Intraventricular murine Aß infusion elicits hippocampal inflammation and disrupts the consolidation, but not retrieval, of conditioned fear in C57BL6/J mice.

Although one of the defining characteristics of Alzheimer's disease is the presence of amyloid-beta (Aß) plaques, the early accumulation of soluble Aß oligomers (AßOs) may disrupt synaptic function and trigger cognitive impairments long before the appearance of plaques. Furthermore, murine models aimed at understanding how AßOs alter formation and retrieval of associative memories are conducted using human Aß species, which are more neurotoxic in the mouse brain than the native murine species. Unfortunately, there is currently a lack of attention in the literature as to what the murine version of the peptide (mAß) does to synaptic function and how it impacts the consolidation and retrieval of associative memories. In the current study, adult mice were infused with mAß 0, 2, 6, or 46 h after contextual-fear conditioning, and were tested 2-48 h later. Interestingly, only mAß infusions within 2 h of training reduced freezing behavior at test, indicating that mAß disrupted the consolidation, but not retrieval of fear memory. This consolidation deficit coincided with increased IL-1ß and reduced synaptophysin mRNA levels, without disrupting other synaptic signaling-related genes here examined. Despite differences between murine and human Aß, the deleterious functional outcomes of early-stage synaptic oligomer presence are similar. Thus, models utilizing or inducing the production of mAß in non-transgenic animals are useful in exploring the role of dysregulated synaptic plasticity and resultant learning deficits induced by Aß oligomers.

Prolonged isolation stress accelerates the onset of Alzheimer's disease-related pathology in 5xFAD mice despite running wheels and environmental enrichment.

Research has demonstrated that stress can exacerbate AD pathology in transgenic mouse models of AD. The purpose of the present studies was to extend this work by determining whether a social stressor, isolation stress, would increase the number of Aß plaques in 5xFAD + transgenic mice in comparison to group-housed controls, and accelerate the onset of cognitive deficits in contextual fear-conditioning. Additionally, we aimed to determine whether the pathological impact of isolation stress could be prevented through exposure to exercise alone or to exercise and an enriched environment throughout the isolation period. Two-month-old 5xFAD + and 5xFAD- animals were isolated or group-housed for two and three months. An additional subset of 5xFAD + mice were housed in isolation, housed in isolation with an exercise wheel, or housed in isolation with an exercise wheel and an enriched environment. Both two and three months of isolation stress significantly increased the number of plaques in the hippocampus of 5xFAD + mice, and three months of isolation increased hippocampal BACE1 expression. Isolated animals also displayed a significant cognitive deficit in contextual fear-conditioning, independent of genotype. Furthermore, neither exercise nor an enriched environment were able to prevent these isolation-induced effects. Understanding how stress impacts the onset and progression of AD is critical, as many individuals endure significant stress over their lifespan, including prolonged social isolation, a societal trend likely to worsen with time.

The α5-GABAAR inverse agonist MRK-016 upregulates hippocampal BDNF expression and prevents cognitive deficits in LPS-treated mice, despite elevations in hippocampal Aß.

Alzheimer's disease is marked by the presence of amyloid-beta (Aß) plaques, elevated central cytokine levels, dysregulation of BDNF-related gene expression, and cognitive decline. Previously, our laboratory has demonstrated that repeated administration of peripheral LPS is sufficient to significantly increase the presence of central Aß in the hippocampus, and that this upregulation corresponds with deficits in learning and memory. We have also previously demonstrated that the inverse benzodiazepine agonist MRK-016 (MRK) can protect against memory acquisition and consolidation errors in mice. To extend these findings, the current study explored the protective effects of MRK in the context of LPS-induced hippocampal Aß accumulation. Hippocampal Aß was significantly elevated, relative to saline-treated animals, following seven days of peripheral LPS injections. Animals were then trained in a contextual fear conditioning paradigm and were immediately treated with MRK or saline once training was complete. Behavioral testing occurred the day after training. Results from this study demonstrate that repeated injections of LPS significantly elevate hippocampal Aß, and inhibit acquisition of contextual fear. Post-training treatment with MRK restored behavioral expression of fear in LPS-treated animals, despite elevated hippocampal Aß, an effect that may be attributed to increased BDNF mRNA expression. Therefore, our data indicate that MRK can prevent LPS- induced cognitive deficits associated with elevated Aß, and restore hippocampal BDNF expression.

Hippocampal Aß expression, but not phosphorylated tau, predicts cognitive deficits following repeated peripheral poly I:C administration.

Alzheimer's disease is marked by the accumulation of the amyloid-beta (Aß) peptide, and increases in phosphorylation of the microtubule associated protein, tau. Changes in these proteins are considered responsible, in part, for the progressive neuronal degeneration and cognitive deficits seen in AD. We examined the effect of repeated consecutive peripheral poly I:C injections on cognitive deficits, central Aß, and phosphorylated tau accumulation, following three treatment durations: 7, 14, and 21 days. Forty-eight hours after the final injection, animals were trained in a contextual fear-conditioning paradigm, and tested 24h later. Immediately after testing, the hippocampus was collected to quantify Aß and phosphorylated tau accumulation. Results showed that, although poly I:C-induced Aß was significantly elevated at all time points examined, poly I:C only disrupted cognition after 14 and 21 days of administration. Moreover, elevations in phosphorylated tau were not seen until the 14-day time point. Interestingly, phosphorylated tau expression then declined at the 21-day time point. Finally, we demonstrated that Aß levels are a stronger predictor of cognitive dysfunction, explaining 37% of the variance, whereas phosphorylated tau levels only accounted for 0.2%. Taken together, these results support the hypothesis that inflammation-induced elevation in Aß disrupts cognition, independently of phosphorylated tau, and suggest that long-term administration of poly I:C may provide a model to investigate the contribution of long-term inflammation toward the development of Alzheimer's-like pathology.

Imatinib methanesulfonate reduces hyperphosphorylation of tau following repeated peripheral exposure to lipopolysaccharide.

For years, the prevailing hypothesis for Alzheimer's Disease (AD) has proposed a mechanism by which deposition of amyloid-beta (Aß) in the brain is independent of tau-pathologies and cognitive decline. However, despite extensive research on the disease, the mechanisms underlying the etiology of tau-pathology remain unknown. Previous research in our lab has shown that imatinib methanesulfonate (IM) blocks the peripheral production of Aß in response to LPS, thereby preventing the buildup of Aß in the hippocampus, and rescuing the cognitive dysfunction that normally follows. The present study aimed to examine the link between Aß and tau following inflammation, and to expand our understanding of how IM affects AD pathology. Specifically, we hypothesized that the IM-mediated inhibition of Aß production following inflammation would successfully protect against the hyperphosphorylation of tau (ptau). Here we show that 7days of LPS treatment in male C57BL/6J mice, which normally produces elevations in peripheral and central Aß, also produces hyperphosphorylation of tau. However, just as pre-treatment and concurrent treatment with IM blocks Aß production, it also blocks the phosphorylation of tau. In addition, 7days of LPS-induced inflammation and Aß production also leads to elevated total tau protein expression. Our results may provide support for the hypothesis that enhanced expression of tau following LPS administration is a protective measure by hippocampal neurons to compensate for the loss of the microtubule-stabilizing protein due to phosphorylation. More importantly, our results support the hypothesis that blocking the production of Aß that follows inflammation also leads to reduced tau phosphorylation, lending credence to a model in which Aß initiates tau phosphorylation.

Pre-treatment of C57BL6/J mice with the TLR4 agonist monophosphoryl lipid A prevents LPS-induced sickness behaviors and elevations in dorsal hippocampus interleukin-1ß, independent of interleukin-4 expression.

Peripheral administration of lipopolysaccharide (LPS) elevates production of pro-inflammatory cytokines, and motivates the expression of sickness behaviors. In this study, we tested the ability of an LPS-derived adjuvant, monophosphoryl lipid A (MPLA), to prevent LPS-induced sickness behaviors in a burrowing paradigm. Testing occurred over a three-day period. Animals received a single injection of either MPLA or saline the first two days of testing. On day three, animals received either LPS or saline. Tissue from the dorsal hippocampus was collected for qRT-PCR to assess expression of IL-1ß and IL-4. Results indicate that, during the pre-treatment phase, administration of MPLA induces an immune response sufficient to trigger sickness behaviors. However, we observed that animals pre-treated with MPLA for two days were resistant to LPS-induced sickness behaviors on day three. Results from the qRT-PCR analysis indicated that LPS-treated animals pre-treated with MPLA expressed significantly less IL-1ß compared to LPS-treated animals pre-treated with saline. However, we did not observe a significant difference in IL-4 expression between groups. Therefore, results indicate that under the given parameters of the study, MPLA pre-treatment protects against LPS-induced sickness behaviors, at least in part, by decreasing expression of the pro-inflammatory cytokine IL-1ß.

Administration of the inverse benzodiazepine agonist MRK-016 rescues acquisition and memory consolidation following peripheral administration of bacterial endotoxin.

Recent evidence suggests that inflammation-induced decrements in cognitive function can be mitigated via manipulation of excitatory or inhibitory transmission. We tested the ability of the inverse benzodiazepine agonist, MRK-016 (MRK) to protect against LPS-induced deficits in memory acquisition and consolidation, using a contextual fear conditioning (CFC) paradigm. In Experiment One, mice received lipopolysaccharide (LPS) and/or MRK injections prior to CFC training, and were then tested 24h after training. In Experiment Two, animals received similar treatment injections immediately after training, and were tested 24h later. Additionally, hippocampal samples were collected 4h after LPS injections and immediately after testing, to evaluate brain-derived neurotrophic factor (BDNF) and insulin-like growth factor 1 (IGF-1) mRNA expression. Results indicate that MRK can protect against LPS-induced learning/memory decrements in both paradigms. We also found, in both paradigms, that animals treated with LPS/Saline expressed significantly less BDNF mRNA when compared to Saline/Saline-treated animals 4h after LPS administration, but that MRK did not restore BDNF expression levels. Further, treatment administrations had no effect on IGF-1 mRNA expression at any collection time-point. In summary, MRK-016 can protect against LPS-induced deficits in memory acquisition and consolidation, in this hippocampus-dependent paradigm, though this protection occurs independently of recovery of BDNF expression.

Alleviation of the effects of endotoxin exposure on behavior and hippocampal IL-1beta by a selective non-peptide antagonist of corticotropin-releasing factor receptors.

Previous research has shown that lipopolysaccharide (LPS) or interleukin-1beta (IL-1beta) administration produces learning/memory deficits in a variety of paradigms. In our laboratory, we have consistently observed LPS-induced behavioral alterations in a two-way active avoidance conditioning paradigm. Following LPS administration, one factor that affects cytokine production is corticotropin-releasing factor (CRF). CRF has well known anti-inflammatory effects, via stimulation of ACTH and corticosterone release. However, CRF acting directly on immune cells or within the CNS may potentiate proinflammatory effects. The current experiments explored the potential of antalarmin, a CRF-R1 non-peptide antagonist, to diminish or negate deficits observed with LPS administration. On the first day of testing, four-month-old male C57BL/6J mice received an intraperitoneal (i.p.) injection of antalarmin, followed 90min later by a second i.p. injection of LPS 4h prior to two-way active avoidance conditioning testing. As hypothesized, LPS administration altered performance. However, pretreatment with antalarmin attenuated the adverse effects of LPS administration. Moreover, evidence indicates that antalarmin attenuated hippocampal, but not peripheral, cytokine release. The behavioral results cannot be explained by alterations in the HPA axis, as antalarmin did not affect the LPS-induced rise in corticosterone. The current research contributes preliminary evidence that CRF may be an important factor in the development of LPS-induced behavioral effects, and that blocking the activity of CRF may be sufficient to alleviate some of the effects of endotoxin exposure, possibly due to diminished LPS-induced IL-1beta release in the dorsal hippocampus.

Chemical sympathectomy increases numbers of inflammatory cells in the peritoneum early in murine listeriosis.

Here, we investigated the effects of sympathectomy on systemic bacterial loads following infection with Listeria monocytogenes, and on innate and specific immune responses in the peritoneum. Sympathectomy decreased systemic bacterial loads, and increased the number of peritoneal leukocytes and the percentage of peritoneal macrophages three days postinfection. This suggests that sympathectomy-induced decreases systemic bacterial loads are associated with increased recruitment of inflammatory cells into tissues during the innate immune response.

Chemical sympathectomy alters numbers of splenic and peritoneal leukocytes.

Sympathectomy of BALB/c mice that were injected with either Listeria monocytogenes or saline did not affect the total number of splenic leukocytes measured 1-3 days after injection, but sympathectomy did increase the percentages of neutrophils in the spleens of both infected and uninfected mice. By contrast, sympathectomy was associated with increased numbers of peritoneal exudate cells (PEC) and peritoneal macrophages in both groups of mice. Sympathectomy did not affect tumor necrosis factor-alpha, interleukin-12, or interferon-gamma production in cultured splenocytes or PEC in either infected or uninfected mice.

Moderate exercise is associated with enhanced antigen-specific cytokine, but not IgM antibody production in aged mice.

It has been suggested that moderate exercise may modulate the immune response in the elderly. We investigated whether moderate exercise had an effect on the immune response to viral infection in both young (2-4 months) and older (16-18 months) male BALB/cJ mice. Exercised (EX) mice ran on a treadmill for 8 weeks at a gradually increasing speed and duration whereas control (CON) mice were only handled briefly during each exercise session and then returned to their cages. Mice were infected with herpes simplex virus type 1 (HSV-1) 24 h post-exercise. Serum IgM anti-HSV antibody, HSV-1 specific Th1/Th2 cytokine production by spleen cells, and cytokine production by alveolar cells were measured 7 days post-infection. In the aged mice, exercise was associated with an enhanced production of the HSV-1 specific Th1-associated cytokines, interleukin (IL)-2 and interferon (IFN)-gamma, but had no effect on the Th2-associated cytokine IL-10 or IgM antibody. No effect of exercise was observed in young mice. IL-12 production was not altered by exercise, but aging was associated with altered IL-12 production in a tissue-specific manner. In conclusion, moderate exercise was associated with increased antigen-specific IL-2 and IFN-gamma production in response to viral challenge in older mice.

Chemical sympathectomy increases the innate immune response and decreases the specific immune response in the spleen to infection with Listeria monocytogenes.

Many investigators have shown that ablation of the sympathetic nervous system (SNS) with 6-hydroxydopamine (6-OHDA) can alter cell-mediated and humoral immune responses to antigenic challenge. Fewer studies have examined 6-OHDA-induced changes in natural immunity. In this study, we have examined the effect of chemical sympathectomy on the nonspecific and specific phases of the response to infection with Listeria monocytogenes. Sympathectomy decreased splenic bacterial loads 3 and 5 days post-infection and increased splenic neutrophils 3 days post-infection. Sympathectomy decreased splenocyte numbers and antigen-stimulated cytokine secretion from splenocytes. These results suggest that the SNS influences specific responses by modulating innate responses.

One-trial conditioning of the antibody response to hen egg lysozyme in rats.

In Wistar rats, reexposure to a novel conditioned stimulus (CS) previously paired with a protein antigen, hen egg lysozyme (HEL) on a single conditioning trial increased anti-HEL IgG levels relative to conditioned rats that were not reexposed to the CS, conditioned rats that were preexposed to the CS, and nonconditioned rats. These results confirm previous findings that a single exposure to a CS associated with immunization is sufficient to elicit an antibody response upon subsequent reexposure to the CS in the absence of exogenous antigen.

Prolonged exercise suppresses antigen-specific cytokine response to upper respiratory infection.

Fatiguing exercise has been associated with an increased susceptibility to infection. This study examined the antigen-specific T-helper (Th) type 1 and Th type 2 cytokine response to herpes simplex virus (HSV) infection after an acute bout of fatiguing exercise. Male BALB/cJ mice ran on a treadmill (Ex) until voluntary fatigue (approximately 2.5 h), and control mice were handled and remained next to the treadmill. Mice were infected with HSV 20 min after exercise. Mice were killed 2 or 7 days postinfection, and sera and spleens were taken for the determination of HSV-specific serum IgM, splenocyte cytokine production during culture with HSV, and splenocyte natural killer cell cytotoxicity. Both Th type 1 [interleukin (IL)-2, interferon-gamma, IL-12] and Th type 2 (IL-10) cytokine production in spleen cell cultures, as well as natural killer cell cytotoxicity, decreased in Ex on day 2 postinfection. On day 7 postinfection, there was no difference in HSV-specific serum IgM or cytokine production by cells from control and Ex mice, with the exception of decreased IL-12 in Ex mice. These findings suggest that fatiguing exercise may alter the kinetics of antigen-specific cytokine production.

Learning in year-old female autoimmune BXSB mice.

BXSB/ MpJ-Yaa and NZB/BINJ mice have been used as animal models for both developmental learning disability and systemic autoimmune disease. Approximately 40-60% of these animals show ectopic clusters of neurons in Layer I of cortex similar to those found in postmortem analyses of human dyslexics, and all exhibit an autoimmune condition similar to systemic lupus erythematosus (SLE) in humans. The expression of immune disease in the BXSB strain, unlike in humans, is more severe in males than females. Most previous studies have examined the behavioral sequelae of neocortical ectopias at a relatively young age, when the BXSB females (unlike the male BXSB and female and male NZBs) are not yet showing high titers of autoantibodies associated with their lupus-like form of autoimmune disease. This study examined the behavior of BXSB females at an age subsequent to autoimmune disease onset. When contrasted with younger animals, year-old BXSB females showed good learning behavior, with no differences in Lashley maze learning and remarkably good performance in a visual discrimination learning task. These results are consistent with other data which indicate that many types of learning behavior are apparently unperturbed by systemic autoimmune disease. Results also showed significant interactions between a measure of lateral paw preference and the presence or absence of ectopias in Lashley maze learning. Animals without ectopias that exhibited a right lateral paw preference showed the greatest number of errors on a number of test measures. These findings support previous results indicating that behavioral effects associated with ectopias may vary based upon the behavioral laterality of affected animals.

A computer-aided procedure for measuring Hebb-Williams maze performance.

We describe a shortened procedure for testing mice over a 5-day interval on a swimming version of the Hebb-Williams maze. The mice are given 1 day of adaptation training, and are tested over the next 4 days on Hebb-Williams problems 1, 6, 12, and 5, in that order. As an animal swims through one of the maze problems, the computer screen shows the maze pattern, and an observer traces the path taken with a computer mouse. The computer program, Observe Software, stores the path sequence, determines the error score for that trial, and sends the information to a spreadsheet where it is available for statistical analyses.

Neocortical ectopias in BXSB mice: effects upon reference and working memory systems.

BXSB mice have an approximately 40-60% incidence of neocortical ectopias in layer I of the prefrontal/motor cortex. Prior studies have found major behavioral differences between those with ectopias and their non-ectopic littermates. Some of these findings indicate that the two groups differ with respect to spatial reference and working memory. The purpose of this study was to measure reference and working memory in the same animals to test the hypothesis that the ectopics would have better reference memory but less effective working memory. The Lashley III maze has cul-de-sacs which must be eliminated, and T-choices where the animal has to decide whether to go left or right. Ectopic and non-ectopic mice were equally able to learn the maze and did not differ on cul-entry or T-choice errors. Then the maze was inverted and the animals were retested. Turning the maze upside down did not change the relative status of the blind alleys. Therefore, the reference memory knowledge from the prior week's training could be used to avoid entering the culs. However, inverting the maze caused a left-right mirror image reversal of the T-choices. Therefore, prior reference memory information would interfere with learning the new path through the maze, whereas working memory would enable the mouse to eliminate T-choice errors. Ectopic mice made less cul-entry errors and more T-choice errors than their non-ectopic littermates, as predicted.

Learning and memory in the autoimmune BXSB mouse: effects of neocortical ectopias and environmental enrichment.

Approximately 40-60% of BXSB mice have ectopic cell clusters in layer 1 of neocortex. Prior studies have shown distinct behavioral differences between those with ectopias and their non-ectopic littermates. In this study, female BXSB mice were reared after weaning in either enriched environments or standard cages. Following an initial round of behavioral testing, all mice were housed in standard cages and retested. Enriched cage mice (both ectopic and non-ectopic) showed increased activity, greater speed, and enhanced learning scores across a variety of tests. Additionally, prior test experience itself had significant positive effects on Hebb-Williams maze learning. The presence of ectopias resulted in better Morris maze learning for standard cage reared mice. Further, ectopic mice, regardless of their housing condition, showed better long-term retention in the Morris maze than did their non-ectopic counterparts. These findings show that abnormalities in corticogenesis need not always result in functional deficit.

Effects of embryo transfer and cortical ectopias upon the behavior of BXSB-Yaa and BXSB-Yaa + mice.

The BXSB-Yaa and BXSB-Yaa + inbred strains of mice differ primarily with respect to the Y chromosome, although there is evidence that they differ on several autosomal genes as well. Each strain has ectopic collections of neurons in neocortical layer I (ectopias), with a higher occurrence in males (58%) than females (42%). Conventionally reared mice from these strains were compared to mice that were transferred, as 8-cell embryos, into the uteri of non-autoimmune recipients, who gave birth to and reared the offspring. The transfer procedure did not change the incidence of ectopias in either sex. There were, however, major differences in behavior. Compared to conventionally reared controls, embryo transfer mice had greater behavioral asymmetry, poorer performance in a black-white discrimination, poorer Morris maze learning, better Lashley maze learning, and better performance in a two-way shuttlebox. Within the transfer groups, females differed as much as males, confirming our prior findings and supporting our thesis that the two strains differ on several autosomal genes in addition to the Y chromosome. These findings show that the intra-uterine environment can powerfully and selectively affect later behavior. When ectopic and non-ectopic mice were compared, BXSB-Yaa mice with neocortical ectopias were better able to learn the Morris spatial maze than non-ectopic controls; this was true whether the mice were conventionally reared or embryo transferred. In contrast, BXSB-Yaa + ectopic mice did not differ from their controls if conventionally reared, but were much worse than controls if embryo transferred.

Sex differences in the distribution of axon types within the genu of the rat corpus callosum.

Neuroanatomical sex differences have been documented in the rat neocortex, including dimorphism of its predominant commissure, the corpus callosum (CC). In particular, CC sex differences have been reported in the ultrastructure of the posterior callosal region, the splenium. Since the CC is a heterogeneous fiber tract with its axons arising from distinct cortical areas and passing through restricted regions along its length, it became of interest to ascertain whether cellular sexual dimorphisms may also be present in another division of the CC. To test this hypothesis, electron microscopy was used to examine axon composition in adult male and female rats in the anterior portion, the genu. The number and size of axons, the thickness of the myelin sheath, and the area within the genu occupied by these constituents, were quantified. Results showed a significant sex difference in the ratio of unmyelinated to myelinated axons, with females having a larger proportion of unmyelinated fibers. This effect was present for both (1) the number of axons, and (2) the area taken up by axonal fibers. No differences were found in the size of either axon type, or for myelin thickness. Comparison of these results with those from the splenium and possible mechanisms underlying this dimorphism are discussed.