The dynamic strategy shifting task: Optimisation of an operant task for assessing cognitive flexibility in rats.

Introduction: Although schizophrenia is associated with a broad range of symptoms including hallucinations, delusions, and reduced motivation, measures of cognitive dysfunction, including cognitive flexibility and executive function, are the strongest predictors of functional outcomes. Antipsychotic medications are useful for reducing psychotic symptoms, but they are ineffective at improving cognitive deficits. Despite extensive investment by industry, the transition from preclinical to clinical trials has not been successful for developing precognitive medications for individuals with schizophrenia. Here, we describe the optimisation of a novel dynamic strategy shifting task (DSST) using standard operant chambers to investigate the optimal stimuli required to limit the extensive training times required in previous tasks. Methods: We determined that optimal learning by male and female Sprague Dawley rats for the flexibility task incorporated dynamic strategy shifts between spatial rules, such as following a visual cue or responding at one location, and non-spatial rules, such as responding to a central visual or auditory cue. A minimum of 6 correct consecutive responses were required to make a within-session change in the behavioural strategies. As a proof of concept, we trained and tested 84 Sprague Dawley rats on the DSST, and then assessed their cognitive flexibility using a within-subject design after an acute dose of ketamine (0, 3, 10 mg/kg). Rats made fewer premature and more perseverant responses to initiate a trial following ketamine. The effects of ketamine on trials to criterion was dependent on the rule. Results: Ketamine induced a significant improvement on the reversal of a non-spatial visual discrimination rule. There was no significant effect of ketamine on the spatial visual or response discrimination rules. Discussion: The DSST is a novel assay for studying distinct forms of cognitive flexibility and offers a rapid and adaptable means of assessing the ability to shift between increasingly challenging rule conditions. The DSST has potential utility in advancing our understanding of cognitive processes and the underlying neurobiological mechanisms related to flexibility in neuropsychiatric and neurological conditions where executive dysfunctions occur.>.

The DDHD2-STXBP1 interaction mediates long-term memory via generation of saturated free fatty acids.

The phospholipid and free fatty acid (FFA) composition of neuronal membranes plays a crucial role in learning and memory, but the mechanisms through which neuronal activity affects the brain's lipid landscape remain largely unexplored. The levels of saturated FFAs, particularly of myristic acid (C14:0), strongly increase during neuronal stimulation and memory acquisition, suggesting the involvement of phospholipase A1 (PLA1) activity in synaptic plasticity. Here, we show that genetic ablation of the PLA1 isoform DDHD2 in mice dramatically reduces saturated FFA responses to memory acquisition across the brain. Furthermore, DDHD2 loss also decreases memory performance in reward-based learning and spatial memory models prior to the development of neuromuscular deficits that mirror human spastic paraplegia. Via pulldown-mass spectrometry analyses, we find that DDHD2 binds to the key synaptic protein STXBP1. Using STXBP1/2 knockout neurosecretory cells and a haploinsufficient STXBP1+/- mouse model of human early infantile encephalopathy associated with intellectual disability and motor dysfunction, we show that STXBP1 controls targeting of DDHD2 to the plasma membrane and generation of saturated FFAs in the brain. These findings suggest key roles for DDHD2 and STXBP1 in lipid metabolism and in the processes of synaptic plasticity, learning, and memory.

Activity in the Dorsomedial Striatum Underlies Serial Reversal Learning Performance Under Probabilistic Uncertainty.

Background: Corticostriatal circuits, particularly the dorsomedial striatum (DMS) and lateral orbitofrontal cortex, are critical for navigating reversal learning under probabilistic uncertainty. These same areas are implicated in the reversal learning impairments observed in individuals with psychosis as well as their psychotic symptoms, suggesting that they may share a common neurobiological substrate. To address this question, we used psychostimulant exposure and specific activation of the DMS during reversal learning in mice to assess corticostriatal activity. Methods: We used amphetamine treatment to induce psychosis-relevant neurobiology in male mice during reversal learning and to examine pathway-specific corticostriatal activation. To determine the causal role of DMS activity, we used chemogenetics to drive midbrain inputs during a range of probabilistic contingencies. Results: Mice treated with amphetamine showed altered punishment learning, which was associated with decreased shifting after losses and increased perseverative errors after reversals. Reversal learning performance and strategies were dependent on increased activity in lateral orbitofrontal cortex to DMS circuits as well as in the DMS itself. Specific activation of midbrain to DMS circuits also decreased shifting after losses and reversal learning performance. However, these alterations were dependent on the probabilistic contingency. Conclusions: Our work suggests that the DMS plays a multifaceted role in reversal learning. Increasing DMS activity impairs multiple reversal learning processes dependent on the level of uncertainty, confirming its role in the maintenance and selection of incoming cortical inputs. Together, these outcomes suggest that elevated dopamine levels in the DMS could contribute to decision-making impairments in individuals with psychosis.

Synthetic Anti-Cocaine Nanoaccine Successfully Prevents Cocaine-Induced Hyperlocomotion.

Cocaine is one of the most widely used and increasingly popular illicit psychoactive drugs. Unlike other commonly used substances of abuse, cocaine has no pharmacological therapies to treat addiction or aid in rehabilitation. Immunopharmacology has long been touted as a possible avenue to develop effective anticocaine therapies; however, lack of efficacy and designs which are not consistent with simple large-scale production have hindered vaccine translation. We have designed and synthesized a peptide-based anti-cocaine immunogen which we have shown is capable of inducing physiologically relevant immune responses in mice as part of a self-adjuvanting delivery system or in combination with the human-approved commercial adjuvant MF59. We have demonstrated that immunization with the reported vaccine elicits high titers of anti-cocaine IgG and prevents cocaine-induced hyperlocomotion in an in vivo murine model. This peptide-hapten immunogen along with self-adjuvanting liposomal-based delivery system provides a platform for the development of effective anti-drug vaccines.

Antagonism of D2 receptors via raclopride ameliorates amphetamine-induced associative learning deficits in male mice.

Dopamine levels in the dorsomedial striatum (DMS) are highly dynamic and are thought to underly the encoding of action-outcome associations. Although it is known that amphetamine disrupts the learning that is required for goal-directed action, the role of D1 and D2 receptors in this process has not been established. In this study, we examined the role of D1 and D2 receptor antagonists on learning in response to amphetamine. We used the outcome-specific devaluation task to examine goal-directed action in male C57BL6/J mice treated systemically with either a D1 antagonist (SCH-23990; 0.01 mg/kg) or a D2 antagonist (raclopride; 0.5 mg/kg) and then administered amphetamine (1 mg/kg). The mice were injected repeatedly throughout the instrumental training phase of the task to assess the impact on the learning of action-outcomes, and the subsequent choice test assessing performance of goal-directed action was conducted drug free. Effects of chronic drug administration on locomotor behaviour was assessed before and after the choice test. Treatment during learning with either amphetamine, or the D1 or D2 antagonists, impaired the subsequent performance of goal-directed action. The amphetamine-induced impairment in goal-directed action was reversed in mice treated with raclopride, but not when treated with SCH-23990. By contrast, amphetamine-induced hyperactivity was reversed in mice treated with SCH-23990, but not in mice treated with raclopride. Taken together, these data support the role of a balance of dopamine receptor signalling after amphetamine treatment. While overall D1 receptor availability is necessary to promote learning, in a state of elevated dopamine, modifying D2 receptor function can ameliorate learning deficits.

Developmental vitamin D-deficiency produces autism-relevant behaviours and gut-health associated alterations in a rat model.

Developmental vitamin D (DVD)-deficiency is an epidemiologically established risk factor for autism. Emerging studies also highlight the involvement of gut microbiome/gut physiology in autism. The current study aims to examine the effect of DVD-deficiency on a broad range of autism-relevant behavioural phenotypes and gut health. Vitamin D deficient rat dams exhibited altered maternal care, DVD-deficient pups showed increased ultrasonic vocalizations and as adolescents, social behaviour impairments and increased repetitive self-grooming behaviour. There were significant impacts of DVD-deficiency on gut health demonstrated by alterations to the microbiome, decreased villi length and increased ileal propionate levels. Overall, our animal model of this epidemiologically validated risk exposure for autism shows an expanded range of autism-related behavioural phenotypes and now alterations in gut microbiome that correlate with social behavioural deficits raising the possibility that DVD-deficiency induced ASD-like behaviours are due to alterations in gut health.

Activating the dorsomedial and ventral midbrain projections to the striatum differentially impairs goal-directed action in male mice.

The cognitive symptoms of schizophrenia are wide ranging and include impaired goal-directed action. This could be driven by an increase in dopamine transmission in the dorsomedial striatum, a pathophysiological hallmark of schizophrenia. Although commonly associated with psychotic symptoms, dopamine signalling in this region also modulates associative learning that aids in the execution of actions. To gain a better understanding of the role of subcortical dopamine in learning and decision-making, we assessed goal-directed action in male mice using the cross-species outcome-specific devaluation task (ODT). First, we administered systemic amphetamine during training to determine the impact of altered dopaminergic signaling on associative learning. Second, we used pathway-specific chemogenetic approaches to activate the dorsomedial and ventral striatal pathways (that originate in the midbrain) to separately assess learning and performance. Amphetamine treatment during learning led to a dose-dependent impairment in goal-directed action. Activation of both striatal pathways during learning also impaired performance. However, when these pathways were activated during choice, only activation of the ventral pathway impaired goal-directed action. This suggests that elevated transmission in the dorsomedial striatal pathway impairs associative learning processes that guide the goal-directed execution of actions. By contrast, elevated transmission of the ventral striatal pathway disrupts the encoding of outcome values that are important for both associative learning and choice performance. These findings highlight the differential roles of the dorsomedial and ventral inputs into the striatum in goal-directed action and provides insight into how striatal dopamine signaling may contribute to the cognitive problems in those with schizophrenia.

Impaired spatial memory in adult vitamin D deficient BALB/c mice is associated with reductions in spine density, nitric oxide, and neural nitric oxide synthase in the hippocampus.

Vitamin D deficiency is prevalent in adults and is associated with cognitive impairment. However, the mechanism by which adult vitamin D (AVD) deficiency affects cognitive function remains unclear. We examined spatial memory impairment in AVD-deficient BALB/c mice and its underlying mechanism by measuring spine density, long term potentiation (LTP), nitric oxide (NO), neuronal nitric oxide synthase (nNOS), and endothelial NOS (eNOS) in the hippocampus. Adult male BALB/c mice were fed a control or vitamin D deficient diet for 20 weeks. Spatial memory performance was measured using an active place avoidance (APA) task, where AVD-deficient mice had reduced latency entering the shock zone compared to controls. We characterised hippocampal spine morphology in the CA1 and dentate gyrus (DG) and made electrophysiological recordings in the hippocampus of behaviourally naïve mice to measure LTP. We next measured NO, as well as glutathione, lipid peroxidation and oxidation of protein products and quantified hippocampal immunoreactivity for nNOS and eNOS. Spine morphology analysis revealed a significant reduction in the number of mushroom spines in the CA1 dendrites but not in the DG. There was no effect of diet on LTP. However, hippocampal NO levels were depleted whereas other oxidation markers were unaltered by AVD deficiency. We also showed a reduced nNOS, but not eNOS, immunoreactivity. Finally, vitamin D supplementation for 10 weeks to AVD-deficient mice restored nNOS immunoreactivity to that seen in in control mice. Our results suggest that lower levels of NO and reduced nNOS immunostaining contribute to hippocampal-dependent spatial learning deficits in AVD-deficient mice.

Partial Loss of USP9X Function Leads to a Male Neurodevelopmental and Behavioral Disorder Converging on Transforming Growth Factor ß Signaling.

BACKGROUND: The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative. METHODS: We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology. RESULTS: Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor ß signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory. CONCLUSIONS: Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor ß signaling and hippocampal function.

Developmental Vitamin D Deficiency in the Rat Impairs Recognition Memory, but Has No Effect on Social Approach or Hedonia.

Developmental vitamin D (DVD) deficiency is a risk factor for schizophrenia. In rodents we show that DVD-deficiency alters brain development and produces behavioral phenotypes in the offspring of relevance to the positive symptoms of schizophrenia. The aims of this study are to examine behavioral phenotypes specific to the cognitive and negative symptoms of schizophrenia in this model, and to vary the duration of vitamin D deficiency during gestation and beyond birth. We hypothesize that a longer duration of DVD-deficiency would result in greater behavioral impairments. Female vitamin D-deficient Sprague Dawley dams were mated at 10 weeks of age. Dietary vitamin D was reintroduced to dams and/or pups at different developmental time-points: Conception, Birth, Post-natal day (PND) 6 and PND21. Adult male and female offspring were assessed on a battery of behavioral tests, including sucrose preference, open field, novel object recognition (NOR), social approach and social novelty. We find that all windows of DVD-deficiency impaired NOR a cognitive measure that requires intact recognition memory. Sucrose consumption, social approach and social memory negative symptom-like phenotypes were unaffected by any maternal dietary manipulation. In addition, contrary to our hypothesis, we find that rats in the Conception group, that is the shortest duration of vitamin D deficiency, demonstrate increased locomotor activity, and decreased interaction time with novel objects. These findings have implications for the increasing number of studies examining the preclinical consequences of maternal vitamin D deficiency, and continue to suggest that adequate levels of maternal vitamin D are required for normal brain development.

Functional and molecular changes in the nucleus accumbens of MK-801-sensitized rats.

Behavioural sensitization is a putative mechanism in the pathophysiology of drug addiction and neuropsychiatric disorders such as schizophrenia. In rodents, drug-induced behavioural sensitization has been described for several different drug classes. The N-methyl-D-aspartate receptor antagonist MK-801 can inhibit sensitization to other drugs of abuse. However, MK-801 also produces behavioural sensitization to its own hyperlocomotor inducing effects, suggesting that MK-801 sensitization has a distinctive mechanism of action. The aim of this study was to carry out a functional and molecular analysis of the nucleus accumbens (NAc) of adult male Sprague-Dawley rats sensitized to MK-801 (seven daily injections of 0.25 mg/kg, 5 days of withdrawal and subsequent 0.25 mg/kg challenge), or following acute MK-801 (0.25 mg/kg), or naive rats as controls. Locomotor activity was the primary measure of sensitization. Ex-vivo slice electrophysiology showed a decrease in the excitatory synaptic strength in the NAc of rats sensitized to MK-801 compared with acute MK-801 treatment or naive controls. An LC-MS/MS SWATH proteomics approach showed that proteins altered by MK-801 sensitization were predominantly related to functions including calcium and glutamate signalling, and mitochondrial dysfunction. These results shed some light on neural changes in the NAc after sensitization to MK-801. This model could prove useful for studying the role of N-methyl-D-aspartate receptors in the pathophysiology of drug addiction and schizophrenia.

Effect of the glucocorticoid receptor antagonist RU486 on MK-801 induced behavioural sensitisation.

Stress is known to modulate sensitisation to repeated psychostimulant exposure. However, there is no direct evidence linking glucocorticoids and sensitisation achieved by repeated administration of the NMDA receptor antagonist MK-801. We tested the hypothesis that co-administration of RU486, a glucocorticoid receptor (GR) antagonist, prior to repeated daily MK-801 injections would block the expression of locomotor sensitisation due to its dual effects on corticosterone and dopamine. We employed a repeated MK-801 administration locomotor sensitisation paradigm in male Sprague Dawley rats. RU486 or a dimethyl sulfoxide (DMSO) vehicle was co-administered with MK-801 or saline during the induction phase. Subsequent to withdrawal, rats were challenged with MK-801 alone to test for the expression of sensitisation. In a separate cohort of rats, plasma corticosterone levels were quantified from blood samples taken on the 1st, 4th and 7th day of induction and at expression. One day after challenge, nucleus accumbens tissue levels of dopamine and its metabolites DOPAC and HVA were measured. During the induction phase, RU486 progressively enhanced locomotor sensitisation to MK-801. RU486 and MK-801 both showed stimulatory effects on corticosterone levels and this was further augmented when given in combination. Contrary to our hypothesis, RU486 did not block the expression of locomotor sensitisation to MK-801 and actually increased levels of dopamine, DOPAC and HVA in nucleus accumbens tissue. Our results showed that RU486 has augmentative rather than inhibitory effects on MK-801-induced sensitisation. This study indicates a divergent role for glucocorticoids in sensitisation to MK-801 compared to sensitisation with other psychostimulants.

MK-801-induced behavioural sensitisation alters dopamine release and turnover in rat prefrontal cortex.

RATIONALE: Repeated exposure to psychostimulants that either increase dopamine (DA) release or target N-methyl-D-aspartate (NMDA) receptors can induce behavioural sensitisation, a phenomenon that may be important for the processes of addiction and even psychosis. A critical component of behavioural sensitisation is an increase in DA release within mesocorticolimbic circuits. In particular, sensitisation to amphetamine leads to increased DA release within well-known sub-cortical brain regions and also regulatory regions such as prefrontal cortex (PFC). However, it is unknown how DA release within the PFC of animals is altered by sensitisation to NMDA receptor antagonists. OBJECTIVES: The aims of the present study were twofold, firstly to examine whether a single dose of dizocilpine maleate (MK-801) could induce long-term behavioural sensitisation and secondly to examine DA release in the PFC of sensitised rats. MATERIALS AND METHODS: Behavioural sensitisation was assessed by measuring locomotion after drug exposure. DA release in the PFC was measured using freely moving microdialysis. RESULTS: We show that a single dose of MK-801 can induce sensitisation to subsequent MK-801 exposure in a high percentage of rats (66 %). Furthermore, rats sensitised to MK-801 have altered DA release and turnover in the PFC compared with non-sensitised rats. CONCLUSION: Schizophrenia patients have been postulated to have 'endogenous sensitisation' to psychostimulants. MK-801-induced sensitised rats, in particular when compared with non-sensitised rats, provide a useful model for studying PFC dysfunction in schizophrenia.

Spectrum of childhood Epstein-Barr virus-associated T-cell proliferations and bone marrow findings.

Systemic Epstein-Barr virus-positive (EBV+) T-cell lymphoproliferative disorder of childhood is a recently described entity. The majority of such cases have been reported from Asia, which suggests an underlying genetic predisposition. We analyzed the clinicopathologic characteristics of 5 children with EBV+ T-cell lymphoid proliferations evaluated and treated at our institute over a 2-year period. There were 3 males and 2 females of Latino (n  =  4) or Caucasian (n  =  1) heritage with a median age of 5 years (age range 2-18 years). All patients presented with EBV infection (acute, n  =  4) with elevated serum EBV viral loads at the time of diagnosis and had systemic manifestations, including fever, hepatosplenomegaly, and pancytopenia. The bone marrow biopsies showed EBV+/CD8+ T-cell lymphocytosis in all patients, with variable degrees of histiocytosis, plasmacytosis, and hemophagocytosis. Interestingly, there was marked and consistent depletion of mature and precursor B cells in the marrow (<1% of total marrow cellularity) in all patients. Three of the patients died of disease-associated complications 2 to 12 weeks after initial diagnosis. Our study describes the detailed bone marrow findings, contributes to the growing number of cases of systemic EBV+ T-cell lymphoproliferative disorder of childhood occurring in the Western hemisphere, and documents this disorder in patients from the Caribbean countries.

Nodular pattern of bone marrow infiltration: frequent finding in immunosuppression-related EBV-associated large B-cell lymphomas.

Different patterns of bone marrow (BM) infiltration by diffuse large B cell lymphomas (DLBCL) have been described. A pure nodular pattern is uncommon, and the pathologic features, as well as the clinical correlates of DLBCL manifesting this pattern in the BM have not been well characterized. We evaluated BM biopsies involved by large B cell lymphomas diagnosed at our institute over an 11-year period to assess the morphology, phenotype, cytogenetic abnormalities, and clinical features of cases associated with a nodular pattern. A distinct nodular pattern of BM involvement was noted in 14 out of 55 (25%) cases. Although both EBV+ and EBV- DLBCL with this pattern were identified, a pure nodular pattern was significantly more common in EBV+ DLBCL compared to EBV- DLBCL (8/9, 89% versus 6/46, 13%; P = 0.00002). The majority of EBV+ DLBCL associated with a nodular pattern had distinctive morphologic features (polymorphic cellular infiltrate and pleomorphic cytology), and CD30 expression was more commonly observed in this group (P = 0.0163). All EBV+ DLBCL and two out of six (33%) EBV- DLBCL had nongerminal center phenotypes. No recurrent cytogenetic abnormalities were detected in either group. Importantly, all EBV+ DLBCL occurred in individuals with immune dysfunction (organ transplant recipients, HIV infection) or in those >50 years of age. Our study indicates a much higher predilection for EBV+ DLBCL to involve the marrow in a nodular pattern compared to EBV- cases and highlights similarities in the morphologic pattern of BM involvement by previously recognized subsets of immunodeficiency-related EBV + lymphomas and the newer entity of "EBV+ DLBCL of the elderly."

Diffuse large B-cell lymphoma with TEL/ETV6 translocation.

Cytogenetic abnormalities of chromosome 12p involving the TEL/ETV6 gene are observed in a variety of hematopoietic neoplasms including acute leukemias, myelodysplastic syndromes, and myeloproliferative disorders. Karyotypic aberrations, including rearrangements, deletions, and amplifications of chromosome 12p, have been documented in B-cell non-Hodgkin lymphoma; however, rearrangements targeting TEL have rarely been reported. Here we describe a diffuse large B-cell lymphoma that had a complex karyotype including t(9;12)(q22;p13), which was confirmed by fluorescence in situ hybridization to represent rearrangement of TEL. Additional cytogenetic abnormalities included t(3;14)(q27;q32) involving the variant, alternative breakpoint region of the BCL6 gene and del(6)(q13q23), resulting in the loss of 1 allele of BLIMP1. This case reiterates the importance of correlating morphologic and phenotypic findings with the results of cytogenetic analysis to avoid errors in diagnosing hematologic neoplasms and highlights the rare association of B-cell non-Hodgkin lymphoma with aberrations of TEL.

Differences in cyclin D2 and D1 protein expression distinguish forebrain progenitor subsets.

Regulation of neural proliferation is an essential component of brain formation and is driven by both intrinsic cell cycle and extrinsic growth and trophic molecules. Among the cell cycle proteins, understanding of the relative roles of the G1-phase active cyclins D2 and D1 (cD2 and cD1) has been hampered by lack of data regarding their expression patterns. In this study, cD2 immunoreactivity was examined in the neocortex, ganglionic eminences/striatum, and hippocampal formation from embryonic day 12.5 until postnatal day 60 to more precisely characterize the expression of this protein during forebrain development. The localization of cD1 was also immunohistologically mapped for comparison. Throughout forebrain development, both overlapping and nonoverlapping protein expression of these cyclins suggests the presence of shared and unique cell cycle requirements for neurogenesis that distinguishes progenitor pools.