GABAA Alpha 2,3 Modulation Improves Select Phenotypes in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability. FXS is caused by functional loss of the Fragile X Protein (FXP), also known as Fragile X Mental Retardation Protein (FMRP). In humans and animal models, loss of FXP leads to sensory hypersensitivity, increased susceptibility to seizures and cortical hyperactivity. Several components of the GABAergic system, the major inhibitory system in the brain, are dysregulated in FXS, and thus modulation of GABAergic transmission was suggested and tested as a treatment strategy. However, so far, clinical trials using broad spectrum GABAA or GABAB receptor-specific agonists have not yielded broad improvement of FXS phenotypes in humans. Here, we tested a more selective strategy in Fmr1 knockout (KO) mice using the experimental drug BAER-101, which is a selective GABAA α2/α3 agonist. Our results suggest that BAER-101 reduces hyperexcitability of cortical circuits, partially corrects increased frequency-specific baseline cortical EEG power, reduces susceptibility to audiogenic seizures and improves novel object memory. Other Fmr1 KO-specific phenotypes were not improved by the drug, such as increased hippocampal dendritic spine density, open field activity and marble burying. Overall, this work shows that BAER-101 improves select phenotypes in Fmr1 KO mice and encourages further studies into the efficacy of GABAA-receptor subunit-selective agonists for the treatment of FXS.

The potassium channel Kv4.2 regulates dendritic spine morphology, electroencephalographic characteristics and seizure susceptibility in mice.

The voltage-gated potassium channel Kv4.2 is a critical regulator of dendritic excitability in the hippocampus and is crucial for dendritic signal integration. Kv4.2 mRNA and protein expression as well as function are reduced in several genetic and pharmacologically induced rodent models of epilepsy and autism. It is not known, however, whether reduced Kv4.2 is just an epiphenomenon or a disease-contributing cause of neuronal hyperexcitability and behavioral impairments in these neurological disorders. To address this question, we used male and female mice heterozygous for a Kv.2 deletion and adult-onset manipulation of hippocampal Kv4.2 expression in male mice to assess the role of Kv4.2 in regulating neuronal network excitability, morphology and anxiety-related behaviors. We observed a reduction in dendritic spine density and reduced proportions of thin and stubby spines but no changes in anxiety, overall activity, or retention of conditioned freezing memory in Kv4.2 heterozygous mice compared with wildtype littermates. Using EEG analyses, we showed elevated theta power and increased spike frequency in Kv4.2 heterozygous mice under basal conditions. In addition, the latency to onset of kainic acid-induced seizures was significantly shortened in Kv4.2 heterozygous mice compared with wildtype littermates, which was accompanied by a significant increase in theta power. By contrast, overexpressing Kv4.2 in wildtype mice through intrahippocampal injection of Kv4.2-expressing lentivirus delayed seizure onset and reduced EEG power. These results suggest that Kv4.2 is an important regulator of neuronal network excitability and dendritic spine morphology, but not anxiety-related behaviors. In the future, manipulation of Kv4.2 expression could be used to alter seizure susceptibility in epilepsy.

Acamprosate in a mouse model of fragile X syndrome: modulation of spontaneous cortical activity, ERK1/2 activation, locomotor behavior, and anxiety.

BACKGROUND: Fragile X Syndrome (FXS) occurs as a result of a silenced fragile X mental retardation 1 gene (FMR1) and subsequent loss of fragile X mental retardation protein (FMRP) expression. Loss of FMRP alters excitatory/inhibitory signaling balance, leading to increased neuronal hyperexcitability and altered behavior. Acamprosate (the calcium salt of N-acetylhomotaurinate), a drug FDA-approved for relapse prevention in the treatment of alcohol dependence in adults, is a novel agent with multiple mechanisms that may be beneficial for people with FXS. There are questions regarding the neuroactive effects of acamprosate and the significance of the molecule's calcium moiety. Therefore, the electrophysiological, cellular, molecular, and behavioral effects of acamprosate were assessed in the Fmr1-/y (knock out; KO) mouse model of FXS controlling for the calcium salt in several experiments. METHODS: Fmr1 KO mice and their wild-type (WT) littermates were utilized to assess acamprosate treatment on cortical UP state parameters, dendritic spine density, and seizure susceptibility. Brain extracellular-signal regulated kinase 1/2 (ERK1/2) activation was used to investigate this signaling molecule as a potential biomarker for treatment response. Additional adult mice were used to assess chronic acamprosate treatment and any potential effects of the calcium moiety using CaCl2 treatment on behavior and nuclear ERK1/2 activation. RESULTS: Acamprosate attenuated prolonged cortical UP state duration, decreased elevated ERK1/2 activation in brain tissue, and reduced nuclear ERK1/2 activation in the dentate gyrus in KO mice. Acamprosate treatment modified behavior in anxiety and locomotor tests in Fmr1 KO mice in which control-treated KO mice were shown to deviate from control-treated WT mice. Mice treated with CaCl2 were not different from saline-treated mice in the adult behavior battery or nuclear ERK1/2 activation. CONCLUSIONS: These data indicate that acamprosate, and not calcium, improves function reminiscent of reduced anxiety-like behavior and hyperactivity in Fmr1 KO mice and that acamprosate attenuates select electrophysiological and molecular dysregulation that may play a role in the pathophysiology of FXS. Differences between control-treated KO and WT mice were not evident in a recognition memory test or in examination of acoustic startle response/prepulse inhibition which impeded conclusions from being made about the treatment effects of acamprosate in these instances.

Fragile X targeted pharmacotherapy: lessons learned and future directions.

Our understanding of fragile X syndrome (FXS) pathophysiology continues to improve and numerous potential drug targets have been identified. Yet, current prescribing practices are only symptom-based in order to manage difficult behaviors, as no drug to date is approved for the treatment of FXS. Drugs impacting a diversity of targets in the brain have been studied in recent FXS-specific clinical trials. While many drugs have focused on regulation of enhanced glutamatergic or deficient GABAergic neurotransmission, compounds studied have not been limited to these mechanisms. As a single-gene disorder, it was thought that FXS would have consistent drug targets that could be modulated with pharmacotherapy and lead to significant improvement. Unfortunately, despite promising results in FXS animal models, translational drug treatment development in FXS has largely failed. Future success in this field will depend on learning from past challenges to improve clinical trial design, choose appropriate outcome measures and age range choices, and find readily modulated drug targets. Even with many negative placebo-controlled study results, the field continues to move forward exploring both the new mechanistic drug approaches combined with ways to improve trial execution. This review summarizes the known phenotype and pathophysiology of FXS and past clinical trial rationale and results, and discusses current challenges facing the field and lessons from which to learn for future treatment development efforts.

d-Cycloserine enhances durability of social skills training in autism spectrum disorder.

BACKGROUND: d-Cycloserine (DCS) enhances extinction learning across species, but it has proven challenging to identify consistent benefit of DCS when added to therapeutic interventions. We conducted a placebo-controlled trial of DCS to potentiate social skills training in autism spectrum disorder (ASD) but found substantial improvement in both the DCS and placebo groups at the conclusion of active treatment. Here, we assess the impact of DCS 11 weeks following active treatment to evaluate the impact of DCS on treatment response durability. METHODS: Study participants included 60 outpatient youth with ASD, ages 5-11 years, all with IQ above 70, and significantly impaired social functioning who completed a 10-week active treatment phase during which they received weekly single doses of 50 mg of DCS or placebo administered 30 min prior to group social skills training. Following the 10-week active treatment phase, blinded follow-up assessments occurred at week 11 and week 22. The primary outcome measure for our durability of treatment evaluation was the parent-rated social responsiveness scale (SRS) total raw score at week 22. RESULTS: Analysis of the SRS total raw score demonstrated significant decrease for the DCS group compared to the placebo group (p = 0.042) indicating greater maintenance of treatment effect in the DCS group. DCS was well tolerated, with irritability being the most frequently reported adverse effect in both groups. CONCLUSIONS: The findings of this study suggest that DCS may help youth with ASD to maintain skills gained during sort-term social skills training. Larger-scale studies with longer follow-up will be necessary to further understand the long-term impact of DCS paired with structured social skills training. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01086475.

Initial analysis of peripheral lymphocytic extracellular signal related kinase activation in autism.

BACKGROUND: Dysregulation of extracellular signal-related kinase (ERK) activity has been potentially implicated in the pathophysiology of autistic disorder (autism). ERK is part of a central intracellular signaling cascade responsible for a myriad of cellular functions. ERK is expressed in peripheral blood lymphocytes, and measurement of activated (phosphorylated) lymphocytic ERK is commonly executed in many areas of medicine. We sought to conduct the first study of ERK activation in humans with autism by utilizing a lymphocytic ERK activation assay. We hypothesized that ERK activation would be enhanced in peripheral blood lymphocytes from persons with autism compared to those of neurotypical control subjects. METHOD: We conducted an initial study of peripheral lymphocyte ERK activation in 45 subjects with autism and 26 age- and gender-matched control subjects (total n = 71). ERK activation was measured using a lymphocyte counting method (primary outcome expressed as lymphocytes staining positive for cytosolic phosphorylated ERK divided by total cells counted) and additional Western blot analysis of whole cell phosphorylated ERK adjusted for total ERK present in the lymphocyte lysate sample. RESULTS: Cytosolic/nuclear localization of pERK activated cells were increased by almost two-fold in the autism subject group compared to matched neurotypical control subjects (cell count ratio of 0.064 ± 0.044 versus 0.034 ± 0.031; p = 0.002). Elevated phosphorylated ERK levels in whole cell lysates also showed increased activated ERK in the autism group compared to controls (n = 54 total) in Western blot analysis. CONCLUSIONS: The results of this first in human ERK activation study are consistent with enhanced peripheral lymphocytic ERK activation in autism, as well as suggesting that cellular compartmentalization of activated ERK may be altered in this disorder. Future work will be required to explore the impact of concomitant medication use and other subject characteristics such as level of cognitive functioning on ERK activation. TRIAL REGISTRATION: Not applicable.

Analysis of peripheral amyloid precursor protein in Angelman Syndrome.

Angelman Syndrome is a rare neurodevelopmental disorder associated with significant developmental and communication delays, high risk for epilepsy, motor dysfunction, and a characteristic behavioral profile. While Angelman Syndrome is known to be associated with the loss of maternal expression of the ubiquitin-protein ligase E3A gene, the molecular sequelae of this loss remain to be fully understood. Amyloid precursor protein (APP) is involved in neuronal development and APP dysregulation has been implicated in the pathophysiology of other developmental disorders including fragile X syndrome and idiopathic autism. APP dysregulation has been noted in preclinical model of chromosome 15q13 duplication, a disorder whose genetic abnormality results in duplication of the region that is epigenetically silenced in Angelman Syndrome. In this duplication model, APP levels have been shown to be significantly reduced leading to the hypothesis that enhanced ubiquitin-protein ligase E3A expression may be associated with this phenomena. We tested the hypothesis that ubiquitin-protein ligase E3A regulates APP protein levels by comparing peripheral APP and APP derivative levels in humans with Angelman Syndrome to those with neurotypical development. We report that APP total, APP alpha (sAPPα) and A Beta 40 and 42 are elevated in the plasma of humans with Angelman Syndrome compared to neurotypical matched human samples. Additionally, we found that elevations in APP total and sAPPα correlated positively with peripheral brain derived neurotrophic factor levels previously reported in this same patient cohort. Our pilot report on APP protein levels in Angelman Syndrome warrants additional exploration and may provide a molecular target of treatment for the disorder. © 2016 Wiley Periodicals, Inc.

Developmental stress and lead (Pb): Effects of maternal separation and/or Pb on corticosterone, monoamines, and blood Pb in rats.

The level of lead (Pb) exposure in children has decreased dramatically since restrictions on its use were implemented. However, even with restrictions, children are exposed to Pb and still present with cognitive and behavioral deficits. One prominent aspect of the exposome of these children is that many come from low social economic status (SES) conditions, and low SES is associated with stress. In order to compare the combined effects of early stress and Pb, Sprague-Dawley rats were exposed to vehicle or Pb either alone or in combination with maternal separation stress during brain development (i.e., postnatal day (P)4-P11, P19, or P28). Maternally separated/isolated pups had lower body and thymus weights during exposure and had increased levels of blood Pb compared with vehicle controls. Isolation, but not Pb, affected the response to an acute stressor (standing in shallow water) when assessed on P19 and P29, but not earlier on P11. Interactions of Pb and isolation were found on monoamines in the neostriatum, hippocampus, and hypothalamus on turnover but not on levels, and most changes were on dopamine turnover. Isolation had greater short-term effects than Pb. Interactions were dependent on age, sex, and acute stress.

Pharmacotherapy for Fragile X Syndrome: Progress to Date.

To date, no drug is approved for the treatment of Fragile X Syndrome (FXS) although many drugs are used to manage challenging behaviors from a symptomatic perspective in this population. While our understanding of FXS pathophysiology is expanding, efforts to devise targeted FXS-specific treatments have had limited success in placebo-controlled trials. Compounds aimed at rectifying excessive glutamate and deficient gamma-aminobutyric acid (GABA) neurotransmission, as well as other signaling pathways known to be affected by Fragile X Mental Retardation Protein (FMRP) are under various phases of development in FXS. With the failure of several metabotropic glutamate receptor subtype 5 (mGlur5) selective antagonists under clinical investigation, no clear single treatment appears to be greatly effective. These recent challenges call into question various aspects of clinical study design in FXS. More objective outcome measures are under development and validation. Future trials will likely be aimed at correcting multiple pathways known to be disrupted by the loss of FMRP. This review offers a brief summary of the prevalence, phenotypic characteristics, genetic causes and molecular functions of FMRP in the brain (as these have been extensively reviewed elsewhere), discusses the most recent finding in FXS drug development, and summarizes FXS trials utilizing symptomatic treatment.

The neurobehavioral and molecular phenotype of Angelman Syndrome.

Angelman Syndrome (AS) is a rare neurodevelopmental disorder associated with developmental delay, speech impairment, gait ataxia, and a unique behavioral profile. AS is caused by loss of maternal expression of the paternally imprinted UBE3A gene. In this study we aim to contribute to understanding of the neurobehavioral phenotype of AS with particular focus on the neuropsychiatric presentation of the disorder. We also undertake initial exploration of brain-derived neurotrophic factor (BDNF) plasma levels in AS. Twelve individuals ages 3 years or older with a confirmed genetic diagnosis of AS underwent detailed medical history, phenotypic characterization, and BDNF plasma sampling. The results of this study demonstrate that individuals with AS suffer from significant developmental delay, impaired adaptive behavior, and sleep disruption. Additionally, hyperactivity/impulsivity appears to be the primary behavioral domain noted in these individuals. The majority of individuals in this project met criteria for autism spectrum disorder on the Autism Diagnostic Observation Schedule (ADOS); however, a negative correlation was noted between ADOS score and developmental age. BDNF plasma levels in AS individuals were significantly elevated compared to neurotypical controls. This is the first report of abnormal BDNF levels in AS, and one that necessitates larger future studies. The results provide a clue to understanding abnormal neuronal development in AS and may help guide future AS research.

Emerging pharmacologic treatment options for fragile X syndrome.

Fragile X syndrome (FXS) is the most common single gene cause of intellectual disability and autism spectrum disorder. Caused by a silenced fragile X mental retardation 1 gene and the subsequent deficiency in fragile X mental retardation protein, patients with FXS experience a range of physical, behavioral, and intellectual debilitations. The FXS field, as a whole, has recently met with some challenges, as several targeted clinical trials with high expectations of success have failed to elucidate significant improvements in a variety of symptom domains. As new clinical trials in FXS are planned, there has been much discussion about the use of the commonly used clinical outcome measures, as well as study design considerations, patient stratification, and optimal age range for treatment. The evidence that modification of these drug targets and use of these failed compounds would prove to be efficacious in human clinical study were rooted in years of basic and translational research. There are questions arising as to the use of the mouse models for studying FXS treatment development. This issue is twofold: many of the symptom domains and molecular and biochemical changes assessed and indicative of efficacy in mouse model study are not easily amenable to clinical trials in people with FXS because of the intolerability of the testing paradigm or a lack of noninvasive techniques (prepulse inhibition, sensory hypersensitivity, startle reactivity, or electrophysiologic, biochemical, or structural changes in the brain); and capturing subtle yet meaningful changes in symptom domains such as sociability, anxiety, and hyperactivity in human FXS clinical trials is challenging with the currently used measures (typically parent/caregiver rating scales). Clinicians, researchers, and the pharmaceutical industry have all had to take a step back and critically evaluate the way we think about how to best optimize future investigations into pharmacologic FXS treatments. As new clinical trials are coming down the drug discovery pipeline, it is clear that the field is moving in a direction that values the development of molecular biomarkers, less subjective quantitative measures of symptom improvement, and rating scales developed specifically for use in FXS in conjunction with drug safety. While summarizing preclinical evidence, where applicable, and discussing challenges in FXS treatment development, this review details both completed clinical trials for the targeted and symptomatic treatment of FXS and introduces novel projects on the cusp of clinical trial investigation.

Prenatal immune challenge in rats: effects of polyinosinic-polycytidylic acid on spatial learning, prepulse inhibition, conditioned fear, and responses to MK-801 and amphetamine.

Prenatal maternal immune activation increases risk for schizophrenia and/or autism. Previous data suggest that maternal weight change in response to the immune activator polyinosinic-polycytidylic (Poly IC) in rats influences the severity of effect in the offspring as does the exposure period. We treated gravid Sprague-Dawley rats from E14 to 18 with 8mg/kg/day Poly IC or saline. The Poly IC group was divided into those that gained the least weight or lost (Poly IC (L)) and those that gained the most (Poly IC (H)) weight. There were no effects of Poly IC on anxiety (elevated zero-maze, open-field, object burying), or Morris water maze cued learning or working memory or Cincinnati water maze egocentric learning. The Poly IC (H) group males had decreased acoustic startle whereas Poly IC (L) females had reduced startle and increased PPI. Poly IC offspring showed exaggerated hyperactivity in response to amphetamine (primarily in the Poly IC (H) group) and attenuated hyperactivity in response to MK-801 challenge (primarily in the Poly IC (L) group). Poly IC (L) males showed reduced cued conditioned freezing; both sexes showed less time in the dark in a light-dark test, and the Poly IC groups showed impaired Morris water maze hidden platform acquisition and probe performance. The data demonstrate that offspring from the most affected dams were more affected than those from less reactive dams indicating that degree of maternal immune activation predicts severity of effects on offspring behavior.

Impact of acamprosate on plasma amyloid-ß precursor protein in youth: a pilot analysis in fragile X syndrome-associated and idiopathic autism spectrum disorder suggests a pharmacodynamic protein marker.

BACKGROUND: Understanding of the pathophysiology of autism spectrum disorder (ASD) remains limited. Brain overgrowth has been hypothesized to be associated with the development of ASD. A derivative of amyloid-ß precursor protein (APP), secreted APPα (sAPPα), has neuroproliferative effects and has been shown to be elevated in the plasma of persons with ASD compared to control subjects. Reduction in sAPPα holds promise as a novel molecular target of treatment in ASD. Research into the neurochemistry of ASD has repeatedly implicated excessive glutamatergic and deficient GABAergic neurotransmission in the disorder. With this in mind, acamprosate, a novel modulator of glutamate and GABA function, has been studied in ASD. No data is available on the impact of glutamate or GABA modulation on sAPPα function. METHODS: Plasma APP derivative levels pre- and post-treatment with acamprosate were determined in two pilot studies involving youth with idiopathic and fragile X syndrome (FXS)-associated ASD. We additionally compared baseline APP derivative levels between youth with FXS-associated or idiopathic ASD. RESULTS: Acamprosate use was associated with a significant reduction in plasma sAPP(total) and sAPPα levels but no change occurred in Aß40 or Aß42 levels in 15 youth with ASD (mean age: 11.1 years). Youth with FXS-associated ASD (n = 12) showed increased sAPPα processing compared to age-, gender- and IQ-match youth with idiopathic ASD (n = 11). CONCLUSIONS: Plasma APP derivative analysis holds promise as a potential biomarker for use in ASD targeted treatment. Reduction in sAPP (total) and sAPPα may be a novel pharmacodynamic property of acamprosate. Future study is required to address limitations of the current study to determine if baseline APP derivative analysis may predict subgroups of persons with idiopathic or FXS-associated ASD who may respond best to acamprosate or to potentially other modulators of glutamate and/or GABA neurotransmission.

Effects of developmental manganese, stress, and the combination of both on monoamines, growth, and corticosterone.

Developmental exposure to manganese (Mn) or stress can each be detrimental to brain development. Here, Sprague-Dawley rats were exposed to two housing conditions and Mn from postnatal day (P)4-28. Within each litter two males and 2 females were assigned to the following groups: 0 (vehicle), 50, or 100 mg/kg Mn by oral gavage every other day. Half the litters were reared in cages with standard bedding and half with no bedding. One pair/group in each litter had an acute shallow water stressor before tissue collection (i.e., standing in shallow water). Separate litters were assessed at P11, 19, or 29. Mn-treated rats raised in standard cages showed no change in baseline corticosterone but following acute stress increased more than controls on P19; no Mn effects were seen on P11 or P29. Mn increased neostriatal dopamine in females at P19 and norepinephrine at P11 and P29. Mn increased hippocampal dopamine at P11 and P29 and 5-HT at P29 regardless of housing or sex. Mn had no effect on hypothalamic dopamine, but increased norepinephrine in males at P29 and 5-HT in males at all ages irrespective of rearing condition. Barren reared rats showed no or opposite effects of Mn, i.e., barren rearing + Mn attenuated corticosterone increases to acute stress. Barren rearing also altered the Mn-induced changes in dopamine and norepinephrine in the neostriatum, but not in the hippocampus. Barren rearing caused a Mn-associated increase in hypothalamic dopamine at P19 and P29 not seen in standard reared Mn-treated groups. Developmental Mn alters monoamines and corticosterone as a function of age, stress (acute and chronic), and sex.

Cognitive impairments from developmental exposure to serotonergic drugs: citalopram and MDMA.

We previously showed that developmental 3,4-methylenedioxymethamphetamine (MDMA) treatment induces long-term spatial and egocentric learning and memory deficits and serotonin (5-HT) reductions. During brain development, 5-HT is a neurotrophic factor influencing neurogenesis, synaptogenesis, migration, and target field organization. MDMA (10 mg/kg × 4/d at 2 h intervals) given on post-natal day (PD) 11-20 in rats (a period of limbic system development that approximates human third trimester brain development) induces 50% reductions in 5-HT during treatment and 20% reductions when assessed as adults. To determine whether the 5-HT reduction is responsible for the cognitive deficits, we used citalopram (Cit) pretreatment to inhibit the effects of MDMA on 5-HT reuptake in a companion study. Cit attenuated MDMA-induced 5-HT reductions by 50% (Schaefer et al., 2012). Here we tested whether Cit (5 or 7.5 mg/kg × 2/d) pretreatment attenuates the cognitive effects of MDMA. Within each litter, different offspring were treated on PD11-20 with saline (Sal) + MDMA, Cit + MDMA, Cit + Sal or Sal + Sal. Neither spatial nor egocentric learning/memory was improved by Cit pretreatment. Unexpectedly, Cit + Sal (at both doses) produced spatial and egocentric learning deficits as severe as those caused by Sal + MDMA. These are the first data showing cognitive deficits resulting from developmental exposure to a selective serotonin reuptake inhibitor. These data indicate the need for further research on the long-term safety of antidepressants during pregnancy.

Neonatal +-methamphetamine exposure in rats alters adult locomotor responses to dopamine D1 and D2 agonists and to a glutamate NMDA receptor antagonist, but not to serotonin agonists.

Neonatal exposure to (+)-methamphetamine (Meth) results in long-term behavioural abnormalities but its developmental mechanisms are unknown. In a series of experiments, rats were treated from post-natal days (PD) 11-20 (stage that approximates human development from the second to third trimester) with Meth or saline and assessed using locomotor activity as the readout following pharmacological challenge doses with dopamine, serotonin and glutamate agonists or antagonists during adulthood. Exposure to Meth early in life resulted in an exaggerated adult locomotor hyperactivity response to the dopamine D1 agonist SKF-82958 at multiple doses, a high dose only under-response activating effect of the D2 agonist quinpirole, and an exaggerated under-response to the activating effect of the N-methyl-d-aspartic acid (NMDA) receptor antagonist, MK-801. No change in locomotor response was seen following challenge with the 5-HT releaser p-chloroamphetamine or the 5-HT2/3 receptor agonist, quipazine. These are the first data to show that PD 11-20 Meth exposure induces long-lasting alterations to dopamine D1, D2 and glutamate NMDA receptor function and may suggest how developmental Meth exposure leads to many of its long-term adverse effects.

Neonatal citalopram treatment inhibits the 5-HT depleting effects of MDMA exposure in rats.

Neonatal exposure to 3,4-methylenedioxymethamphetamine (MDMA) produces long-term learning and memory deficits and increased anxiety-like behavior. The mechanism underlying these behavioral changes is unknown but we hypothesized that it involves perturbations to the serotonergic system as this is the principle mode of action of MDMA in the adult brain. During development 5-HT is a neurotrophic factor involved in neurogenesis, synaptogenesis, migration, and target region specification. We have previously showed that MDMA exposure (4×10 mg/kg/day) from P11-20 (analogous to human third trimester exposure) induces ~50% decreases in hippocampal 5-HT throughout treatment. To determine whether MDMA-induced 5-HT changes are determinative, we tested if these changes could be prevented by treatment with a selective serotonin reuptake inhibitor (citalopram: CIT). In a series of experiments we evaluated the effects of different doses and dose regimens of CIT on MDMA-induced 5-HT depletions in three brain regions (hippocampus, entorhinal cortex, and neostriatum) at three time-points (P12, P16, P21) during the treatment interval (P11-20) known to induce behavioral alterations when animals are tested as adults. We found that 5 mg/kg CIT administered twice daily significantly attenuated MDMA-induced 5-HT depletions in all three regions at all three ages but that the protection was not complete at all ages. Striatal dopamine was unaffected. We also found increases in hippocampal NGF and plasma corticosterone following MDMA treatment on P16 and P21, respectively. No changes in BDNF were observed. CIT treatment may be a useful means of interfering with MDMA-induced 5-HT reductions and thus permit tests of the hypothesis that the drug's cognitive and/or anxiety effects are mediated through early disruptions to 5-HT dependent developmental processes.

Prenatal immune challenge in rats: altered responses to dopaminergic and glutamatergic agents, prepulse inhibition of acoustic startle, and reduced route-based learning as a function of maternal body weight gain after prenatal exposure to poly IC.

Prenatal maternal immune activation has been used to test the neurodevelopmental hypothesis of schizophrenia. Most of the data are in mouse models; far less is available for rats. We previously showed that maternal weight change in response to the immune activator polyinosinic-polycytidylic acid (Poly IC) in rats differentially affects offspring. Therefore, we treated gravid Harlan Sprague-Dawley rats i.p. on embryonic day 14 with 8 mg/kg of Poly IC or Saline. The Poly IC group was divided into those that lost or gained the least weight, Poly IC (L), versus those that gained the most weight, Poly IC (H), following treatment. The study design controlled for litter size, litter sampling, sex distribution, and test experience. We found no effects of Poly IC on elevated zero maze, open-field activity, object burying, light-dark test, straight channel swimming, Morris water maze spatial acquisition, reversal, or shift navigation or spatial working or reference memory, or conditioned contextual or cued fear or latent inhibition. The Poly IC (H) group showed a significant decrease in the rate of route-based learning when visible cues were unavailable in the Cincinnati water maze and reduced prepulse inhibition of acoustic startle in females, but not males. The Poly IC (L) group exhibited altered responses to acute pharmacological challenges: exaggerated hyperactivity in response to (+)-amphetamine and an attenuated hyperactivity in response to MK-801. This model did not exhibit the cognitive, or latent inhibition deficits reported in Poly IC-treated rats but showed changes in response to drugs acting on neurotransmitter systems implicated in the pathophysiology of schizophrenia (dopaminergic hyperfunction and glutamatergic hypofunction).

Dorsal striatal dopamine depletion impairs both allocentric and egocentric navigation in rats.

Successful navigation requires interactions among multiple but overlapping neural pathways mediating distinct capabilities, including egocentric (self-oriented, route-based) and allocentric (spatial, map-based) learning. Route-based navigation has been shown to be impaired following acute exposure to the dopaminergic (DA) drugs (+)-methamphetamine and (+)-amphetamine, but not the serotoninergic (5-HT) drugs (±)-3,4-methylenedioxymethamphetamine or (±)-fenfluramine. The dopaminergic-rich neostriatum is involved in both allocentric and egocentric navigation. This experiment tested whether dorsal striatal DA loss using bilateral 6-hydroxydopamine (6-OHDA) injections impaired one or both types of navigation. Two weeks following 6-OHDA injections, rats began testing in the Cincinnati water maze (CWM) followed by the Morris water maze (MWM) for route-based and spatial navigation, respectively. 6-OHDA treatment significantly increased latency and errors in the CWM and path length, latency, and cumulative distance in the MWM with no difference on cued MWM trials. Neostriatal DA levels were reduced by 80% at 2 and 7 weeks post-treatment. In addition, 6-OHDA increased DA turnover and decreased norepinephrine (NE) levels. 6-OHDA injections did not alter monoamine levels in the prefrontal cortex. The data support that neostriatal DA modulates both types of navigation.

(±)3,4-methylenedioxymethamphetamine ("ecstasy") treatment modulates expression of neurotrophins and their receptors in multiple regions of adult rat brain.

(±)3,4-Methylenedioxymethamphetamine (MDMA), a widely used drug of abuse, rapidly reduces serotonin levels in the brain when ingested or administered in sufficient quantities, resulting in deficits in complex route-based learning, spatial learning, and reference memory. Neurotrophins are important for survival and preservation of neurons in the adult brain, including serotonergic neurons. In this study, we examined the effects of MDMA on the expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and their respective high-affinity receptors, tropomyosin receptor kinase (trk)B and trkC, in multiple regions of the rat brain. A serotonergic-depleting dose of MDMA (10 mg/kg × 4 at 2-hour intervals on a single day) was administered to adult Sprague-Dawley rats, and brains were examined 1, 7, or 24 hours after the last dose. Messenger RNA levels of BDNF, NT-3, trkB, and trkC were analyzed by using in situ hybridization with cRNA probes. The prefrontal cortex was particularly vulnerable to MDMA-induced alterations in that BDNF, NT-3, trkB, and trkC mRNAs were all upregulated at multiple time points. MDMA-treated animals had increased BDNF expression in the frontal, parietal, piriform, and entorhinal cortices, increased NT-3 expression in the anterior cingulate cortex, and elevated trkC in the entorhinal cortex. In the nigrostriatal system, BDNF expression was upregulated in the substantia nigra pars compacta, and trkB was elevated in the striatum in MDMA-treated animals. Both neurotrophins and trkB were differentially regulated in several regions of the hippocampal formation. These findings suggest a possible role for neurotrophin signaling in the learning and memory deficits seen following MDMA treatment.