GABAergic terminals are a source of galanin to modulate cholinergic neuron development in the neonatal forebrain.

The distribution and (patho-)physiological role of neuropeptides in the adult and aging brain have been extensively studied. Galanin is an inhibitory neuropeptide that can coexist with γ-aminobutyric acid (GABA) in the adult forebrain. However, galanin's expression sites, mode of signaling, impact on neuronal morphology, and colocalization with amino acid neurotransmitters during brain development are less well understood. Here, we show that galaninergic innervation of cholinergic projection neurons, which preferentially express galanin receptor 2 (GalR2) in the neonatal mouse basal forebrain, develops by birth. Nerve growth factor (NGF), known to modulate cholinergic morphogenesis, increases GalR2 expression. GalR2 antagonism (M871) in neonates reduces the in vivo expression and axonal targeting of the vesicular acetylcholine transporter (VAChT), indispensable for cholinergic neurotransmission. During cholinergic neuritogenesis in vitro, GalR2 can recruit Rho-family GTPases to induce the extension of a VAChT-containing primary neurite, the prospective axon. In doing so, GalR2 signaling dose-dependently modulates directional filopodial growth and antagonizes NGF-induced growth cone differentiation. Galanin accumulates in GABA-containing nerve terminals in the neonatal basal forebrain, suggesting its contribution to activity-driven cholinergic development during the perinatal period. Overall, our data define the cellular specificity and molecular complexity of galanin action in the developing basal forebrain.

Whole body vibration ameliorates anxiety-like behavior and memory functions in 30 months old senescent male rats.

Whole body vibration (WBV) is a form of passive exercise that offers an alternative physical training to aged individuals with limitations in their physical and mental capabilities. The aim of the present study was to explore the therapeutic potential of five weeks of WBV on anxiety-like behaviors as well as learning and memory abilities in senescent thirty months old rats. Animals were exposed to 5 min vibration twice per day, five times per week during the five consecutive weeks. Pseudo WBV treated animals served as controls. After five weeks of WBV treatment, animals were tested for anxiety-like behavior by the open field test and for spatial and object memory functions by the novel and spatial object recognition tests, respectively. As a result, anxiety-like and exploratory behaviors were significantly improved in the WBV treated group compared to the pseudo WBV group. Furthermore, WBV treatment increased discrimination performance in both spatial and object memory function testing. These results indicate that WBV treatment in thirty months old rats seems to have comparable beneficial effects on age-related emotional and cognitive performance as what has been reported in younger age groups.

Lipocalin 2: novel component of proinflammatory signaling in Alzheimer's disease.

Alzheimer's disease (AD) is associated with an altered immune response, resulting in chronic increased inflammatory cytokine production with a prominent role of TNF-α. TNF-α signals are mediated by two receptors: TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Signaling through TNFR2 is associated with neuroprotection, whereas signaling through TNFR1 is generally proinflammatory and proapoptotic. Here, we have identified a TNF-α-induced proinflammatory agent, lipocalin 2 (Lcn2) via gene array in murine primary cortical neurons. Further investigation showed that Lcn2 protein production and secretion were activated solely upon TNFR1 stimulation when primary murine neurons, astrocytes, and microglia were treated with TNFR1 and TNFR2 agonistic antibodies. Lcn2 was found to be significantly decreased in CSF of human patients with mild cognitive impairment and AD and increased in brain regions associated with AD pathology in human postmortem brain tissue. Mechanistic studies in cultures of primary cortical neurons showed that Lcn2 sensitizes nerve cells to ß-amyloid toxicity. Moreover, Lcn2 silences a TNFR2-mediated protective neuronal signaling cascade in neurons, pivotal for TNF-α-mediated neuroprotection. The present study introduces Lcn2 as a molecular actor in neuroinflammation in early clinical stages of AD.

Braak staging in mouse models of Alzheimer's disease.

In the present paper by David E. Hurtado and colleagues report on a new mouse model for AD bearing Aß and MAPT pathology by crossing PS19 and PDAPP Tg mice. Here, we tried to highlight the importance and necessity of the critical and systematic analysis of models such as the Braak like staging in AD mouse models.

Measuring serotonin synthesis: from conventional methods to PET tracers and their (pre)clinical implications.

The serotonergic system of the brain is complex, with an extensive innervation pattern covering all brain regions and endowed with at least 15 different receptors (each with their particular distribution patterns), specific reuptake mechanisms and synthetic processes. Many aspects of the functioning of the serotonergic system are still unclear, partially because of the difficulty of measuring physiological processes in the living brain. In this review we give an overview of the conventional methods of measuring serotonin synthesis and methods using positron emission tomography (PET) tracers, more specifically with respect to serotonergic function in affective disorders. Conventional methods are invasive and do not directly measure synthesis rates. Although they may give insight into turnover rates, a more direct measurement may be preferred. PET is a noninvasive technique which can trace metabolic processes, like serotonin synthesis. Tracers developed for this purpose are α-[(11)C]methyltryptophan ([(11)C]AMT) and 5-hydroxy-L-[ß-(11)C]tryptophan ([(11)C]5-HTP). Both tracers have advantages and disadvantages. [(11)C]AMT can enter the kynurenine pathway under inflammatory conditions (and thus provide a false signal), but this tracer has been used in many studies leading to novel insights regarding antidepressant action. [(11)C]5-HTP is difficult to produce, but trapping of this compound may better represent serotonin synthesis. AMT and 5-HTP kinetics are differently affected by tryptophan depletion and changes of mood. This may indicate that both tracers are associated with different enzymatic processes. In conclusion, PET with radiolabelled substrates for the serotonergic pathway is the only direct way to detect changes of serotonin synthesis in the living brain.

New small-size peptides possessing antifungal activity.

The synthesis, in vitro evaluation, and conformational study of a new series of small-size peptides acting as antifungal agents are reported. In a first step of our study we performed a conformational analysis using Molecular Mechanics calculations. The electronic study was carried out using Molecular electrostatic potentials (MEPs) obtained from RHF/6-31G calculations. On the basis of the theoretical predictions three small-size peptides, RQWKKWWQWRR-NH(2), RQIRRWWQWRR-NH(2), and RQIRRWWQW-NH(2) were synthesized and tested. These peptides displayed a significant antifungal activity against human pathogenic strains including Candida albicans and Cryptococcus neoformans. Our experimental and theoretical results allow the identification of a topographical template which can serve as a guide for the design of new compounds with antifungal properties for potential therapeutic applications against these pathogenic fungi.

In silico study of full-length amyloid beta 1-42 tri- and penta-oligomers in solution.

Amyloid oligomers are considered to play causal roles in the pathogenesis of amyloid-related degenerative diseases including Alzheimer's disease. Using MD simulation techniques, we explored the contributions of the different structural elements of trimeric and pentameric full-length Abeta1-42 aggregates in solution to their stability and conformational dynamics. We found that our models are stable at a temperature of 310 K, and converge toward an interdigitated side-chain packing for intermolecular contacts within the two beta-sheet regions of the aggregates: beta1 (residues 18-26) and beta2 (residues 31-42). MD simulations reveal that the beta-strand twist is a characteristic element of Abeta-aggregates, permitting a compact, interdigitated packing of side chains from neighboring beta-sheets. The beta2 portion formed a tightly organized beta-helix, whereas the beta1 portion did not show such a firm structural organization, although it maintained its beta-sheet conformation. Our simulations indicate that the hydrophobic core comprising the beta2 portion of the aggregate is a crucial stabilizing element in the Abeta aggregation process. On the basis of these structure-stability findings, the beta2 portion emerges as an optimal target for further antiamyloid drug design.

Neuronal AKAP150 coordinates PKA and Epac-mediated PKB/Akt phosphorylation.

In diverse neuronal processes ranging from neuronal survival to synaptic plasticity cyclic adenosine monophosphate (cAMP)-dependent signaling is tightly connected with the protein kinase B (PKB)/Akt pathway but the precise nature of this connection remains unknown. In the current study we investigated the effect of two mainstream pathways initiated by cAMP, cAMP-dependent protein kinase (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) on PKB/Akt phosphorylation in primary cortical neurons and HT-4 cells. We demonstrate that PKA activation leads to a reduction of PKB/Akt phosphorylation, whereas activation of Epac has the opposite effect. This effect of Epac on PKB/Akt phosphorylation was mediated by Rap activation. The increase in PKB/Akt phosphorylation after Epac activation could be blocked by pretreatment with Epac2 siRNA and to a somewhat smaller extent by Epac1 siRNA. PKA, PKB/Akt and Epac were all shown to establish complexes with neuronal A-kinase anchoring protein150 (AKAP150). Interestingly, activation of Epac increased phosphorylation of PKB/Akt complexed to AKAP150. From experiments using PKA-binding deficient AKAP150 and peptides disrupting PKA anchoring to AKAPs, we conclude that AKAP150 acts as a key regulator in the two cAMP pathways to control PKB/Akt phosphorylation.

Effects of Long-Term Moderate Intensity Exercise on Cognitive Behaviors and Cholinergic Forebrain in the Aging Rat.

Physical exercise is now generally considered as a strategy to maintain cognitive abilities and to prevent age-related cognitive decline. In the present study, Wistar rats were subjected to moderate intensity treadmill exercise for 6 months prior to sacrifice at 12-, 24- and 32-month of age. This chronic physical intervention was tested on motility in the Open field (OF). Cognitive functions were measured in the Morris water maze (MWM) for spatial learning and in the Novel object recognition (NOR) tests. Since learning and memory are closely associated with cholinergic forebrain function ChAT fiber density after exercise training was assessed in hippocampus, and motor- and somatosensory cortical areas. Furthermore, quantification of ChAT-positive fiber aberrations as a neuropathological marker was also carried out in these brain areas. Our results show that in OF chronic exercise maintained horizontal locomotor activity in all age groups. Rearing activity, MWM and notably NOR performance were improved only in the 32-months old animals. Regarding cholinergic neuronal innervation, apart from a general age-related decline, exercise increased ChAT fiber density in the hippocampus CA1 area and in the motor cortex notably in the 32-months group. Massive ChAT fiber aberrations in all investigated areas which developed in senescence were clearly attenuated by exercise. The results suggest that moderate intensity chronic exercise in the rat is especially beneficial in advanced age. In conclusion, chronic exercise attenuates the age-related decline in cognitive and motor behaviors as well as age-related cholinergic fiber reduction, reduces malformations of cholinergic forebrain innervation.

Major depressive disorder is associated with changes in a cluster of serum and urine biomarkers.

Major Depressive Disorder (MDD) is a heterogeneous disorder with a considerable symptomatic overlap with other psychiatric and somatic disorders. This study aims at providing evidence for association of a set of serum and urine biomarkers with MDD. We analyzed urine and serum samples of 40 MDD patients and 47 age- and sex-matched controls using 40 potential MDD biomarkers (21 serum biomarkers and 19 urine biomarkers). All participants were of Caucasian origin. We developed an algorithm to combine the heterogeneity at biomarker level. This method enabled the identification of correlating biomarkers based on differences in variation and distribution between groups, combined the outcome of the selected biomarkers, and calculated depression probability scores (the "bio depression score"). Phenotype permutation analysis showed a significant discrimination between MDD and euthymic (control) subjects for biomarkers in urine (P < .001), in serum (P = .02) and in the combined serum plus urine result (P < .001). Based on this algorithm, a combination of 8 urine biomarkers and 9 serum biomarkers were identified to correlate with MDD, enabling an area under the curve (AUC) of 0.955 in a Receiver Operating Characteristic (ROC) analysis. Selection of either urine biomarkers or serum biomarkers resulted in AUC values of 0.907 and 0.853, respectively. Internal cross-validation (5-fold) confirmed the association of this set of biomarkers with MDD.

TNF-alpha-mediates neuroprotection against glutamate-induced excitotoxicity via NF-kappaB-dependent up-regulation of K2.2 channels.

Previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) induces neuroprotection against excitotoxic damage in primary cortical neurons via sustained nuclear factor-kappa B (NF-kappaB) activation. The transcription factor NF-kappaB can regulate the expression of small conductance calcium-activated potassium (K(Ca)) channels. These channels reduce neuronal excitability and as such may yield neuroprotection against neuronal overstimulation. In the present study we investigated whether TNF-alpha-mediated neuroprotective signaling is inducing changes in the expression of small conductance K(Ca) channels. Interestingly, the expression of K(Ca)2.2 channel was up-regulated by TNF-alpha treatment in a time-dependent manner whereas the expression of K(Ca)2.1 and K(Ca)2.3 channels was not altered. The increase in K(Ca)2.2 channel expression after TNF-alpha treatment was shown to be dependent on TNF-R2 and NF-kappaB activation. Furthermore, activation of small conductance K(Ca) channels by 6,7-dichloro-1H-indole-2,3-dione 3-oxime or cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine-induced neuroprotection against a glutamate challenge. Treatment with the small conductance K(Ca) channel blocker apamin or K(Ca)2.2 channel siRNA reverted the neuroprotective effect elicited by TNF-alpha. We conclude that treatment of primary cortical neurons with TNF-alpha leads to increased K(Ca)2.2 channel expression which renders neurons more resistant to excitotoxic cell death.

Chronically restricted sleep leads to depression-like changes in neurotransmitter receptor sensitivity and neuroendocrine stress reactivity in rats.

STUDY OBJECTIVES: Frequently disrupted and restricted sleep is a common problem for many people in our Western society. In the long run, insufficient sleep may have repercussions for health and may sensitize individuals to psychiatric diseases. In this context, we applied an animal model of chronic sleep restriction to study effects of sleep loss on neurobiological and neuroendocrine systems that have been implied in the pathophysiology of depression, particularly the serotonergic system and the hypothalamic-pituitary-adrenal (HPA) axis. DESIGN: Adult rats were exposed to a schedule of chronic partial sleep deprivation allowing them only 4 h of sleep per day. Sleep restriction was achieved by placing the animals in slowly rotating drums. To examine the regulation and reactivity of the HPA axis, blood samples were collected to measure adrenocorticotropin (ACTH) and corticosterone (CORT) responses. MEASUREMENTS AND RESULTS: While one day of restricted sleep had no significant effect on HPA axis stress reactivity, sleep restriction for a week caused a blunted pituitary ACTH response in a conditioned fear paradigm. Despite this lower ACTH response, adrenal CORT release was normal. The blunted pituitary response may be related to reduced sensitivity of serotonin-1A receptors and/or receptors for corticotropin-releasing hormone (CRH), since sleep restricted rats showed similar reductions in ACTH release to direct pharmacological stimulation with a serotonin-1A agonist or CRH. CONCLUSIONS: Chronic sleep restriction may lead to changes in neurotransmitter receptor systems and neuroendocrine reactivity in a manner similar to that seen in depression. This experimental study thus supports the hypothesis that disrupted and restricted sleep may contribute to the symptomatology of psychiatric disorders.

Lovastatin induces neuroprotection through tumor necrosis factor receptor 2 signaling pathways.

Statins are widely used as medication to lower cholesterol levels in human patients. In addition, it was recently reported that they also reduce the incidence of stroke and progression of Alzheimer's disease when prophylactically administered. To date there is only limited information available on how statins exert this beneficial effect. In this study we investigated the neuroprotective effect of lovastatin in primary cortical neurons. We found that lovastatin protects cortical neurons in a concentration-dependent manner against glutamate-mediated excitotoxicity. Interestingly, lovastatin with or without glutamate and/or tumor necrosis factor-alpha (TNF-alpha) increased TNF receptor 2 (TNF-R2) expression in cortical neurons. It was previously shown that activation of TNF-R2 signaling, which includes phosphorylation of protein kinase B (PKB)/Akt and activation of nuclear factor-kappa B (NF-kappaB), protects neurons against ischemic or excitotoxic insults. To investigate if lovastatin-induced neuroprotection was mediated by TNF-R2 signaling, primary cortical neurons were isolated from TNF-R1(-/-) or TNF-R2(-/-) mice. We could show that lovastatin is neuroprotective in TNF-R1(-/-) neurons, while protection is completely absent in TNF-R2(-/-) neurons. Furthermore, lovastatin-mediated neuroprotection led to an increase in PKB/Akt and NF-kappaB phosphorylation, whereas inhibition of PKB/Akt activation entirely abolished lovastatin-induced neuroprotection. Thus, lovastatin-induced neuroprotection against glutamate-excitotoxicity via activation of TNF-R2-signaling pathways.

Inhibition of PKA anchoring to A-kinase anchoring proteins impairs consolidation and facilitates extinction of contextual fear memories.

Both genetic and pharmacological studies demonstrated that contextual fear conditioning is critically regulated by cyclic AMP-dependent protein kinase (PKA). Since PKA is a broad range protein kinase, a mechanism for confining its activity is required. It has been shown that intracellular spatial compartmentalization of PKA signaling is mediated by A-kinase anchoring proteins (AKAPs). Here, we investigated the role of PKA anchoring to AKAPs in different stages of the memory process (acquisition, consolidation, retrieval and extinction) using contextual fear conditioning, a hippocampus-dependent learning task. Mice were injected intracerebroventricularly or intrahippocampally with the membrane permeable PKA anchoring disrupting peptides St-Ht31 or St-superAKAP-IS at different time points during the memory process. Blocking PKA anchoring to AKAPs resulted in an impairment of fear memory consolidation. Moreover, disrupted PKA anchoring promoted contextual fear extinction in the mouse hippocampus. We conclude that the temporal and spatial compartmentalization of hippocampal PKA signaling pathways, as achieved by anchoring of PKA to AKAPs, is specifically instrumental in long-term contextual fear memory consolidation and extinction, but not in acquisition and retrieval.

Repetitive stimulation of adenosine A1 receptors in vivo: changes in receptor numbers, G-proteins and A1 receptor agonist-induced hypothermia.

Adenosine is an important neuromodulator and neuroprotective molecule, which is produced in the brain as a function of neuronal activity, coupling energy expenditure to energy supply. Under conditions of increased need and reduced availability of energy, including hypoxia and prolonged wakefulness, there is an increase in adenosine turnover and adenosine receptor stimulation. The aim of the present study was to examine how repetitive adenosine receptor stimulation affects receptor function and adenosinergic signaling in the brain. Adult male Wistar rats received daily intraperitoneal injections of the adenosine A1 receptor agonist N(6)-cyclopentyladenosine (CPA; 0.25 mg/kg; once per day) and effects on adenosine signaling were established with receptor and G-protein autoradiography. Injections of CPA for 5 consecutive days caused a significant decrease in adenosine A1 receptor numbers in the hippocampus and somatosensory cortex. In contrast, while the amount of adenosine A1 receptor-activated G-proteins was not affected in most regions, a significant increase was found in the somatosensory cortex. On the level of physiological output, CPA-induced hypothermia was significantly attenuated, suggesting a functional desensitization of the A1 receptor system. Taken together, the present findings suggest that repetitive stimulation of the A1 receptors can affect elements of the adenosinergic signaling cascade in the rat brain in a region-specific manner.

Permanent, bilateral common carotid artery occlusion in the rat: a model for chronic cerebral hypoperfusion-related neurodegenerative diseases.

Chronic cerebral hypoperfusion has been associated with cognitive decline in aging and Alzheimer's disease. Moreover, the pattern of cerebral blood flow in mild cognitive impairment has emerged as a predictive marker for the progression into Alzheimer's disease. The reconstruction of a pathological condition in animal models is a suitable approach to the unraveling of causal relationships. For this reason, permanent, bilateral occlusion of the common carotid arteries (2VO) in rats has been established as a procedure to investigate the effects of chronic cerebral hypoperfusion on cognitive dysfunction and neurodegenerative processes. Over the years, the 2VO model has generated a large amount of data, revealing the 2VO-related pattern of cerebral hypoperfusion and metabolic changes, learning and memory disturbances, failure of neuronal signaling, and the neuropathological changes in the hippocampus. In addition, the model has been introduced in research into ischemic white matter injury and ischemic eye disease. The present survey sets out to provide a comprehensive summary of the achievements made with the 2VO model, and a critical evaluation and integration of the various results, and to relate the experimental data to human diseases. The data that have accumulated from use of the 2VO model in the rat permit an understanding of the causative role played by cerebral hypoperfusion in neurodegenerative diseases. Thorough characterization of the model suggests that 2VO in the rat is suitable for the development of potentially neuroprotective strategies in neurodegenerative diseases.

A-kinase anchoring protein 150 in the mouse brain is concentrated in areas involved in learning and memory.

A-kinase anchoring proteins (AKAPs) form large macromolecular signaling complexes that specifically target cAMP-dependent protein kinase (PKA) to unique subcellular compartments and thus, provide high specificity to PKA signaling. For example, the AKAP79/150 family tethers PKA, PKC and PP2B to neuronal membranes and postsynaptic densities and plays an important role in synaptic function. Several studies suggested that AKAP79/150 anchored PKA contributes to mechanisms associated with synaptic plasticity and memory processes, but the precise role of AKAPs in these processes is still unknown. In this study we established the mouse brain distribution of AKAP150 using two well-characterized AKAP150 antibodies. Using Western blotting and immunohistochemistry we showed that AKAP150 is widely distributed throughout the mouse brain. The highest AKAP150 expression levels were observed in striatum, cerebral cortex and several other forebrain regions (e.g. olfactory tubercle), relatively high expression was found in hippocampus and olfactory bulb and low/no expression in cerebellum, hypothalamus, thalamus and brain stem. Although there were some minor differences in mouse AKAP150 brain distribution compared to the distribution in rat brain, our data suggested that rodents have a characteristic AKAP150 brain distribution pattern. In general we observed that AKAP150 is strongly expressed in mouse brain regions involved in learning and memory. These data support its suggested role in synaptic plasticity and memory processes.

Sigma-1 Agonist Binding in the Aging Rat Brain: a MicroPET Study with [(11)C]SA4503.

PURPOSE: Sigma-1 receptor ligands modulate the release of several neurotransmitters and intracellular calcium signaling. We examined the binding of a radiolabeled sigma-1 agonist in the aging rat brain with positron emission tomography (PET). PROCEDURES: Time-dependent uptake of [(11)C]SA4503 was measured in the brain of young (1.5 to 3 months) and aged (18 to 32 months) Wistar Hannover rats, and tracer-kinetic models were fitted to this data, using metabolite-corrected plasma radioactivity as input function. RESULTS: In aged animals, the injected probe was less rapidly metabolized and cleared. Logan graphical analysis and a 2-tissue compartment model (2-TCM) fit indicated changes of total distribution volume (V T) and binding potential (BP ND) of the tracer. BP ND was reduced particularly in the (hypo)thalamus, pons, and medulla. CONCLUSIONS: Some areas showed reductions of ligand binding with aging whereas binding in other areas (cortex) was not significantly affected.

Too little sleep gradually desensitizes the serotonin 1A receptor system.

STUDY OBJECTIVES: In our 24-hour society, frequently disrupted and restricted sleep is a rapidly increasing problem that may contribute to the development of diseases such as depression. One of the proposed neurobiological mechanisms underlying depression is a disturbance in the brain's serotonergic neurotransmission, particularly a desensitization of the serotonin (5-HT)1A receptor system. However, a relationship between chronic sleep loss and changes in (5-HT)1A receptors has not been established yet. Therefore, in the present study, we experimentally tested the hypothesis that chronic sleep restriction leads to desensitization of the (5-HT)1A receptor system. DESIGN: Rats were subjected to a schedule of restricted sleep allowing them 4 hours of sleep per day. Sleep restriction was achieved by placing the animals in slowly rotating wheels. The sensitivity of the (5-HT)1A receptor system was examined by measuring the hypothermic response to a standard injection of a 1A agonist. RESULTS: After 2 days of restricted sleep, the sensitivity of the (5-HT)1A receptor system was not yet affected; however, after 8 days of sleep restriction, it was desensitized. Control experiments indicated that the effect of sleep restriction was not due to forced activity or stress. Importantly, the desensitization of the (5-HT)1A system persisted for many days even with unlimited recovery sleep. Normalization occurred gradually but required at least 7 days. CONCLUSIONS: Chronic sleep restriction causes a gradual and persistent desensitization of the (5-HT)1A receptor system. This finding provides a link between chronic sleep loss and sensitivity for disorders that are associated with altered serotonergic neurotransmission.