Sex-dependent alterations in the physiology of entorhinal cortex neurons in old heterozygous 3xTg-AD mice.

While the higher prevalence of Alzheimer's disease (AD) in women is clear, studies suggest that biological sex may also influence AD pathogenesis. However, mechanisms behind these differences are not clear. To investigate physiological differences between sexes at the cellular level in the brain, we investigated the intrinsic and synaptic properties of entorhinal cortex neurons in heterozygous 3xTg-AD mice of both sexes at the age of 20 months. This brain region was selected because of its early association with AD symptoms. First, we found physiological differences between male and female non-transgenic mice, providing indirect evidence of axonal alterations in old females. Second, we observed a transgene-dependent elevation of the firing activity, post-burst afterhyperpolarization (AHP), and spontaneous excitatory postsynaptic current (EPSC) activity, without any effect of sex. Third, the passive properties and the hyperpolarization-activated current (Ih) were altered by transgene expression only in female mice, whereas the paired-pulse ratio (PPR) of evoked EPSC was changed only in males. Fourth, both sex and transgene expression were associated with changes in action potential properties. Consistent with previous work, higher levels of Aß neuropathology were detected in 3xTg-AD females, whereas tau deposition was similar. In summary, our results support the idea that aging and AD neuropathology differentially alter the physiology of entorhinal cortex neurons in males and females.

A novel wireless brain stimulation device for long-term use in freely moving mice.

Deep brain stimulation (DBS) has been used in clinical settings for many years despite a paucity of knowledge related to the anatomical and functional substrates that lead to benefits and/or side-effects in various disease contexts. In order to maximize the potential of this approach in humans, a better understanding of its mechanisms of action is absolutely necessary. However, the existing micro-stimulators available for pre-clinical models, are limited by the lack of relevant small size devices. This absence prevents sustained chronic stimulation and real time monitoring of animals during stimulation, parameters that are critical for comparison to clinical findings. We therefore sought to develop and refine a novel small wireless micro-stimulator as a means by which to study consequent behavioural to molecular changes in experimental animals. Building on previous work from our group, we refined our implantable micro-stimulator prototype, to be easily combined with intravital 2-photon imaging. Using our prototype we were able to replicate the well described clinical benefits on motor impairment in a mouse model of Parkinson's disease in addition to capturing microglia dynamics live during stimulation. We believe this new device represents a useful tool for performing pre-clinical studies as well as dissecting brain circuitry and function.

Transgenic autoinhibition of p21-activated kinase exacerbates synaptic impairments and fronto-dependent behavioral deficits in an animal model of Alzheimer's disease.

Defects in p21-activated kinase (PAK) lead to dendritic spine abnormalities and are sufficient to cause cognition impairment. The decrease in PAK in the brain of Alzheimer's disease (AD) patients is suspected to underlie synaptic and dendritic disturbances associated with its clinical expression, particularly with symptoms related to frontal cortex dysfunction. To investigate the role of PAK combined with Aß and tau pathologies (3xTg-AD mice) in the frontal cortex, we generated a transgenic model of AD with a deficit in PAK activity (3xTg-AD-dnPAK mice). PAK inactivation had no effect on Aß40 and Aß42 levels, but increased the phosphorylation ratio of tau in detergent-insoluble protein fractions in the frontal cortex of 18-month-old heterozygous 3xTg-AD mice. Morphometric analyses of layer II/III pyramidal neurons in the frontal cortex showed that 3xTg-AD-dnPAK neurons exhibited significant dendritic attrition, lower spine density and longer spines compared to NonTg and 3xTg-AD mice. Finally, behavioral assessments revealed that 3xTg-AD-dnPAK mice exhibited pronounced anxious traits and disturbances in social behaviors, reminiscent of fronto-dependent symptoms observed in AD. Our results substantiate a critical role for PAK in the genesis of neuronal abnormalities in the frontal cortex underlying the emergence of psychiatric-like symptoms in AD.

Loss of Metabotropic Glutamate Receptor 5 Function on Peripheral Benzodiazepine Receptor in Mice Prenatally Exposed to LPS.

Parental microglial induced neuroinflammation, triggered by bacterial- or viral infections, can induce neuropsychiatric disorders like schizophrenia and autism to offspring in animal models. Recent investigations suggest that microglia, the resident immune cells of the brain, provides a link between neurotransmission, immune cell activation, brain inflammation and neuronal dysfunction seen with the offspring. Relatively little is known about how reduction of brain inflammation and restoration of glial function are associated with diminution of brain degeneration and behavioral deficits in offspring. Increased mGluR5 expression and the long-lasting excitotoxic effects of the neurotoxin during brain development are associated with the glial dysfunctions. We investigated the relationship of mGluR5 and PBR and how they regulate glial function and inflammatory processes in mice prenatally exposed to LPS (120µg/kg, between gestational days 15 and 17), an inflammatory model of a psychiatric disorder. Using PET imaging, we showed that pharmacological activation of mGluR5 during 5 weeks reduced expression of classic inflammation marker PBR in many brain areas and that this molecular association was not present in LPS-exposed offspring. The post-mortem analysis revealed that the down regulation of PBR was mediated through activation of mGluR5 in astrocytes. In addition, we demonstrated that this interaction is defective in a mouse model of the psychiatric deficit offering a novel insight of mGluR5 involvement to brain related disorders and PBR related imaging studies. In conclusion, mGluR5 driven glutamatergic activity regulates astrocytic functions associated with PBR (cholesterol transport, neurosteroidogenesis, glial phenotype) during maturation and could be associated with neuropsychiatric disorders in offspring.

A novel combinational approach of microstimulation and bioluminescence imaging to study the mechanisms of action of cerebral electrical stimulation in mice.

Deep brain stimulation (DBS) is used to treat a number of neurological conditions and is currently being tested to intervene in neuropsychiatric conditions. However, a better understanding of how it works would ensure that side effects could be minimized and benefits optimized. We have thus developed a unique device to perform brain stimulation (BS) in mice and to address fundamental issues related to this methodology in the pre-clinical setting. This new microstimulator prototype was specifically designed to allow simultaneous live bioluminescence imaging of the mouse brain, allowing real time assessment of the impact of stimulation on cerebral tissue. We validated the authenticity of this tool in vivo by analysing the expression of toll-like receptor 2 (TLR2), corresponding to the microglial response, in the stimulated brain regions of TLR2-fluc-GFP transgenic mice, which we further corroborated with post-mortem analyses in these animals as well as in human brains of patients who underwent DBS to treat their Parkinson's disease. In the present study, we report on the development of the first BS device that allows for simultaneous live in vivo imaging in mice. This tool opens up a whole new range of possibilities that allow a better understanding of BS and how to optimize its effects through its use in murine models of disease.

The morphological and molecular changes of brain cells exposed to direct current electric field stimulation.

BACKGROUND: The application of low-intensity direct current electric fields has been experimentally used in the clinic to treat a number of brain disorders, predominantly using transcranial direct current stimulation approaches. However, the cellular and molecular changes induced by such treatment remain largely unknown. METHODS: Here, we tested various intensities of direct current electric fields (0, 25, 50, and 100V/m) in a well-controlled in vitro environment in order to investigate the responses of neurons, microglia, and astrocytes to this type of stimulation. This included morphological assessments of the cells, viability, as well as shape and fiber outgrowth relative to the orientation of the direct current electric field. We also undertook enzyme-linked immunosorbent assays and western immunoblotting to identify which molecular pathways were affected by direct current electric fields. RESULTS: In response to direct current electric field, neurons developed an elongated cell body shape with neurite outgrowth that was associated with a significant increase in growth associated protein-43. Fetal midbrain dopaminergic explants grown in a collagen gel matrix also showed a reorientation of their neurites towards the cathode. BV2 microglial cells adopted distinct morphological changes with an increase in cyclooxygenase-2 expression, but these were dependent on whether they had already been activated with lipopolysaccharide. Finally, astrocytes displayed elongated cell bodies with cellular filopodia that were oriented perpendicularly to the direct current electric field. CONCLUSION: We show that cells of the central nervous system can respond to direct current electric fields both in terms of their morphological shape and molecular expression of certain proteins, and this in turn can help us to begin understand the mechanisms underlying the clinical benefits of direct current electric field.

Hypo-anxious phenotype of adolescent offspring prenatally exposed to LPS is associated with reduced mGluR5 expression in hippocampus.

Many studies have reported long-term modulation of metabotropic glutamate receptor 5 (mGluR5) by inflammatory processes and a pharmacological modulation of mGluR5 is known to regulate anxiety level. However, it is not known if non-pharmacological modulation of mGluR5 by inflammation impaired the unconditional level of anxiety. In this study, we investigated this relation in LPS prenatal immune challenge (120µg/kg, 3x i.p. injection in late gestation), a developmental model of neuroinflammation in which some studies have reported hypo-anxious phenotype. Using positron emission tomographic imaging (PET) approaches, we have demonstrated a decrease in the binding potential of [18F]fluoro-5-(2-pyridinylethynyl)benzonitrile ([18F]FPEB, a radioligand for mGluR5) in hippocampus of adolescent offspring prenatally exposed to LPS, without significant change in the binding of [11C]peripheral benzodiazepine receptor 28 ([11C]PBR28), an inflammatory marker. In addition, dark-light box emergence test revealed a lower level of anxiety in LPS-exposed offspring and this behavioural phenotype was associated with the binding potential of [18F]FPEB in hippocampus. These results confirm that neuroinflammation during developmental phase modulates the physiology of mGluR5 and this alteration can be associated with behavioural phenotype related to anxiety. In addition, this study supports a hypotheses that mGluR5 could be used as a diagnostic target in anxiety.

The different effects of LPS and poly I:C prenatal immune challenges on the behavior, development and inflammatory responses in pregnant mice and their offspring.

In recent years, in vivo animal models of prenatal infection have been developed in an attempt to recreate behavioral and neuropathological features associated to a number of neurological and neuropsychiatric disorders. However, these models are still in their emerging phase and a better understanding of how these types of infections relate to adult-onset of brain-related disorders is needed. Here, we undertook an extensive behavioral characterization of both pregnant females and their pups following late gestational exposure (from gestational days (GD) 15-17) to either lipopolysaccharide (LPS; 120µg/kg i.p.) or polyinosinic:polycytidylic acid (poly I:C; 5mg/kg i.v.). We observed that both LPS and poly I:C treatments produced anxiety-like behaviors in treated pregnant females, although to a lesser extent with LPS. LPS injections, but not poly I:C, led to reduced food intake and consequently decreased weight gain in pregnant dams. In pups, poly I:C treatments triggered a delay in growth and sensorimotor development, as evaluated by righting, geotaxis and grasping reflexes. At the cellular level, both toxins induced an initial inflammatory response while only LPS reduced the expression of brain cell markers in foetuses (GFAP and NeuN), which was no longer observable at postnatal day (PnD) 10. Higher levels of IL-2, IL-5 and IL-6 in plasma and an upregulation of the metabotropic receptor 5 (mGluR5) in foetal brains of 10-day-old offspring prenatally exposed to poly I:C was also observed. Interestingly, the increased mGluR5 expression correlated with impairments of the righting reflex. This study is the first to directly compare reflex development following LPS and poly I:C prenatal immune challenges in mice and sheds light onto the different patterns of behavior and pathology in dams and their offspring.

PAK inactivation impairs social recognition in 3xTg-AD Mice without increasing brain deposition of tau and Aß.

Defects in p21-activated kinase (PAK) are suspected to play a role in cognitive symptoms of Alzheimer's disease (AD). Dysfunction in PAK leads to cofilin activation, drebrin displacement from its actin-binding site, actin depolymerization/severing, and, ultimately, defects in spine dynamics and cognitive impairment in mice. To determine the role of PAK in AD, we first quantified PAK by immunoblotting in homogenates from the parietal neocortex of subjects with a clinical diagnosis of no cognitive impairment (n = 12), mild cognitive impairment (n = 12), or AD (n = 12). A loss of total PAK, detected in the cortex of AD patients (-39% versus controls), was correlated with cognitive impairment (r(2) = 0.148, p = 0.027) and deposition of total and phosphorylated tau (r(2) = 0.235 and r(2) = 0.206, respectively), but not with Aß42 (r(2) = 0.056). Accordingly, we found a decrease of total PAK in the cortex of 12- and 20-month-old 3xTg-AD mice, an animal model of AD-like Aß and tau neuropathologies. To determine whether PAK dysfunction aggravates AD phenotype, 3xTg-AD mice were crossed with dominant-negative PAK mice. PAK inactivation led to obliteration of social recognition in old 3xTg-AD mice, which was associated with a decrease in cortical drebrin (-25%), but without enhancement of Aß/tau pathology or any clear electrophysiological signature. Overall, our data suggest that PAK decrease is a consequence of AD neuropathology and that therapeutic activation of PAK may exert symptomatic benefits on high brain function.

Dietary intake of unsaturated fatty acids modulates physiological properties of entorhinal cortex neurons in mice.

Dietary lipids modify brain fatty acid profile, but evidence of their direct effect on neuronal function is sparse. The enthorinal cortex (EC) neurons connecting to the hippocampus play a critical role in learning and memory. Here, we have exposed mice to diets based on canola:soybean oils (40 : 10, g/kg) or safflower : corn oils (25 : 25, g/kg) to investigate the relationship between the lipid profile of brain fatty acids and the intrinsic properties of EC neurons. Consumption of canola : soybean oil-enriched diet led to the increase of the monounsaturated fatty acid oleic acid and to a decrease of arachidonic acid in ethanolamine glycerophospholipids of the white matter. We also found an important rise in docosahexaenoic acid (DHA) within ethanolamine glycerophospholipids and phosphatidylserine of gray matter. The canola:soybean oil treatment led to a shorter duration of action potential (-21%), a reduction in the duration of postsynaptic response (-21%) and increased firing activity (+43%). Data from additional experiments with animals fed DHA alone or DHA with canola oil suggested that dietary monounsaturated fatty acid may have contributed to these effects on EC neuron physiology. Since neuronal function within the enthorhinal-hippocampal loop is critical to learning and memory processes, the present data may provide a functional basis for the beneficial cognitive effects of canola oil-based diets.

Chronic dietary intake of α-linolenic acid does not replicate the effects of DHA on passive properties of entorhinal cortex neurons.

n-3 PUFA are receiving growing attention for their therapeutic potential in central nervous system (CNS) disorders. We have recently shown that long-term treatment with DHA alters the physiology of entorhinal cortex (EC) neurons. In the present study, we investigated by patch-clamp the effect of another major dietary n-3 PUFA, α-linolenic acid (LNA), on the intrinsic properties of EC neurons. Mice were chronically exposed to isoenergetic diets deficient in n-3 PUFA or enriched in either DHA or LNA on an equimolar basis. GC analyses revealed an increase in DHA (34%) and a decrease in arachidonic acid (AA, - 23%) in brain fatty acid concentrations after consumption of the DHA-enriched diet. Dietary intake of LNA similarly affected brain fatty acid profiles, but at a lower magnitude (DHA: 23%, AA: - 13%). Compared to the n-3 PUFA-deficient diet, consumption of DHA, but not LNA, induced membrane hyperpolarisation ( -60 to -70 mV), increased cellular capacitance (32%) and spontaneous excitatory postsynaptic current frequency (50%). We propose that the inefficiency of LNA to modulate cellular capacitance was related to its inability to increase the brain DHA:AA ratio over the threshold necessary to up-regulate syntaxin-3 (46%) and translocate drebrin (40% membrane:cytosol ratio). In summary, our present study shows that the increase in brain DHA content following chronic administration of LNA was not sufficient to alter the passive and synaptic properties of EC neurons, compared to direct dietary intake of DHA. These diverging results have important implications for the therapeutic use of n-3 PUFA in CNS disease, favouring the use of preformed DHA.

DHA improves cognition and prevents dysfunction of entorhinal cortex neurons in 3xTg-AD mice.

Defects in neuronal activity of the entorhinal cortex (EC) are suspected to underlie the symptoms of Alzheimer's disease (AD). Whereas neuroprotective effects of docosahexaenoic acid (DHA) have been described, the effects of DHA on the physiology of EC neurons remain unexplored in animal models of AD. Here, we show that DHA consumption improved object recognition (↑12%), preventing deficits observed in old 3xTg-AD mice (↓12%). Moreover, 3xTg-AD mice displayed seizure-like akinetic episodes, not detected in NonTg littermates and partly prevented by DHA (↓50%). Patch-clamp recording revealed that 3xTg-AD EC neurons displayed (i) loss of cell capacitance (CC), suggesting reduced membrane surface area; (ii) increase of firing rate versus injected current (F-I) curve associated with modified action potentials, and (iii) overactivation of glutamatergic synapses, without changes in synaptophysin levels. DHA consumption increased CC (↑12%) and decreased F-I slopes (↓21%), thereby preventing the opposite alterations observed in 3xTg-AD mice. Our results indicate that cognitive performance and basic physiology of EC neurons depend on DHA intake in a mouse model of AD.

Developmental remodelling of the lemniscal synapse in the ventral basal thalamus of the mouse.

Synapse elimination occurs throughout the nervous system during development, and is essential for the formation of neural circuits. The mechanisms underlying synapse elimination in the brain, however, remain largely unknown. Using whole-cell patch-clamp recording in a slice preparation, we examined synaptic refinement at the somatosensory relay synapse (lemniscal synapse) in the ventral basal thalamus of the mouse during postnatal development. At 1 week old, each neuron in the ventral basal thalamus is innervated by multiple lemniscal fibres, as revealed by multiple increments of the synaptic response. By 16 days after birth (P16), the majority of neurons showed an all-or-none response, suggesting a single fibre innervation. In addition to synapse elimination, extensive modifications in synaptic properties occur during the second week after birth. The ratio of AMPA to NMDA component of the synaptic current tripled between P7 and P17. The decay constant of the NMDA component decreased by about 70% between P7 and P17; ifenprodil (3 microm) reduced the NMDA component by about 40% in neurons at P7-9, but was much less effective at P20-24. On the other hand, there was little change in the inward rectification of AMPA component between P11 and P24. Paired-pulse ratios, measured at -70 and +40 mV, were stable between P7 and P24. Whisker deprivation from P5 through P19 had no effect on the elimination or the maturation of the lemniscal synapse. These results suggest that the lemniscal synapse in the ventral basal thalamus undergoes extensive refinement during the second week, and that sensory experience has a rather limited role in this process.

Gain modulation by serotonin in pyramidal neurones of the rat prefrontal cortex.

Serotonin (5-HT) is widely implicated in brain functions and diseases. The vertebrate brain is extensively innervated by 5-HT fibres originating from the brain stem, and 5-HT axon terminals interact with other neurones in complex ways. The cellular mechanisms underlying 5-HT function in the brain are not well understood. The present study examined the effect of 5-HT on the responsiveness of neurones in the neocortex. Using patch-clamp recording in acute slices, we showed that 5-HT substantially increased the slope (gain) of the firing rate-current curve in layer 5 pyramidal neurones of the rat prefrontal cortex. The effect of 5-HT on gain is confined to the range of firing rate (0-10 Hz) that is known to be behaviourally relevant. 5-HT also changed current threshold for spike train generation, but this effect was inconsistent, and was independent of the effect on gain. The gain modulation by 5-HT was mediated by 5-HT2 receptors, and involved postsynaptic mechanisms. 5-HT2-mediated gain increase could not be attributed to changes in the membrane potential, the input resistance or the properties of action potentials, but was associated with a reduction of the afterhyperpolarization and an induction of the slow afterdepolarization. Blocking Ca2+ entry with Cd2+ increased the gain by itself and blocked 5-HT2- mediated gain increase. Buffering [Ca2+](i) with 25 mM EGTA also substantially reduced 5-HT2- mediated gain increase. Noradrenaline, which blocked the afterhyperpolarization, also induced a moderate increase in gain. Together, our results suggest that 5-HT may regulate the dynamics of cortical circuits through multiplicative scaling.