeta overproduction inside the sprouting/dystrophic axonal terminals coincide with the onset and accumulation of extracellular amyloid deposition during the development of neuritic plaques in transgenic models of AD. Our findings appear to be in harmony with an early hypothesis that axonal pathogenesis plays a key or leading role in plaque formation.

A novel population of cells that express typical immature neuronal markers including doublecortin (DCX+) has been recently identified throughout the adult cerebral cortex of relatively large mammals (guinea pig, rabbit, cat, monkey and human). These cells are more common in the associative relative to primary cortical areas and appear to develop into interneurons including type II nitrinergic neurons. Here we further describe these cells in the cerebral cortex and amygdala, in comparison with DCX+ cells in the hippocampal dentate gyrus, in three age groups of rhesus monkeys: young adult (12.3 +/- 0.2 years, n = 3), mid-age (21.2 +/- 1.9 years, n = 3) and aged (31.3 +/- 1.8 years, n = 4). DCX+ cells with a heterogeneous morphology persisted in layers II/III primarily over the associative cortex and amygdala in all groups (including in two old animals with cerebral amyloid pathology), showing a parallel decline in cell density with age across regions. In contrast to the cortex and amygdala, DCX+ cells in the subgranular zone diminished in the mid-age and aged groups. DCX+ cortical cells might arrange as long tangential migratory chains in the mid-age and aged animals, with apparently distorted cell clusters seen in the aged group. Cortical DCX+ cells colocalized commonly with polysialylated neural cell adhesion molecule and partially with neuron-specific nuclear protein and gamma-aminobutyric acid, suggesting a potential differentiation of these cells into interneuron phenotype. These data suggest a life-long role for immature interneuron-like cells in the associative cerebral cortex and amygdala in nonhuman primates.

DCX-immunoreactive (DCX+) cells occur in the piriform cortex in adult mice and rats, but also in the neocortex in adult guinea pigs and rabbits. Here we describe these cells in adult domestic cats and primates. In cats and rhesus monkeys, DCX+ cells existed across the allo- and neocortex, with an overall ventrodorsal high to low gradient at a given frontal plane. Labeled cells formed a cellular band in layers II and upper III, exhibiting dramatic differences in somal size (5-20 microm), shape (unipolar, bipolar, multipolar and irregular), neuritic complexity and labeling intensity. Cell clusters were also seen in this band, and those in the entorhinal cortex extended into deeper layers as chain-like structures. Densitometry revealed a parallel decline of the cells across regions with age in cats. Besides the cellular band, medium-sized cells with weak DCX reactivity resided sparsely in other layers. Throughout the cortex, virtually all DCX+ cells co-expressed polysialylated neural cell adhesion molecule. Medium to large mature-looking DCX+ cells frequently colocalized with neuron-specific nuclear protein and gamma-aminobutyric acid (GABA), and those with a reduced DCX expression also partially co-labeled for glutamic acid decarboxylase, parvalbumin, calbindin, beta-nicotinamide adenine dinucleotide phosphate diaphorase and neuronal nitric oxide synthase. Similar to cats and monkeys, small and larger DCX+ cells were detected in surgically removed human frontal and temporal cortices. These data suggest that immature neurons persist into adulthood in many cortical areas in cats and primates, and that these cells appear to undergo development and differentiation to become functional subgroups of GABAergic interneurons.

Doublecortin-immunoreactive (DCX+) cells were detected across the allo- and neo-cortical regions in the adult guinea pig cerebrum, localized to layer II specifically at its border with layer I. The density of labeled cells declined with age, whereas no apparent apoptotic activity was detectable over the cortex including layer II. DCX+ cells varied in somal size, labeling intensity, nuclear appearance, and complexity of processes. These cells were often arranged in clusters with cells of similar morphology sometimes packed tightly together. They exhibited complete colocalization with polysialylated neural cell adhesion molecule (PSA-NCAM) and neuron-specific type III beta-tubulin (TuJ1). Medium to large-sized DCX+ cells had well-developed neuritic processes, and expressed neuron-specific nuclear protein (NeuN). Large mature-looking cells with weak DCX reactivity invariably displayed heavy NeuN reactivity, implicating a transitional stage of these labeled cells. These "transitional" cells also consistently exhibited weak reactivity for gamma-aminobutyric acid (GABA), glutamate decarboxylase (GAD67), beta-nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) and neuronal nitric oxide synthase (nNOS), suggestive of them being young GABAergic/nitrinergic interneurons. Our data indicate that DCX+ cells exist widely in the adult guinea pig cerebral cortex, with a predominant localization in upper layer II. The morphological variation and differential expression of neuronal markers in these cells implicate that they might be developing neurons, and that they are probably differentiating into GABAergic interneurons. This population of cells might be involved in interneuron plasticity in the adult mammalian cerebral cortex.

Purpose: The elderly have an increased incidence and prevalence for seizure disorders. Further, since up to 50% of these cases have no identifiable antecedent, it has been hypothesized that aging of the central nervous system itself may be epileptogenic. Aged rats, compared to adults, exhibit a greater susceptibility to and severity of seizures associated with hippocampal activation. Whether this aging-related change reflects proconvulsive changes in limbic circuitry is unknown and thus was the focus of this study., Methods: Hippocampal slices from adult and aged Fischer 344 rats were examined using electrophysiological techniques. The dentate gyrus was our model region since it is involved with both wet-dog shakes and limbic seizures, and it is affected preferentially with age., Results: No differences were noted between groups in field potential activity elicited with low frequency stimulation. In contrast, 5-Hz molecular layer stimulation could evoke multiple population spikes in approximately 40% of aged versus 0% of adult slices. Further, recording in CA3 revealed that this stimulation paradigm could elicit multiple spikes in aged, but not adult, slices that frequently evolved into spontaneous epileptiform bursts. This change in the capacity of the dentate to respond to and filter afferent input was associated with an aging-related decrease in the frequency of spontaneous IPSPs and an increased propensity for large amplitude prolonged EPSPs following disinhibition., Conclusions: These epileptogenic changes in dentate function and circuitry could contribute to the exacerbated susceptibility for hippocampal seizures in aged rodents, as well as the aging-related decline in spatial learning and memory.

The central nervous system (CNS) undergoes a variety of anatomic, physiologic, and behavioral changes during aging. One region that has received a great deal of attention is the hippocampal formation due to the increased incidence of impaired spatial learning and memory with age. The hippocampal formation is also highly susceptible to Alzheimer's disease, ischemia/hypoxia, and seizure generation, the three most common aging-related neurological disorders. While data reveal that the dentate gyrus plays a key role in hippocampal function and dysfunction, the majority of electrophysiological studies that have examined the effects of age on the hippocampal formation have focused on CA3 and CA1. We perceive this to be an oversight and consequently will highlight data in this review which demonstrate an age-related disruption in dentate circuitry and function, and propose that these changes contribute to the decline in hippocampal-dependent behavior seen with "normal" aging.

The availability of human hippocampi obtained through surgery (usually for treatment of temporal lobe epilepsy) has allowed us to investigate the properties of the human dentate in a way that cannot be done with other brain regions. The dentate has been the primary focus of these studies because of its relative preservation in all patient specimens. Moreover, there is extensive synaptic reorganization of numerous neurotransmitter systems in this the fascia dentate (dentate gyrus and the hilus) in humans with specific forms of TLE. These changes are not evident in tissue from patients with seizure that begin outside the hippocampus, and, as a result, this tissue provides an invaluable resource for comparisons. Physiological data using both slices and acutely dissociated cells demonstrate that the granule cells have membrane properties similar to those of rodents although there are specific changes that appear to be associated with seizures. Similarly, in the non-sclerotic hippocampi, the synaptic properties are similar to those reported in rodents. There are also a number of parallels between the findings in humans and in status animal models of temporal lobe epilepsy. This review will cover analyses of membrane properties as well as of glutamatergic, GABAergic, and neuromodulatory systems. Thus, while there are a number of issues that invariably arise with studies of pathological human tissue, this tissue is ideally suited to verify and refine animal models of temporal lobe epilepsy. In addition, one can argue that human tissue provides the only resource to evaluate the ways that granule cells recorded from laboratory animals approximate human granule cell physiology.

Although cortical malformations (CMs) are often associated with epilepsy, the underlying mechanisms are unknown. The reeler mouse is a model of CM with enhanced susceptibility to epileptiform activity, including the in vitro dentate gyrus, a region normally resistant to seizures. In this study, field potential recordings in hippocampal slices and the Timm stain were used to examine mossy fiber distribution in the dentate gyrus. In artificial cerebrospinal fluid containing bicuculline, 100% of reeler slices and 0% of control slices had spontaneous and antidromic evoked prolonged negative field potential shifts that were blocked by glutamate receptor antagonists. Sections from reeler mice, but not controls, exhibited a dark band of Timm's stain at the molecular layer/granule cell layer border. These data reveal that mossy fiber distribution is altered in reeler mice and coincides with the presence of an abnormal proconvulsive glutamatergic circuit.

The outcome of patients with traumatic brain injury (TBI) can be predicted by the extracellular potassium concentration and the change in energy homeostasis. In this study, the authors investigated the effects of high potassium concentrations on extracellular levels of glucose, pyruvate and lactate in the rat striatum. Applying artificial cerebrospinal fluid (ACSF) enriched with 120 mM potassium by reverse microdialysis leads to an increase in lactate and reduction in glucose and pyruvate. Consequently, the lactate to pyruvate ratio was also increased. These data are discussed in the context of recent studies on lactate/pyruvate conversion and the potential mechanisms whereby high potassium could affect this equilibrium. We conclude that ischemic-like events are unlikely to explain these K(+)-induced changes.

Reeler mice are a model of cortical malformation with enhanced seizure susceptibility. Data suggest that the propensity to anesthesia-induced seizures may be enhanced in animal models with developmental anomalies. Consequently, reeler mice were monitored behaviorally before, during and after isoflurane anesthesia. During recovery, 12% of reeler homozygotes had class I/II seizures while the remaining 88% exhibited convulsive seizures entailing opisthotonus and forepaw drumming. Similar behavior was not observed in controls. These data reveal that reeler mice display isoflurane-induced seizures and provide support for the hypothesis that developmental anomalies may predispose the central nervous system to anesthesia-induced seizures.

Purpose: Seizures are observed frequently in humans with diffuse neuronal migration disorders. The reeler mutant mouse also exhibits a diffuse disruption of migration, yet no pro-epileptic phenotype has been reported for this model. Whether this disparity reflects a phenotypic difference that can be used to delineate the mechanisms associated with increasing seizure susceptibility or reflects a paucity of knowledge is unclear. Consequently, this study examined whether seizure susceptibility is altered in reeler mutant mice., Methods: In vivo (minimal electroshock delivered transcorneally) and in vitro techniques (field-potential recordings in neocortical and hippocampal brain slice preparations exposed to bicuculline methiodide) were used to determine whether the susceptibility to epileptiform activity is enhanced in reeler homozygous mice relative to controls. Adult (3-7 months) male reeler homozygotes (rl/rl) and controls (+/?) were identified based on their behavioral phenotype and were used in all experiments., Results: Minimal electroshock revealed that rl/rl mice, compared with controls, exhibited a lower threshold for electroshock-induced seizures (4.5 +/- 0.52 vs. 6.7 +/- 0.35 mA), and a higher incidence of behavioral seizures (median seizure score, class 4 vs. class 0) when animals were subjected to a 5-mA electroshock stimulus. Additionally, neocortical and hippocampal slices from rl/rl mice were more likely to generate spontaneous epileptiform activity after bicuculline application, compared with controls, and the duration of the epileptiform events elicited in 10-30 muM bicuculline was longer in slices from rl/rl mice., Conclusions: These data demonstrate that rl/rl mice have enhanced seizure susceptibility that is in part intrinsic to the malformed neocortex and hippocampus. Thus in contrast to prior belief, most animal models of diffuse neuronal migration disorders do exhibit a pro-epileptic phenotype.

A PubMed search of the years 1965 to 2003 found only 30 articles that were directly related to modeling seizures or epilepsy in aged animals. This lack of research is disturbing but explainable because of the high cost of aged animals and their increasing infirmity. Many changes occur in the older brain: cell loss in the hippocampal formation, changes in long-term potentiation maintenance, alteration in kindling, increased susceptibility to status epilepticus, and neuronal damage from stroke. The effect of aging on voltage-gated sodium and calcium channels has not been studied sufficiently. With increasing numbers of elderly persons with epilepsy needing appropriate treatment, the need to better understand the basic mechanisms of epilepsy is crucial.

Human temporal lobe epilepsy (TLE) is associated with bioenergetic abnormalities including decreased phosphocreatine (PCr) normalized to ATP. The physiological consequences of these metabolic alterations have not been established. We hypothesized that impaired bioenergetics would correlate with alterations in physiological functions under conditions that strongly activate neural metabolism. We correlated several physiological variables obtained from epileptic human dentate granule cells studied in slices with hippocampal PCr/ATP measured using in vivo magnetic resonance spectroscopy. The physiological variables included: the ability to fire multiple action potentials in response to single stimuli, the inhibitory postsynaptic potential (IPSP) conductance and the responses to a 10 Hz, 10 s stimulus train. We noted a significant negative correlation between the ability to fire multiple spikes in response to single synaptic stimulation and PCr/ATP (P < 0.03) and a positive correlation between the IPSP conductance and PCr/ATP (P < 0.05). Finally, there was a strong correlation between PCr/ATP and the recovery of the membrane potential following a stimulus train (P < 0.01), with low PCr/ATP being associated with prolonged recovery times. These data suggest that the bioenergetic impairment seen in this tissue is associated with specific changes in excitatory and inhibitory neuronal responses to synchronized synaptic inputs.

Purpose: The elderly exhibit an increased risk for developing status epilepticus and status-related morbidity and mortality. However, it is unclear how aging alters the progression of electroencephalographic (EEG) activity and behavioral manifestations during status epilepticus., Methods: A repetitive low-dose kainate treatment protocol (2.5 mg/kg/h; i.p.) was used in this study in conjunction with EEG and behavioral monitoring from freely behaving adult (7-8 months) and aged (22-25 months) Fischer 344 rats to assess the effects of aging on status epilepticus., Results: During kainate treatment, both groups exhibited an increase in EEG power that corresponded with the time course of kainate treatment. However, visual inspection and spectral analysis revealed a reduction of the faster frequencies (12.5-35 Hz) in the EEGs of aged rodents. A similar progression of behavioral manifestations was observed in adult and aged rodents during kainate treatment, although the frequency of preseizure manifestations (e.g., wet-dog shakes; aged rats, 110 events/h vs. adults, 25 events/h; median values) was greater, and latency to onset for any given behavioral manifestation (e.g., class V seizures; aged median, 60 min, vs. adult median, 145 min) was consistently shorter within the aged group., Conclusions: These data reveal that aged Fischer 344 rats exhibit altered EEG activity (reduction of higher frequencies) and clinical manifestations during kainate-induced status epilepticus. Taken together, these data indicate an age-related change in seizure onset and spread after exposure to glutamate analogues.

Purpose: Focal neocortical seizures can be associated with a number of specific pathologies including supratentorial tumors and cavernous malformations (CMs), both of which are highly epileptogenic., Methods: To begin to address the question of whether these lesions have different mechanisms of epileptogenesis, we used intracellular recordings from neurons adjacent to intracerebral neoplasms and cerebral CMs. Developmental anomalies were not included in this study., Results: Neurons adjacent to CMs had a greater propensity to show large (>5 mV), complex spontaneous synaptic events than did neurons neighboring neoplastic substrates (50 vs. 4.7% of cells and 75 and 8% of patients, respectively; p < 0.004; p < 0.05). Both spontaneous excitatory and inhibitory events were noted. In contrast, in tissue adjacent to tumors, low-amplitude (<3 mV) spontaneous excitatory activity predominated. Neurons neighboring CMs also exhibited more excitable responses to synaptic stimulation, with multiple action potentials riding on prolonged excitatory postsynaptic potentials (EPSPs) being evoked in 71% of these cells, versus 32% of cells from the tumor group; p < 0.05. In studies using hippocampal tissue, we noted a similar pattern of spontaneous activity in tissue adjacent to CMs., Conclusions: These data suggest that CMs may induce seizure activity via a different pathophysiologic mechanism(s) than glial tumors.

Hypocretin is a recently discovered peptide that is synthesized by neurons in the lateral hypothalamic area (LH) and is believed to play a role in sleep regulation, arousal, endocrine control, and food intake. These functions are critical for the development of independent survival. We investigated the developmental profile of the hypocretin system in rats. Northern blot analysis showed that the expression of hypocretin mRNA increased from postnatal day 1 to adulthood. Both of the identified hypocretin receptor mRNAs were strongly expressed very early in hypothalamic development, and expression subsequently decreased in the mature brain. Immunocytochemistry revealed hypocretin-2 peptide expression in the cell bodies of LH neurons and in axons in the brain and spinal cord as early as embryonic day 19. Whole-cell patch clamp recordings from postnatal P1-P14 LH slices demonstrated a robust increase in synaptic activity in all LH neurons tested (n = 20) with a 383% increase in the frequency of spontaneous activity upon hypocretin-2 (1.5 microM) application. A similar increase in activity was found with hypocretin-1 application to LH slices. Hypocretin-2 evoked a robust increase in synaptic activity even on the earliest day tested, the day of birth. Furthermore, voltage-clamp recordings and calcium digital imaging experiments using cultured LH cells revealed that both hypocretin-1 and -2 induced enhancement of neuronal activity occurred as early as synaptic activity was detected. Thus, as in the adult central nervous system, hypocretin exerts a profound excitatory influence on neuronal activity early in development, which might contribute to the development of arousal, sleep regulation, feeding, and endocrine control., (Copyright 2001 Wiley-Liss, Inc.)

In vivo dialysis and in vitro electrophysiological studies suggest that GABA uptake is altered in the dentate gyrus of human temporal lobe epileptics characterized with mesial temporal sclerosis (MTLE). Concordantly, anatomical studies have shown that the pattern of GABA-transporter immunoreactivity is also altered in this region. This decrease in GABA uptake, presumably due to a change in the GABA transporter system, may help preserve inhibitory tone interictally. However, transporter reversal can also occur under several conditions, including elevations in [K(+)]o, which occurs during seizures. Thus GABA transporters could contribute to seizure termination and propagation through heterotransport. To test whether GABA transport is compromised in both the forward (uptake) and reverse (heterotransport) direction in the sclerotic epileptic dentate gyrus, the physiological effects of microapplied GABA and nipecotic acid (NPA; a compound that induces heterotransport) were examined in granule cells in hippocampal slices from kainate (KA)-induced epileptic rats and patients with temporal lobe epilepsy (TLE). GABA- and NPA-induced responses were prolonged in granule cells from epileptic rats versus controls (51.3 and 31.3% increase, respectively) while the conductance change evoked with NPA microapplication was reduced by 40%. Furthermore the ratio of GABA/NPA conductance, but not duration, was significantly >1 in epileptic rats but not controls, suggesting a compromise in transporter function in both directions. Similar changes were observed in tissue resected from epileptic patients with medial temporal sclerosis but not in those without the anatomical changes associated with MTLE. These data suggest that the GABA transporter system is functionally compromised in both the forward and reverse directions in the dentate gyrus of chronically epileptic tissue characterized by mesial temporal sclerosis. This alteration may enhance inhibitory tone interically yet be permissive for seizure propagation due to a decreased probability for GABA heterotransport during seizures.

Mossy fiber reorganization has been hypothesized to restore inhibition months after kainate-induced status epilepticus. The time course of recovery of inhibition after kainate treatment, however, is not well established. We tested the hypothesis that if inhibition is decreased after kainate treatment, it is restored within the first week when little or no mossy fiber reorganization has occurred. Chronic in vivo recordings of the septal dentate gyrus were performed in rats before and 1, 4, and 7-8 d after kainate (multiple injections of 5 mg/kg, i.p.; n = 17) or saline (n = 11) treatment. Single and paired-pulse stimuli were used to assess synaptic inhibition. The first day after kainate treatment, only a fraction of rats showed multiple population spikes (35%), prolonged field postsynaptic potentials (76%), and loss of paired-pulse inhibition (29%) to perforant path stimulation. Thus, inhibition was reduced in only some of the kainate-treated rats. By 7-8 d after treatment, nearly all kainate-treated rats showed partial or full recovery in these response characteristics. Histological analysis indicated that kainate-treated rats had a significant decrease in the number of hilar neurons compared to controls, but Timm staining showed little to no mossy fiber reorganization. These results suggest that a decrease in synaptic inhibition in the septal dentate gyrus is not a prerequisite for epileptogenesis and that most of the recovery of inhibition occurs before robust Timm staining in the inner molecular layer.

Recent reports suggest that kainate acting at presynaptic receptors reduces the release of the inhibitory transmitter GABA from hippocampal neurons. In contrast, in the hypothalamus in the presence of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor antagonists [1-(4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466) and D,L-2-amino-5-phosphonopentanoic acid (AP5)], kainate increased GABA release. In the presence of tetrodotoxin, the frequency, but not the amplitude, of GABA-mediated miniature inhibitory postsynaptic currents (IPSCs) was enhanced by kainate, consistent with a presynaptic site of action. Postsynaptic activation of kainate receptors on cell bodies/dendrites was also found. In contrast to the hippocampus where kainate increases excitability by reducing GABA release, in the hypothalamus where a much higher number of GABAergic cells exist, kainate-mediated activation of transmitter release from inhibitory neurons may reduce the level of neuronal activity in the postsynaptic cell.

Previous electrophysiological studies have demonstrated that in a subset of hippocampal slices from tissue resected from patients with mesial temporal lobe epilepsy, perforant path stimulation can elicit prolonged negative field-potential shifts in the dentate granule cell layer (Masukawa et al., 1989. Brain Res. 493, 168-174; Isokawa and Fried, 1996. Neuroscience 72, 31-37). In this investigation, hippocampal slices were prepared from rats: (1) 2-4 days following kainate treatment, when little or no reorganization of the mossy fibers would be present and (2) 3-13 months after kainate treatment, when mossy fiber reorganization would have occurred. In saline-treated controls, perforant path stimulation typically evoked a single population spike. In contrast, perforant path stimulation could evoke 3-12 population spikes in nearly all slices from kainate-injected rats 2-4 days and 3-13 months after treatment. The majority of slices from kainate-injected rats 3-13 months after treatment had qualitatively similar responses to perforant path stimulation as that observed in slices from kainate-injected rats 2-4 days after treatment. However, in 17% of the slices from kainate-treated rats 3-13 months after treatment (29% of rats), the multiple population spikes were followed by a prolonged negative field-potential shift (duration: 140 ms-1.5 s) with variable superimposed population spike activity. This type of epileptiform activity was only observed in slices with robust Timm's staining in the inner molecular layer and similar responses could also be evoked in these slices with hilar stimulation. Furthermore, pharmacological depression of inhibition by adding the GABA(A) receptor antagonist bicuculline unmasked hilar-evoked prolonged negative field-potential shifts in most slices from kainate-treated rats 3-13 months following treatment, and these slices had robust Timm's staining in the inner molecular layer. Such events were not observed in slices from saline-treated controls or kainate-injected rats 2-4 days after treatment. In conclusion, the prolonged negative field-potential shifts evoked to perforant path stimulation in normal ACSF were associated with mossy fiber reorganization, but the relative contribution of altered inhibition, increased synaptic excitation, or even non-synaptic mechanisms is unknown.

Neuropeptide Y (NPY) has been shown to depress hyperexcitable activity that has been acutely induced in the normal rat brain. To test the hypothesis that NPY can also reduce excitability in the chronically epileptic human brain, we recorded intracellularly from dentate granule cells in hippocampal slices from patients with hippocampal seizure onset. NPY had a potent and long-lasting inhibitory action on perforant path-evoked excitatory responses. In comparison, the group 3 metabotropic glutamate receptor agonist L-2-amino-4-phosphonobutyric acid (L-AP4) evoked a mild and transient decrease. NPY-containing axons were found throughout the hippocampus, and in many epileptic patients were reorganized, particularly in the dentate molecular layer. NPY may therefore play a beneficial role in reducing granule cell excitability in chronically epileptic human tissue, and subsequently limit seizure severity.

Alterations in synaptic inhibition are associated with epileptiform activity in several acute animal models; however, it is not clear if there are changes in inhibition in chronically epileptic tissue. We have used intracellular recordings from granule cells of patients with temporal lobe epilepsy to determine whether synaptic inhibition is compromised. Two groups of patients with medial temporal lobe epilepsy were used, those with medial temporal lobe sclerosis (MTLE), and those with extrahippocampal masses (MaTLE) where the cell loss and synaptic reorganization that characterize MTLE are not seen. Although the level of tonic inhibition at the somata was not significantly different in the two patient groups, there was a reduction in the conductance of polysynaptic perforant path-evoked fast and slow inhibitory postsynaptic potentials (IPSPs) (53% and 66%, respectively). We found that there was a comparable decrease in the monosynaptic IPSP conductances examined in the presence of glutamatergic antagonists as that seen for the polysynaptically evoked IPSPs. These data suggest that the decrease in inhibition seen in normal artificial cerebrospinal fluid in MTLE granule cells cannot be solely explained by a decrease in excitatory input onto inhibitory interneurons and may reflect changes at the interneuron-granule cells synapse or in the number of specific inhibitory interneurons.

In contrast to the mature brain, in which GABA is the major inhibitory neurotransmitter, in the developing brain GABA can be excitatory, leading to depolarization, increased cytoplasmic calcium, and action potentials. We find in developing hypothalamic neurons that glutamate can inhibit the excitatory actions of GABA, as revealed with fura-2 digital imaging and whole-cell recording in cultures and brain slices. Several mechanisms for the inhibitory role of glutamate were identified. Glutamate reduced the amplitude of the cytoplasmic calcium rise evoked by GABA, in part by activation of group II metabotropic glutamate receptors (mGluRs). Presynaptically, activation of the group III mGluRs caused a striking inhibition of GABA release in early stages of synapse formation. Similar inhibitory actions of the group III mGluR agonist L-AP4 on depolarizing GABA activity were found in developing hypothalamic, cortical, and spinal cord neurons in vitro, suggesting this may be a widespread mechanism of inhibition in neurons throughout the developing brain. Antagonists of group III mGluRs increased GABA activity, suggesting an ongoing spontaneous glutamate-mediated inhibition of excitatory GABA actions in developing neurons. Northern blots revealed that many mGluRs were expressed early in brain development, including times of synaptogenesis. Together these data suggest that in developing neurons glutamate can inhibit the excitatory actions of GABA at both presynaptic and postsynaptic sites, and this may be one set of mechanisms whereby the actions of two excitatory transmitters, GABA and glutamate, do not lead to runaway excitation in the developing brain. In addition to its independent excitatory role that has been the subject of much attention, our data suggest that glutamate may also play an inhibitory role in modulating the calcium-elevating actions of GABA that may affect neuronal migration, synapse formation, neurite outgrowth, and growth cone guidance during early brain development.

Human temporal lobe epilepsy is associated with complex partial seizures that can produce secondarily generalized seizures and motor convulsions. In some patients with temporal lobe epilepsy, the seizures and convulsions occur following a latent period after an initial injury and may progressively increase in frequency for much of the patient's life. Available animal models of temporal lobe epilepsy are produced by acute treatments that often have high mortality rates and/or are associated with a low proportion of animals developing spontaneous chronic motor seizures. In this study, rats were given multiple low-dose intraperitoneal (i.p.) injections of kainate in order to minimize the mortality rate usually associated with single high-dose injections. We tested the hypothesis that these kainate-treated rats consistently develop a chronic epileptic state (i.e. long-term occurrence of spontaneous, generalized seizures and motor convulsions) following a latent period after the initial treatment. Kainate (5 mg/kg per h, i.p.) was administered to rats every hour for several hours so that class III-V seizures were elicited for > or = 3 h, while control rats were treated similarly with saline. This treatment protocol had a relatively low mortality rate (15%). After acute treatment, rats were observed for the occurrence of motor seizures for 6-8 h/week. Nearly all of the kainate-treated rats (97%) had two or more spontaneous motor seizures months after treatment. With this observation protocol, the average latency for the first spontaneous motor seizure was 77+/-38 (+/-S.D.) days after treatment. Although variability was observed between rats, seizure frequency initially increased with time after treatment, and nearly all of the kainate-treated rats (91%) had spontaneous motor seizures until the time of euthanasia (i.e. 5-22 months after treatment). Therefore, multiple low-dose injections of kainate, which cause recurrent motor seizures for > or = 3 h, lead to the development of a chronic epileptic state that is characterized by (i) a latent period before the onset of chronic motor seizures, and (ii) a high but variable seizure frequency that initially increases with time after the first chronic seizure. This modification of the kainate-treatment protocol is efficient and relatively simple, and the properties of the chronic epileptic state appear similar to severe human temporal lobe epilepsy. Furthermore, the observation that seizure frequency initially increased as a function of time after kainate treatment supports the hypothesis that temporal lobe epilepsy can be a progressive syndrome.