Characterization of an experimental model to determine streptococcal M protein-induced autoimmune cardiac and neurobehavioral abnormalities.

Group A streptococcal (GAS) infection is associated with a spectrum of autoimmune diseases including acute rheumatic fever/rheumatic heart disease (ARF/RHD) and neurobehavioral abnormalities. Antibodies against GAS M proteins cross-react with host tissue proteins in the heart and brain leading to the symptomatology observed in ARF/RHD. As throat carriage of Streptococcus dysgalactiae subspecies equisimilis (SDSE) has been reported to be relatively high in some ARF/RHD endemic regions compared with GAS, and both SDSE and GAS express coiled-coil surface protein called M protein, we hypothesized that streptococci other than GAS can also associated with ARF/RHD and neurobehavioral abnormalities. Neurobehavioral assessments and electrocardiography were performed on Lewis rats before and after exposure to recombinant GAS and SDSE M proteins. Histological assessments were performed to confirm inflammatory changes in cardiac and neuronal tissues. ELISA and Western blot analysis were performed to determine the cross-reactivity of antibodies with host connective, cardiac and neuronal tissue proteins. Lewis rats injected with M proteins either from GAS or SDSE developed significant cardiac functional and neurobehavioral abnormalities in comparison to control rats injected with phosphate-buffered saline. Antibodies against GAS and SDSE M proteins cross-reacted with cardiac, connective and neuronal proteins. Serum from rats injected with streptococcal antigens showed higher immunoglobulin G binding to the striatum and cortex of the brain. Cardiac and neurobehavioral abnormalities observed in our experimental model were comparable to the cardinal symptoms observed in patients with ARF/RHD. Here for the first time, we demonstrate in an experimental model that M proteins from different streptococcal species could initiate and drive the autoimmune-mediated cardiac tissue damage and neurobehavioral abnormalities.

Group A streptococcal antigen exposed rat model to investigate neurobehavioral and cardiac complications associated with post-streptococcal autoimmune sequelae.

Background: The neuropsychiatric disorders due to post-streptococcal autoimmune complications such as Sydenham's chorea (SC) are associated with acute rheumatic fever and rheumatic heart disease (ARF/RHD). An animal model that exhibits characteristics of both cardiac and neurobehavioral defects in ARF/RHD would be an important adjunct for future studies. Since age, gender, strain differences, and genotypes impact on the development of autoimmunity, we investigated the behavior of male and female Wistar and Lewis rat strains in two age cohorts (<6 weeks and >12 weeks) under normal husbandry conditions and following exposure to group A streptococcus (GAS). Methods: Standard behavioral assessments were performed to determine the impairments in fine motor control (food manipulation test), gait and balance (beam walking test), and obsessive-compulsive behavior (grooming and marble burying tests). Furthermore, electrocardiography, histology, and behavioral assessments were performed on male and female Lewis rats injected with GAS antigens. Results: For control Lewis rats there were no significant age and gender dependent differences in marble burying, food manipulation, beam walking and grooming behaviors. In contrast significant age-dependent differences were observed in Wistar rats in all the behavioral tests except for food manipulation. Therefore, Lewis rats were selected for further experiments to determine the effect of GAS. After exposure to GAS, Lewis rats demonstrated neurobehavioral abnormalities and cardiac pathology akin to SC and ARF/RHD, respectively. Conclusion: We have characterised a new model that provides longitudinal stability of age-dependent behavior, to simultaneously investigate both neurobehavioral and cardiac abnormalities associated with post-streptococcal complications.

Requirements for a Robust Animal Model to Investigate the Disease Mechanism of Autoimmune Complications Associated With ARF/RHD.

The pathogenesis of Acute Rheumatic Fever/Rheumatic Heart Disease (ARF/RHD) and associated neurobehavioral complications including Sydenham's chorea (SC) is complex. Disease complications triggered by Group A streptococcal (GAS) infection are confined to human and determining the early events leading to pathology requires a robust animal model that reflects the hallmark features of the disease. However, modeling these conditions in a laboratory animal, of a uniquely human disease is challenging. Animal models including cattle, sheep, pig, dog, cat, guinea pigs rats and mice have been used extensively to dissect molecular mechanisms of the autoimmune inflammatory responses in ARF/RHD. Despite the characteristic limitations of some animal models, several rodent models have significantly contributed to better understanding of the fundamental mechanisms underpinning features of ARF/RHD. In the Lewis rat autoimmune valvulitis model the development of myocarditis and valvulitis with the infiltration of mononuclear cells along with generation of antibodies that cross-react with cardiac tissue proteins following exposure to GAS antigens were found to be similar to ARF/RHD. We have recently shown that Lewis rats injected with recombinant GAS antigens simultaneously developed cardiac and neurobehavioral changes. Since ARF/RHD is multifactorial in origin, an animal model which exhibit the characteristics of several of the cardinal diagnostic criteria observed in ARF/RHD, would be advantageous to determine the early immune responses to facilitate biomarker discovery as well as provide a suitable model to evaluate treatment options, safety and efficacy of vaccine candidates. This review focuses on some of the common small animals and their advantages and limitations.

A review of the antimicrobial side of antidepressants and its putative implications on the gut microbiome.

OBJECTIVE: Serotonin reuptake inhibitors are the predominant treatment for major depressive disorder. In recent years, the diversity of the gut microbiota has emerged to play a significant role in the occurrence of major depressive disorder and other mood and anxiety disorders. Importantly, the role of the gut microbiota in the treatment of such disorders remains to be elucidated. Here, we provide a review of the literature regarding the effects of physiologically relevant concentrations of serotonin reuptake inhibitors on the gut microbiota and the implications this might have on their efficacy in the treatment of mood disorders. METHODS: First, an estimation of gut serotonin reuptake inhibitor concentrations was computed based on pharmacokinetic and gastrointestinal transit properties of serotonin reuptake inhibitors. Literature regarding the in vivo and in vitro antimicrobial properties of serotonin reuptake inhibitors was gathered, and the estimated gut concentrations were examined in the context of these data. Computer-based investigation revealed putative mechanisms for the antimicrobial effects of serotonin reuptake inhibitors. RESULTS: In vivo evidence using animal models shows an antimicrobial effect of serotonin reuptake inhibitors on the gut microbiota. Examination of the estimated physiological concentrations of serotonin reuptake inhibitors in the gastrointestinal tract collected from in vitro studies suggests that the microbial community of both the small intestine and the colon are exposed to serotonin reuptake inhibitors for at least 4 hours per day at concentrations that are likely to exert an antimicrobial effect. The potential mechanisms of the effect of serotonin reuptake inhibitors on the gut microbiota were postulated to include inhibition of efflux pumps and/or amino acid transporters. CONCLUSION: This review raises important issues regarding the role that gut microbiota play in the treatment of mood-related behaviours, which holds substantial potential clinical outcomes for patients suffering from major depressive disorder and other mood-related disorders.

Storage and handling of human faecal samples affect the gut microbiome composition: A feasibility study.

Human gut microbiome analysis through faecal sampling typically involves five stages: sample collection, storage, DNA extraction, next generation sequencing and bioinformatics analysis. Of these, the first three are considered irreversible. This feasibility study describes an assessment of methodologies used for faecal DNA extraction and sample handling, using the parameters DNA yield, purity and resultant microbial profile. Six DNA extraction techniques, including commercially available kits and manual protocols were compared on human faecal samples (n = 3). Different extraction techniques produced significant variance in DNA yield (range 2.7-164 ng/mg faeces) and microbial diversity profiles, with considerable variation in phyla dominance (Firmicutes (P < 0.001), Bacteroidetes (P = 0.003), Actinobacteria (P = 0.003), One-way ANOVA). The most effective method, with the highest DNA yield, was a simple and inexpensive extraction technique named MetaHIT. Using this method, DNA was extracted from separate faecal samples (n = 3) and had been aliquoted to seven storage conditions including three stabilizing buffers and three temperature conditions, for a period of 120-h, with storage at -80 °C as a control treatment. DNA yield and purity was not statistically different between the control and remaining treatments. 16S rDNA-based diversity profile was largely comparable across the treatments with only minor differences in genera between samples stored at room temperature in air and - 80 °C control. Overall these results suggest that the choice of DNA extraction method has a greater influence on the resultant microbial diversity profile than the short-term storage method.

Anticipatory Behavior for a Mealworm Reward in Laying Hens Is Reduced by Opioid Receptor Antagonism but Not Standard Feed Intake.

It is widely accepted that the absence of suffering no longer defines animal welfare and that positive affective experiences are imperative. For example, laying hens may be housed in environments that do not cause chronic stress but may lack particular resources that promote positive affective experiences, such as conspecifics or effective enrichment. Despite a consensus of how important positive affect is for animal welfare, they are difficult to identify objectively. There is a need for valid and reliable indicators of positive affect. Pharmacological interventions can be an effective method to provide insight into affective states and can assist with the investigation of novel indicators such as associated biomarkers. We aimed to validate a pharmacological intervention that blocks the subjective hedonistic phase associated with reward in laying hens via the administration of the non-selective (µ, δ, and κ) opioid receptor antagonist, nalmafene. We hypothesized that nonfood deprived, hens that did not experience a positive affective state when presented with a mealworm food reward due to the administration of nalmefene, would show minimal anticipatory and consummatory behavior when the same food reward was later presented. Hens (n = 80) were allocated to treatment groups, receiving either nalmefene or vehicle (0.9% saline) once or twice daily, for four consecutive days. An anticipatory test (AT) was performed on all days 30 min post-drug administration. Behavioral responses during the appetitive and consummatory phase were assessed on days 1, 3 and 4. Anticipatory behavior did not differ between treatment groups the first time hens were provided with mealworm food rewards. However, antagonism of opioid receptors reduced anticipatory and consummatory behavior on days 3 and 4. Feed intake of standard layer mash was not impacted by treatment, thus nalmefene reduced non-homeostatic food consumption but not homeostatic consumption. Behavioral observations during the AT provided no evidence that nalmefene treated hens were fearful, sedated or nauseous. The results suggest that we successfully blocked the hedonistic subjective component of reward in laying hens and provide evidence that this method could be used to investigate how hens perceive their environment and identify associated novel indicators to assess hen welfare.

Biophenols: Enzymes (ß-secretase, Cholinesterases, histone deacetylase and tyrosinase) inhibitors from olive (Olea europaea L.).

The focus of this study was on inhibition of enzymes involved in the pathogenesis Alzheimer's disease (AD) including prime amyloid beta (Aß) producing enzyme (ß-secretase: BACE-1) and disease progression enzymes including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), histone deacetylase (HDAC), and tyrosinase along with the catecholamine L-DOPA, by using olive biophenols. Here we report the strongest inhibition of BACE-1 from rutin (IC50: 3.8 nM) followed by verbascoside (IC50: 6.3 nM) and olive fruit extract (IC50: 18 ng), respectively. Olive biophenol, quercetin exhibited strongest enzyme inhibitory activity against tyrosinase (IC50: 10.73 µM), BChE (IC50: 19.08 µM), AChE (IC50: 55.44 µM), and HDAC (IC50: 105.1 µM) enzymes. Furthermore, olive biophenol verbascoside (IC50: 188.6 µM), and hydroxytyrosol extreme extract (IC50: 66.22 µg) were showed the highest levels of inhibition against the HDAC enzyme. Neuroprotective capacity against levodopa-induced toxicity in neuroblastoma (SH-SY5Y) cells of olive biophenols were assessed, where rutin indicated the highest neuroprotection (74%), followed by caffeic acid (73%), and extract hydroxytyrosol extreme (97%), respectively. To the best of our knowledge, this is the first in vitro report on the enzymes inhibitory activity of olive biophenols. Taken together, our in vitro results data suggest that olive biophenols could be a promising natural inhibitor, which may reduce the enzyme-induced toxicity associated with the oxidative stress involved in the progression of AD. CHEMICAL COMPOUNDS USED IN THE STUDY: Acetylthiocholine iodide (PubChem CID: 74629); S-Butyrylthiocholine chloride (PubChem CID: 3015121); Caffeic acid (PubChem CID: 689043); Dimethyl sulfoxide (DMSO) (PubChem: 679); L-3,4-Dihydroxyphenylalanine (L-DOPA) (PubChem CID: 6047); 5,5'-Dithiobis (2-nitrobenzoic acid) (DTNB) (PubChem CID: 6254); Epigallocatechin gallate (EGCG) (PubChem CID: 65064); Ethylenediamine tetraacetic acid (EDTA) (PubChem CID: 6049); Galantamine hydrobromide (PubChem CID: 121587); l-Glutamine (PubChem CID: 5961); Hydroxytyrosol (PubChem CID: 82755); Kojic acid (PubChem CID: 3840); Luteolin (PubChem CID: 5280445); Oleuropein (PubChem CID: 5281544); Penicillin-streptomycin (PubChem CID: 131715954); Quercetin (PubChem CID: 5280343); Rutin (PubChem CID: 5280805); Tris-HCl buffer (PubChem: 93573); Trypan blue (PubChem: 9562061).

Olive Biophenols Reduces Alzheimer's Pathology in SH-SY5Y Cells and APPswe Mice.

Alzheimer's disease (AD) is a major neurodegenerative disease, associated with the hallmark proteinacious constituent called amyloid beta (Aß) of senile plaques. Moreover, it is already established that metals (particularly copper, zinc and iron) have a key role in the pathogenesis of AD. In order to reduce the Aß plaque burden and overcome the side effects from the synthetic inhibitors, the current study was designed to focus on direct inhibition of with or without metal-induced Aß fibril formation and aggregation by using olive biophenols. Exposure of neuroblastoma (SH-SY5Y) cells with Aß42 resulted in decrease of cell viability and morphological changes might be due to severe increase in the reactive oxygen species (ROS). The pre-treated SH-SY5Y cells with olive biophenols were able to attenuate cell death caused by Aß42, copper- Aß42, and [laevodihydroxyphenylalanine (l-DOPA)] l-DOPA-Aß42-induced toxicity after 24 h of treatment. Oleuropein, verbascoside and rutin were the major anti-amyloidogenic compounds. Transgenic mice (APPswe/PS1dE9) received 50 mg/kg of oleuropein containing olive leaf extracts (OLE) or control diet from 7 to 23 weeks of age. Treatment mice (OLE) were showed significantly reduced amyloid plaque deposition (p < 0.001) in cortex and hippocampus as compared to control mice. Our findings provide a basis for considering natural and low cost biophenols from olive as a promising candidate drug against AD. Further studies warrant to validate and determine the anti-amyloid mechanism, bioavailability as well as permeability of olive biophenols against blood brain barrier in AD.

Olive (Olea europaea L.) Biophenols: A Nutriceutical against Oxidative Stress in SH-SY5Y Cells.

Plant biophenols have been shown to be effective in the modulation of Alzheimer's disease (AD) pathology resulting from free radical-induced oxidative stress and imbalance of the redox chemistry of transition metal ions (e.g., iron and copper). On the basis of earlier reported pharmacological activities, olive biophenols would also be expected to have anti-Alzheimer's activity. In the present study, the antioxidant activity of individual olive biophenols (viz. caffeic acid, hydroxytyrosol, oleuropein, verbascoside, quercetin, rutin and luteolin) were evaluated using superoxide radical scavenging activity (SOR), hydrogen peroxide (H2O2) scavenging activity, and ferric reducing ability of plasma (FRAP) assays. The identification and antioxidant activities in four commercial olive extracts-Olive leaf extractTM (OLE), Olive fruit extractTM (OFE), Hydroxytyrosol ExtremeTM (HTE), and Olivenol plusTM (OLP)-were evaluated using an on-line HPLC-ABTS•+ assay, and HPLC-DAD-MS analysis. Oleuropein and hydroxytyrosol were the predominant biophenols in all the extracts. Among the single compounds examined, quercetin (EC50: 93.97 µM) and verbascoside (EC50: 0.66 mM) were the most potent SOR and H2O2 scavengers respectively. However, OLE and HTE were the highest SOR (EC50: 1.89 µg/mL) and H2O2 (EC50: 115.8 µg/mL) scavengers among the biophenol extracts. The neuroprotection of the biophenols was evaluated against H2O2-induced oxidative stress and copper (Cu)-induced toxicity in neuroblastoma (SH-SY5Y) cells. The highest neuroprotection values (98% and 92%) against H2O2-induced and Cu-induced toxicities were shown by the commercial extract HTETM. These were followed by the individual biophenols, caffeic acid (77% and 64%) and verbascoside (71% and 72%). Our results suggest that olive biophenols potentially serve as agents for the prevention of neurodegenerative diseases such as AD, and other neurodegenerative ailments that are caused by oxidative stress.

The protective role of plant biophenols in mechanisms of Alzheimer's disease.

Self-assembly of amyloid beta peptide (Aß) into the neurotoxic oligomers followed by fibrillar aggregates is a defining characteristic of Alzheimer's disease (AD). Several lines of proposed hypotheses have suggested the mechanism of AD pathology, though the exact pathophysiological mechanism is not yet elucidated. The poor understanding of AD and multitude of adverse responses reported from the current synthetic drugs are the leading cause of failure in the drug development to treat or halt the progression of AD and mandate the search for safer and more efficient alternatives. A number of natural compounds have shown the ability to prevent the formation of the toxic oligomers and disrupt the aggregates, thus attracted much attention. Referable to the abundancy and multitude of pharmacological activities of the plant active constituents, biophenols that distinguish them from the other phytochemicals as a natural weapon against the neurodegenerative disorders. This review provides a critical assessment of the current literature on in vitro and in vivo mechanistic activities of biophenols associated with the prevention and treatment of AD. We have contended the need for more comprehensive approaches to evaluate the anti-AD activity of biophenols at various pathologic levels and to assess the current evidences. Consequently, we highlighted the various problems and challenges confronting the AD research, and offer recommendations for future research.

Basal Forebrain Atrophy Contributes to Allocentric Navigation Impairment in Alzheimer's Disease Patients.

The basal forebrain degenerates in Alzheimer's disease (AD) and this process is believed to contribute to the cognitive decline observed in AD patients. Impairment in spatial navigation is an early feature of the disease but whether basal forebrain dysfunction in AD is responsible for the impaired navigation skills of AD patients is not known. Our objective was to investigate the relationship between basal forebrain volume and performance in real space as well as computer-based navigation paradigms in an elderly cohort comprising cognitively normal controls, subjects with amnestic mild cognitive impairment and those with AD. We also tested whether basal forebrain volume could predict the participants' ability to perform allocentric- vs. egocentric-based navigation tasks. The basal forebrain volume was calculated from 1.5 T magnetic resonance imaging (MRI) scans, and navigation skills were assessed using the human analog of the Morris water maze employing allocentric, egocentric, and mixed allo/egocentric real space as well as computerized tests. When considering the entire sample, we found that basal forebrain volume correlated with spatial accuracy in allocentric (cued) and mixed allo/egocentric navigation tasks but not the egocentric (uncued) task, demonstrating an important role of the basal forebrain in mediating cue-based spatial navigation capacity. Regression analysis revealed that, although hippocampal volume reflected navigation performance across the entire sample, basal forebrain volume contributed to mixed allo/egocentric navigation performance in the AD group, whereas hippocampal volume did not. This suggests that atrophy of the basal forebrain contributes to aspects of navigation impairment in AD that are independent of hippocampal atrophy.

Comparative studies using the Morris water maze to assess spatial memory deficits in two transgenic mouse models of Alzheimer's disease.

Evaluation of the efficacy of novel therapeutics for potential treatment of Alzheimer's disease (AD) requires an animal model that develops age-related cognitive deficits reproducibly between independent groups of investigators. Herein we assessed comparative temporal changes in spatial memory function in two commercially available transgenic mouse models of AD using the Morris water maze (MWM), incorporating both visible and hidden platform training. Individual cohorts of cDNA-based 'line 85'-derived double-transgenic mice coexpressing the 'Swedish' mutation of amyloid precursor protein (APPSwe) and the presenillin 1 (PS1) 'dE9' mutation were assessed in the MWM at mean ages of 3.6, 9.3 and 14.8 months. We found significant deficits in spatial memory retention in APPSwe/PS1dE9 mice aged 3.6 months and robust deficits in spatial memory acquisition and retention in APPSwe/PS1dE9 mice aged 9.3 months, with a further significant decline by age 14.8 months. ß-Amyloid deposits were present in brain sections by 7.25 months of age. In contrast, MWM studies with individual cohorts (aged 4-21 months) of single-transgenic genomic-based APPSwe mice expressing APPSwe on a yeast artificial chromosomal (YAC) construct showed no significant deficits in spatial memory acquisition until 21 months of age. There were no significant deficits in spatial memory retention up to 21 months of age and ß-amyloid deposits were not present in brain sections up to 24 months of age. These data, generated using comprehensive study designs, show that APPSwe/PS1dE9 but not APPSwe YAC mice appear to provide a suitably robust model of AD for efficacy assessment of novel AD treatments in development.

Lesions of the basal forebrain cholinergic system in mice disrupt idiothetic navigation.

Loss of integrity of the basal forebrain cholinergic neurons is a consistent feature of Alzheimer's disease, and measurement of basal forebrain degeneration by magnetic resonance imaging is emerging as a sensitive diagnostic marker for prodromal disease. It is also known that Alzheimer's disease patients perform poorly on both real space and computerized cued (allothetic) or uncued (idiothetic) recall navigation tasks. Although the hippocampus is required for allothetic navigation, lesions of this region only mildly affect idiothetic navigation. Here we tested the hypothesis that the cholinergic medial septo-hippocampal circuit is important for idiothetic navigation. Basal forebrain cholinergic neurons were selectively lesioned in mice using the toxin saporin conjugated to a basal forebrain cholinergic neuronal marker, the p75 neurotrophin receptor. Control animals were able to learn and remember spatial information when tested on a modified version of the passive place avoidance test where all extramaze cues were removed, and animals had to rely on idiothetic signals. However, the exploratory behaviour of mice with cholinergic basal forebrain lesions was highly disorganized during this test. By contrast, the lesioned animals performed no differently from controls in tasks involving contextual fear conditioning and spatial working memory (Y maze), and displayed no deficits in potentially confounding behaviours such as motor performance, anxiety, or disturbed sleep/wake cycles. These data suggest that the basal forebrain cholinergic system plays a specific role in idiothetic navigation, a modality that is impaired early in Alzheimer's disease.

Diffusion-weighted magnetic resonance imaging detection of basal forebrain cholinergic degeneration in a mouse model.

Loss of basal forebrain cholinergic neurons is an early and key feature of Alzheimer's disease, and magnetic resonance imaging (MRI) volumetric measurement of the basal forebrain has recently gained attention as a potential diagnostic tool for this condition. The aim of this study was to determine whether loss of basal forebrain cholinergic neurons underpins changes which can be detected through diffusion MRI using diffusion tensor imaging (DTI) and probabilistic tractography in a mouse model. To cause selective basal forebrain cholinergic degeneration, the toxin saporin conjugated to a p75 neurotrophin receptor antibody (mu-p75-SAP) was used. This resulted in ~25% loss of the basal forebrain cholinergic neurons and significant loss of terminal cholinergic projections in the hippocampus, as determined by histology. To test whether lesion of cholinergic neurons caused basal forebrain, hippocampal, or whole brain atrophy, we performed manual segmentation analysis, which revealed no significant atrophy in lesioned animals compared to controls (Rb-IgG-SAP). However, analysis by DTI of the basal forebrain area revealed a significant increase in fractional anisotropy (FA; +7.7%), mean diffusivity (MD; +6.1%), axial diffusivity (AD; +8.5%) and radial diffusivity (RD; +4.0%) in lesioned mice compared to control animals. These parameters strongly inversely correlated with the number of choline acetyl transferase-positive neurons, with FA showing the greatest association (r(2)=0.72), followed by MD (r(2)=0.64), AD (r(2)=0.64) and RD (r(2)=0.61). Moreover, probabilistic tractography analysis of the septo-hippocampal tracts originating from the basal forebrain revealed an increase in streamline MD (+5.1%) and RD (+4.3%) in lesioned mice. This study illustrates that moderate loss of basal forebrain cholinergic neurons (representing only a minor proportion of all septo-hippocampal axons) can be detected by measuring either DTI parameters of the basal forebrain nuclei or tractography parameters of the basal forebrain tracts. These findings provide increased support for using DTI and probabilistic tractography as non-invasive tools for diagnosing and/or monitoring the progression of conditions affecting the integrity of the basal forebrain cholinergic system in humans, including Alzheimer's disease.

Prolactin stimulates precursor cells in the adult mouse hippocampus.

In the search for ways to combat degenerative neurological disorders, neurogenesis-stimulating factors are proving to be a promising area of research. In this study, we show that the hormonal factor prolactin (PRL) can activate a pool of latent precursor cells in the adult mouse hippocampus. Using an in vitro neurosphere assay, we found that the addition of exogenous PRL to primary adult hippocampal cells resulted in an approximate 50% increase in neurosphere number. In addition, direct infusion of PRL into the adult dentate gyrus also resulted in a significant increase in neurosphere number. Together these data indicate that exogenous PRL can increase hippocampal precursor numbers both in vitro and in vivo. Conversely, PRL null mice showed a significant reduction (approximately 80%) in the number of hippocampal-derived neurospheres. Interestingly, no deficit in precursor proliferation was observed in vivo, indicating that in this situation other niche factors can compensate for a loss in PRL. The PRL loss resulted in learning and memory deficits in the PRL null mice, as indicated by significant deficits in the standard behavioral tests requiring input from the hippocampus. This behavioral deficit was rescued by direct infusion of recombinant PRL into the hippocampus, indicating that a lack of PRL in the adult mouse hippocampus can be correlated with impaired learning and memory.

Phosphorylation of mitogen-activated protein kinase in the medial prefrontal cortex and the amygdala following memory retrieval or forgetting in developing rats.

We examined neuronal correlates of forgetting in rats by detection of phosphorylated mitogen-activated protein kinase (pMAPK) in the medial prefrontal cortex (mPFC) and amygdala. In Experiment 1, postnatal day (P)23 and P16 rats received paired noise CS-shock US presentations. When tested immediately after conditioning, P23 and P16 rats exhibited similar levels of conditioned fear; when tested after 2 days, however, P16 rats showed poor CS-elicited freezing relative to P23 rats. In Experiment 2, P16 and P23 rats received either paired or unpaired CS-US presentations, and then were tested 48 h later. Consistent with Experiment 1, P16 rats showed forgetting whereas P23 rats exhibited good retention at test. Additionally, unpaired groups showed poor CS-elicited freezing at test. Immunohistochemistry showed that P23 and P16 rats given paired presentations exhibited significant elevation of pMAPK-immunoreactive (ir) neurons in the amygdala compared to rats given unpaired presentations. That is, MAPK phosphorylation in the amygdala tracked learning history rather than behavioral performance at test. In contrast, only the P23-paired group showed an elevated number of pMAPK-ir neurons in mPFC, indicating that MAPK phosphorylation in the mPFC tracks memory expression. Different test-perfusion intervals were employed in Experiment 3, which showed that the developmental dissociation in the pMAPK-ir neurons observed in the mPFC in Experiment 2 was not due to age differences in the rate of phosphorylation of MAPK. These findings provide initial evidence suggesting that while the mPFC is involved in memory retrieval, MAPK phosphorylation in the amygdala may be a persisting neural signature of fear memory.

The role of prefrontal cortex in predictive fear learning.

Pavlovian fear conditioning depends on prediction error, or the discrepancy between actual and expected outcomes. We used immunohistochemistry, neuronal tract tracing, and reversible inactivation to study the role of prefrontal cortex and thalamocortical pathways in predictive fear learning. Unexpected, but not expected, conditioned stimulus (CS)-unconditioned stimulus (US) presentations caused increased c-Fos expression in the prefrontal cortex (PFC), midline thalamus, lateral amygdala, as well as retrograde labeled midline thalamic afferents to PFC. Reversible inactivation of dorsomedial PFC, but not infralimbic PFC, prevented the associative blocking of fear learning. These results suggest a role for dorsomedial PFC (dmPFC), and a thalamic → dmPFC pathway, in signaling whether or not aversive events are expected or unexpected and so whether they are to be learned about.

Norepinephrine directly activates adult hippocampal precursors via beta3-adrenergic receptors.

Adult hippocampal neurogenesis is a critical form of cellular plasticity that is greatly influenced by neural activity. Among the neurotransmitters that are widely implicated in regulating this process are serotonin and norepinephrine, levels of which are modulated by stress, depression and clinical antidepressants. However, studies to date have failed to address a direct role for either neurotransmitter in regulating hippocampal precursor activity. Here we show that norepinephrine but not serotonin directly activates self-renewing and multipotent neural precursors, including stem cells, from the hippocampus of adult mice. Mechanistically, we provide evidence that beta(3)-adrenergic receptors, which are preferentially expressed on a Hes5-expressing precursor population in the subgranular zone (SGZ), mediate this norepinephrine-dependent activation. Moreover, intrahippocampal injection of a selective beta(3)-adrenergic receptor agonist in vivo increases the number of proliferating cells in the SGZ. Similarly, systemic injection of the beta-adrenergic receptor agonist isoproterenol not only results in enhancement of proliferation in the SGZ but also leads to an increase in the percentage of nestin/glial fibrillary acidic protein double-positive neural precursors in vivo. Finally, using a novel ex vivo "slice-sphere" assay that maintains an intact neurogenic niche, we demonstrate that antidepressants that selectively block the reuptake of norepinephrine, but not serotonin, robustly increase hippocampal precursor activity via beta-adrenergic receptors. These findings suggest that the activation of neurogenic precursors and stem cells via beta(3)-adrenergic receptors could be a potent mechanism to increase neuronal production, providing a putative target for the development of novel antidepressants.

Fear extinction across development: the involvement of the medial prefrontal cortex as assessed by temporary inactivation and immunohistochemistry.

Extinction in adult animals, including humans, appears to involve the medial prefrontal cortex (mPFC). However, the role of mPFC in extinction across development has not yet been studied. Given several recent demonstrations of developmental differences in extinction of conditioned fear at a behavioral level, different neural circuitries may mediate fear extinction across development. In all experiments, noise conditioned stimulus (CS) and shock unconditioned stimulus (US) were used. In experiment 1A, temporary unilateral inactivation of the mPFC during extinction training impaired long-term extinction the following day in postnatal day 24 (P24) rats but not in P17 rats. In experiment 1B, bilateral inactivation of the mPFC again failed to disrupt long-term extinction in P17 rats. In experiment 2, extinction training increased phosphorylated mitogen-activated protein kinase (pMAPK) in the mPFC for P24 rats but not for P17 rats, whereas rats of both ages displayed elevated pMAPK in the amygdala. Across both ages, "not trained," "reactivated," and "no extinction" control groups expressed very low numbers of pMAPK-immunoreactive (IR) neurons across both neural structures. This result indicates that the mere conditioning experience, the exposure to the CS, or the expression of CS-elicited fear in and of itself is not sufficient to explain the observed increase in pMAPK-IR neurons in the mPFC and/or the amygdala after extinction. Together, these findings show that extinction in P17 rats does not involve the mPFC, which has important theoretical and clinical implications for the treatment of anxiety disorders in humans.

Induction of Fos proteins in regions of the nucleus accumbens and ventrolateral striatum correlates with catalepsy and stereotypic behaviours induced by morphine.

A history of intermittent exposures to drugs of abuse can cause long-term changes in acute behavioural responses to a subsequent drug exposure. In drug-naive rats, morphine can elicit intermittent cataleptic postures followed by sustained increases in locomotor activity. Chronic intermittent morphine treatment can reduce catalepsy and increase locomotor behaviour and stereotypy induced by morphine, even after prolonged periods of abstinence. The nucleus accumbens and limbic basal ganglia circuitry are implicated in the expression of various morphine-induced motor behaviours and catalepsy. We examined the effect of intermittent morphine exposure on the distribution of Fos proteins in the basal ganglia following a subsequent morphine challenge administered after a period of drug abstinence. We found that such exposures increased c-Fos induced by a morphine challenge in accumbens core regions that were immunoreactive for the micro-opioid receptor, and this correlated with the frequency of stereotypic behaviours displayed by the rats. We also found that a history of morphine exposures increased c-Fos in the ventrolateral striatum in response to a morphine challenge following 14 d but not 24 h of drug abstinence. In contrast, such a history induced acute Fras in the nucleus accumbens in response to a morphine challenge following 24 h but not 14 d of morphine abstinence. These data provide further confirmation that psychomotor sensitisation induced by repetitive morphine exposure involves long-term neuroadaptations in basal ganglia circuitry particularly at the level of the nucleus accumbens.