Amy J. Ramsey

Hyperdopaminergia and NMDA receptor hypofunction disrupt neural phase signaling

Neural phase signaling has gained attention as a putative coding mechanism through which the brain binds the activity of neurons across distributed brain areas to generate thoughts, percepts, and behaviors. Neural phase signaling has been shown to play a role in various cognitive processes, and it has been suggested that altered phase signaling may play a role in mediating the cognitive deficits observed across neuropsychiatric illness. Here, we investigated neural phase signaling in two mouse models of cognitive dysfunction: mice with genetically induced hyperdopaminergia [dopamine transporter knock-out (DAT-KO) mice] and mice with genetically induced NMDA receptor hypofunction [NMDA receptor subunit-1 knockdown (NR1-KD) mice]. Cognitive function in these mice was assessed using a radial-arm maze task, and local field potentials were recorded from dorsal hippocampus and prefrontal cortex as DAT-KO mice, NR1-KD mice, and their littermate controls engaged in behavioral exploration. Our results demonstrate that both DAT-KO and NR1-KD mice display deficits in spatial cognitive performance. Moreover, we show that persistent hyperdopaminergia alters interstructural phase signaling, whereas NMDA receptor hypofunction alters interstructural and intrastructural phase signaling. These results demonstrate that dopamine and NMDA receptor dependent glutamate signaling play a critical role in coordinating neural phase signaling, and encourage further studies to investigate the role that deficits in phase signaling play in mediating cognitive dysfunction.

Increased expression of the dopamine transporter leads to loss of dopamine neurons, oxidative stress and l-DOPA reversible motor deficits

The dopamine transporter is a key protein responsible for regulating dopamine homeostasis. Its function is to transport dopamine from the extracellular space into the presynaptic neuron. Studies have suggested that accumulation of dopamine in the cytosol can trigger oxidative stress and neurotoxicity. Previously, ectopic expression of the dopamine transporter was shown to cause damage in non-dopaminergic neurons due to their inability to handle cytosolic dopamine. However, it is unknown whether increasing dopamine transporter activity will be detrimental to dopamine neurons that are inherently capable of storing and degrading dopamine. To address this issue, we characterized transgenic mice that over-express the dopamine transporter selectively in dopamine neurons. We report that dopamine transporter over-expressing (DAT-tg) mice display spontaneous loss of midbrain dopamine neurons that is accompanied by increases in oxidative stress markers, 5-S-cysteinyl-dopamine and 5-S-cysteinyl-DOPAC. In addition, metabolite-to-dopamine ratios are increased and VMAT2 protein expression is decreased in the striatum of these animals. Furthermore, DAT-tg mice also show fine motor deficits on challenging beam traversal that are reversed with l-DOPA treatment. Collectively, our findings demonstrate that even in neurons that routinely handle dopamine, increased uptake of this neurotransmitter through the dopamine transporter results in oxidative damage, neuronal loss and l-DOPA reversible motor deficits. In addition, DAT over-expressing animals are highly sensitive to MPTP-induced neurotoxicity. The effects of increased dopamine uptake in these transgenic mice could shed light on the unique vulnerability of dopamine neurons in Parkinsons disease.

D1 Dopamine Receptor Coupling to PLCβ Regulates Forward Locomotion in Mice

Several studies have reported the coupling of dopamine signaling to phospholipase C β (PLCβ) both in vitro and in vivo. However, the precise physiological relevance of this signaling pathway in mediating dopamine behaviors is still unclear. Here we report that stimulation of dopamine receptor signaling in vivo with systemic administration of apomorphine, amphetamine, and cocaine leads to increased production of inositol triphosphate (IP3) in the mouse striatum. Using selective antagonists and dopamine D1 and D2 receptor knock-out animals, we show that the production of IP3 is mediated by the D1 receptor, but not the D2 receptor. A selective blocker of PLCβ, U73122, was used to assess the physiological relevance of D1-mediated IP3 production. We show that U73122 inhibits the locomotor-stimulating effects of apomorphine, amphetamine, cocaine, and SKF81297. Furthermore, U73122 also suppresses the spontaneous hyperactivity exhibited by dopamine transporter knock-out mice. Importantly, the effects of U73122 are selective to dopamine-mediated hyperactivity, as this compound does not affect hyperactivity induced by the glutamate NMDA receptor antagonist MK801. Finally, we present evidence showing that an imbalance of D1- and D2-mediated signaling following U73122 treatment modifies the locomotor output of animals from horizontal locomotor activity to vertical activity, further highlighting the importance of the PLCβ pathway in the regulation of forward locomotion via dopamine receptors.

NR1 knockdown mice as a representative model of the glutamate hypothesis of schizophrenia.

N-methyl d-aspartate (NMDA) receptor subunit NR1 knockdown (NR1-KD) mice have a global reduction of NMDA receptors, enabling their use as a genetic model to study the role of NMDA receptors in the pathophysiology of schizophrenia. This targeted mutation results in a spectrum of altered behaviors that are similar to those induced by NMDA receptor antagonists, which have long been used to model schizophrenia in animals. NR1-KD mice serve as a complementary tool to pharmacological models, providing insight into the consequences of sustained NMDA receptor dysfunction in early brain development and throughout the life of the animal. Though in many respects the phenotype of NR1-KD mice mimics that of acute NMDA receptor antagonism, there are also notable differences. In this chapter we highlight some of the molecular, behavioral, and neurophysiological phenotypes of NR1-KD mice and compare these to pharmacological models of NMDA receptor dysfunction. Through the study of these models, our improved understanding of how the brain adapts to persistent NMDA receptor hypofunction may eventually suggest new therapeutic strategies for schizophrenia.

N-Aryl Piperazine Metabotropic Glutamate Receptor 5 Positive Allosteric Modulators Possess Efficacy in Preclinical Models of NMDA Hypofunction and Cognitive Enhancement

Impaired transmission through glutamatergic circuits has been postulated to play a role in the underlying pathophysiology of schizophrenia. Furthermore, inhibition of the N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptors (NMDAR) induces a syndrome that recapitulates many of the symptoms observed in patients with schizophrenia. Selective activation of metabotropic glutamate receptor subtype 5 (mGlu5) may provide a novel therapeutic approach for treatment of symptoms associated with schizophrenia through facilitation of transmission through central glutamatergic circuits. Here, we describe the characterization of two novel N-aryl piperazine mGlu5 positive allosteric modulators (PAMs): 2-(4-(2-(benzyloxy)acetyl)piperazin-1-yl)benzonitrile (VU0364289) and 1-(4-(2,4-difluorophenyl)piperazin-1-yl)-2-((4-fluorobenzyl)oxy)ethanone (DPFE). VU0364289 and DPFE induced robust leftward shifts in the glutamate concentration-response curves for Ca2+ mobilization and extracellular signal-regulated kinases 1 and 2 phosphorylation. Both PAMs displayed micromolar affinity for the common mGlu5 allosteric binding site and high selectivity for mGlu5. VU0364289 and DPFE possessed suitable pharmacokinetic properties for dosing in vivo and produced robust dose-related effects in reversing amphetamine-induced hyperlocomotion, a preclinical model predictive of antipsychotic-like activity. In addition, DPFE enhanced acquisition of contextual fear conditioning in rats and reversed behavioral deficits in a mouse model of NMDAR hypofunction. In contrast, DPFE had no effect on reversing apomorphine-induced disruptions of prepulse inhibition of the acoustic startle reflex. These mGlu5 PAMs also increased monoamine levels in the prefrontal cortex, enhanced performance in a hippocampal-mediated memory task, and elicited changes in electroencephalogram dynamics commensurate with procognitive effects. Collectively, these data support and extend the role for the development of novel mGlu5 PAMs for the treatment of psychosis and cognitive deficits observed in individuals with schizophrenia.

NMDA receptor-deficient mice display sexual dimorphism in the onset and severity of behavioural abnormalities.

N‐methyl‐d‐aspartate (NMDA) receptor‐deficient mice can be used to understand the role that NMDA receptors (NMDARs) play in the pathophysiology of neurodevelopmental disorders such as schizophrenia. Genetically modified mice with low levels of NR1 subunit (NR1 knockdown mice) have reduced receptor levels throughout development, and have robust abnormalities in behaviours that are relevant to schizophrenia. We traced the onset and severity of these behaviours at three developmental stages to understand when in development the underlying circuits depend on intact NMDAR function. We examined social behaviour, working memory, executive function, locomotor activity and stereotypy at 3, 6 and 12 weeks of age in NR1 knockdown mice and their wild‐type littermates. We discovered that each of these behaviours had a unique developmental trajectory in mutant mice, and males showed an earlier onset and severity than females in several behaviours. Hyperlocomotion was most substantial in juvenile mice and plateaued in adult mice, whereas stereotypy progressively worsened with age. Impairments in working memory and sociability were sexually dimorphic, with deficits first detected in peri‐adolescent males but only detected in adult females. Interestingly, executive function was most impaired in peri‐adolescent mice of either sex. Furthermore, while juvenile mutant mice had some ability to problem solve in the puzzle box test, the same mice lost this ability when tested 4 weeks later. Our studies highlight key developmental periods for males and females in the expression of behaviours that are relevant to psychiatric disorders.

Chronic SSRI Treatment Exacerbates Serotonin Deficiency in Humanized Tph2 Mutant Mice

Selective serotonin reuptake inhibitors (SSRIs) are a major class of antidepressants that act by blocking inward transport of serotonin (5-HT) into presynaptic neurons mediated by the serotonin transporter (SERT). Both reuptake and ongoing synthesis are essential in supporting intraneuronal serotonin concentrations in serotonergic neurons. A rare mutation in tryptophan hydroxylase 2 (Tph2), the rate limiting enzyme for 5-HT synthesis, was identified in several patients with major depression, and knock-in mice expressing the analogous mutation (R439H Tph2 KI) show 80% reduction in 5-HT synthesis and tissue levels. Chronic treatment with SSRIs (fluoxetine and paroxetine) resulted in a dramatic further depletion of 5-HT tissue levels in R439H Tph2 KI mice (down to 1-3% of wild type levels) while having little effects in wild-type controls. Treatment with the 5-HT precursor 5-hydroxytryptophan (5-HTP) restored 5-HT tissue content in mutant mice, and cotreatment with 5-HTP and fluoxetine essentially prevented the depleting effect of a chronic SSRI. These data demonstrate that chronic SSRI treatment could further exacerbate the 5-HT deficiency in Tph2 mutation carriers, and this can be prevented by 5-HTP supplementation.

α5GABAA receptor deficiency causes autism-like behaviors

The prevalence of autism spectrum disorders (ASDs), which affect over 1% of the population, has increased twofold in recent years. Reduced expression of GABAA receptors has been observed in postmortem brain tissue and neuroimaging of individuals with ASDs. We found that deletion of the gene for the α5 subunit of the GABAA receptor caused robust autism‐like behaviors in mice, including reduced social contacts and vocalizations. Screening of human exome sequencing data from 396 ASD subjects revealed potential missense mutations in GABRA5 and in RDX, the gene for the α5GABAA receptor‐anchoring protein radixin, further supporting a α5GABAA receptor deficiency in ASDs.

Differential effects of NMDA receptor antagonism on spine density

The authors demonstrate that different NMDAR antagonists (ketamine and MK-801) have varying effects on spine density depending on dose, drug regimen, and brain region. While acute ketamine treatment increases cortical spine density in mice, subchronic exposure to either drug reduces spine density in both the cortex and striatum.

Prefrontal Cortex-Mediated Impairments in a Genetic Model of NMDA Receptor Hypofunction Are Reversed by the Novel M1 PAM VU6004256.

Abnormalities in the signaling of the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) within cortical and limbic brain regions are thought to underlie many of the complex cognitive and affective symptoms observed in individuals with schizophrenia. The M1 muscarinic acetylcholine receptor (mAChR) subtype is a closely coupled signaling partner of the NMDAR. Accumulating evidence suggests that development of selective positive allosteric modulators (PAMs) of the M1 receptor represent an important treatment strategy for the potential normalization of disruptions in NMDAR signaling in patients with schizophrenia. In the present studies, we evaluated the effects of the novel and highly potent M1 PAM, VU6004256, in ameliorating selective prefrontal cortical (PFC)-mediated physiologic and cognitive abnormalities in a genetic mouse model of global reduction in the NR1 subunit of the NMDAR (NR1 knockdown [KD]). Using slice-based extracellular field potential recordings, deficits in muscarinic agonist-induced long-term depression (LTD) in layer V of the PFC in the NR1 KD mice were normalized with bath application of VU6004256. Systemic administration of VU6004256 also reduced excessive pyramidal neuron firing in layer V PFC neurons in awake, freely moving NR1 KD mice. Moreover, selective potentiation of M1 by VU6004256 reversed the performance impairments of NR1 KD mice observed in two preclinical models of PFC-mediated learning, specifically the novel object recognition and cue-mediated fear conditioning tasks. VU6004256 also produced a robust, dose-dependent reduction in the hyperlocomotor activity of NR1 KD mice. Taken together, the current findings provide further support for M1 PAMs as a novel therapeutic approach for the PFC-mediated impairments in schizophrenia.