Lynette C. Daws

Autism gene variant causes hyperserotonemia, serotonin receptor hypersensitivity, social impairment and repetitive behavior

Fifty years ago, increased whole-blood serotonin levels, or hyperserotonemia, first linked disrupted 5-HT homeostasis to Autism Spectrum Disorders (ASDs). The 5-HT transporter (SERT) gene (SLC6A4) has been associated with whole blood 5-HT levels and ASD susceptibility. Previously, we identified multiple gain-of-function SERT coding variants in children with ASD. Here we establish that transgenic mice expressing the most common of these variants, SERT Ala56, exhibit elevated, p38 MAPK-dependent transporter phosphorylation, enhanced 5-HT clearance rates and hyperserotonemia. These effects are accompanied by altered basal firing of raphe 5-HT neurons, as well as 5HT1A and 5HT2A receptor hypersensitivity. Strikingly, SERT Ala56 mice display alterations in social function, communication, and repetitive behavior. Our efforts provide strong support for the hypothesis that altered 5-HT homeostasis can impact risk for ASD traits and provide a model with construct and face validity that can support further analysis of ASD mechanisms and potentially novel treatments.

Calmodulin Kinase II Interacts with the Dopamine Transporter C Terminus to Regulate Amphetamine-Induced Reverse Transport

Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound to the distal C terminus of DAT and colocalized with DAT in dopaminergic neurons. CaMKIIalpha stimulated dopamine efflux via DAT in response to amphetamine in heterologous cells and in dopaminergic neurons. CaMKIIalpha phosphorylated serines in the distal N terminus of DAT in vitro, and mutation of these serines eliminated the stimulatory effects of CaMKIIalpha. A mutation of the DAT C terminus impairing CaMKIIalpha binding also impaired amphetamine-induced dopamine efflux. An in vivo role for CaMKII was supported by chronoamperometry measurements showing reduced amphetamine-induced dopamine efflux in response to the CaMKII inhibitor KN93. Our data suggest that CaMKIIalpha binding to the DAT C terminus facilitates phosphorylation of the DAT N terminus and mediates amphetamine-induced dopamine efflux.

Interleukin-1 Receptor Activation by Systemic Lipopolysaccharide Induces Behavioral Despair Linked to MAPK Regulation of CNS Serotonin Transporters

Serotonin (5-hydroxytryptamine, 5-HT) has long been implicated in regulation of mood. Medications that block the neuronal 5-HT transporter (SERT) are used as major pharmacological treatment for mood disorders. Conversely, stimuli that enhance SERT activity might be predicted to diminish synaptic 5-HT availability and increase the risk for 5-HT-related CNS disorders. We have shown that the inflammatory cytokines enhance brain SERT activity in cultured serotonergic cells and nerve terminal preparations in vitro. In this study, we establish that intraperitoneal injection of the cytokine-inducer lipopolysaccharide (LPS) stimulates brain SERT activity, acting at doses below those required to induce overt motor suppression. SERT stimulation by LPS is paralleled by increased immobility in both the tail suspension test (TST) and the forced swim test (FST); antidepressant-sensitive alterations are thought to model aspects of behavioral despair. Both the stimulation of SERT activity and induced immobility are absent when LPS is administered to interleukin-1 receptor (IL-1R)-deficient mice and in the presence of SB203580, an inhibitor of IL-1R-stimulated p38 MAPK. Moreover, the ability of LPS to enhance immobility in TST is lost in SERT knockout mice. These findings reveal an ability of peripheral inflammatory stimuli to enhance brain SERT activity through IL-1R and p38 MAPK pathways in vivo and identify a requirement for SERT expression in immune-system-modulated despair behaviors. Our studies identify IL-1R- and p38 MAPK-dependent regulation of SERT as one of the mechanisms by which environmentally driven immune system activation can trigger despair-like behavior in an animal model, encouraging future analysis of the pathway for risk factors in neuropsychiatric disorders.

Mice lacking the AMPA GluR1 receptor exhibit striatal hyperdopaminergia and 'schizophrenia-related' behaviors.

There is growing evidence implicating dysfunctional glutamatergic neurotransmission and abnormal interactions between the glutamate and dopamine (DA) systems in the pathophysiology of various neuropsychiatric disorders including schizophrenia. The present study evaluated knockout (KO) mice lacking the L-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) GluR1 receptor subunit for a range of behaviors considered relevant to certain symptoms of schizophrenia. KO showed locomotor hyperactivity during exposure to open field and in response to a novel object, but normal activity in a familiar home cage. Open field locomotor hyperactivity in KO was effectively normalized to WT levels by treatment with the DA antagonist and neuroleptic haloperidol, while locomotor stimulant effects of the NMDA receptor antagonist MK-801 were absent in KO. Social behaviors during a dyadic conspecific encounter were disorganized in KO. KO showed deficits in prepulse inhibition of the acoustic startle response. In vivo chronoamperometric measurement of extracellular DA clearance in striatum demonstrated retarded clearance in KO. These data demonstrate behavioral abnormalities potentially pertinent to schizophrenia in GluR1 KO, together with evidence of dysregulated DA function. Present findings provide novel insight into the potential role of GluR1, AMPA receptors and glutamate × DA interactions in the pathophysiology of schizophrenia and other neuropsychiatric conditions.

Hypoinsulinemia regulates amphetamine-induced reverse transport of dopamine

The behavioral effects of psychomotor stimulants such as amphetamine (AMPH) arise from their ability to elicit increases in extracellular dopamine (DA). These AMPH-induced increases are achieved by DA transporter (DAT)-mediated transmitter efflux. Recently, we have shown that AMPH self-administration is reduced in rats that have been depleted of insulin with the diabetogenic agent streptozotocin (STZ). In vitro studies suggest that hypoinsulinemia may regulate the actions of AMPH by inhibiting the insulin downstream effectors phosphotidylinositol 3-kinase (PI3K) and protein kinase B (PKB, or Akt), which we have previously shown are able to fine-tune DAT cell-surface expression. Here, we demonstrate that striatal Akt function, as well as DAT cell-surface expression, are significantly reduced by STZ. In addition, our data show that the release of DA, determined by high-speed chronoamperometry (HSCA) in the striatum, in response to AMPH, is severely impaired in these insulin-deficient rats. Importantly, selective inhibition of PI3K with LY294002 within the striatum results in a profound reduction in the subsequent potential for AMPH to evoke DA efflux. Consistent with our biochemical and in vivo electrochemical data, findings from functional magnetic resonance imaging experiments reveal that the ability of AMPH to elicit positive blood oxygen level–dependent signal changes in the striatum is significantly blunted in STZ-treated rats. Finally, local infusion of insulin into the striatum of STZ-treated animals significantly recovers the ability of AMPH to stimulate DA release as measured by high-speed chronoamperometry. The present studies establish that PI3K signaling regulates the neurochemical actions of AMPH-like psychomotor stimulants. These data suggest that insulin signaling pathways may represent a novel mechanism for regulating DA transmission, one which may be targeted for the treatment of AMPH abuse and potentially other dopaminergic disorders.

Organic cation transporter 3: Keeping the brake on extracellular serotonin in serotonin-transporter-deficient mice

Mood disorders cause much suffering and are the single greatest cause of lost productivity worldwide. Although multiple medications, along with behavioral therapies, have proven effective for some individuals, millions of people lack an effective therapeutic option. A common serotonin (5-HT) transporter (5-HTT/SERT, SLC6A4) polymorphism is believed to confer lower 5-HTT expression in vivo and elevates risk for multiple mood disorders including anxiety, alcoholism, and major depression. Importantly, this variant is also associated with reduced responsiveness to selective 5-HT reuptake inhibitor antidepressants. We hypothesized that a reduced antidepressant response in individuals with a constitutive reduction in 5-HTT expression could arise because of the compensatory expression of other genes that inactivate 5-HT in the brain. A functionally upregulated alternate transporter for 5-HT may prevent extracellular 5-HT from rising to levels sufficiently high enough to trigger the adaptive neurochemical events necessary for therapeutic benefit. Here we demonstrate that expression of the organic cation transporter type 3 (OCT3, SLC22A3), which also transports 5-HT, is upregulated in the brains of mice with constitutively reduced 5-HTT expression. Moreover, the OCT blocker decynium-22 diminishes 5-HT clearance and exerts antidepressant-like effects in these mice but not in WT animals. OCT3 may be an important transporter mediating serotonergic signaling when 5-HTT expression or function is compromised.

Exaggerated effect of fluvoxamine in heterozygote serotonin transporter knockout mice

Clearance rates for serotonin (5‐HT) in heterozygote (+/–) and homozygote (–/–) serotonin transporter (5‐HTT) knockout (KO) mice have not been determined in vivo. Moreover, the effect of selective serotonin reuptake inhibitors (SSRIs) on 5‐HT clearance in these mice has not been examined. In this study, the rate of clearance of exogenously applied 5‐HT was measured in the CA3 region of the hippocampus of anesthetized mice using high‐speed chronoamperometry. Compared with wild‐type mice, the maximal rate of 5‐HT clearance from extracellular fluid (ECF) was decreased in heterozygotes and more markedly so in KO mice. Heterozygote mice were more sensitive to the 5‐HT uptake inhibitor, fluvoxamine, resulting in longer clearance times for 5‐HT than in wild‐type mice; as expected, the KO mice were completely unresponsive to fluvoxamine. There were no associated changes in norepinephrine transporter density, nor was there an effect of the norepinephrine uptake inhibitor, desipramine, on 5‐HT clearance in any genotype. Thus, adaptive changes in the norepinephrine transport system do not occur in the CA3 region of hippocampus as a consequence of 5‐HTT KO. These data highlight the potential of the heterozygote 5‐HTT mutant mice to model the dynamic in vivo consequences of the human 5‐HTT polymorphism.

Density and function of central serotonin (5-HT) transporters, 5-HT1A and 5-HT2A receptors, and effects of their targeting on BTBR T+tf/J mouse social behavior

J. Neurochem. (2011) 116, 291–303.

5-HT1B Receptor-Mediated Regulation of Serotonin Clearance in Rat Hippocampus In Vivo

Abstract: The 5‐hydroxytryptamine (5‐HT; serotonin) transporter (5‐HTT) is important in terminating serotonergic neurotransmission and is a primary target for many psychotherapeutic drugs. Study of the regulation of 5‐HTT activity is therefore important in understanding the control of serotonergic neurotransmission. Using high‐speed chronoamperometry, we have demonstrated that local application of 5‐HT1B antagonists into the CA3 region of the hippocampus prolongs the clearance of 5‐HT from extracellular fluid (ECF). In the present study, we demonstrate that the 5‐HT1B antagonist cyanopindolol does not produce this effect by increasing release of endogenous 5‐HT or by directly binding to the 5‐HTT. Dose‐response studies showed that the potency of cyanopindolol to inhibit clearance of 5‐HT was equivalent to that of the selective 5‐HT reuptake inhibitor fluvoxamine. Local application of the 5‐HT1A antagonist WAY 100635 did not alter 5‐HT clearance, suggesting that the effect of cyanopindolol to prolong clearance is not via a mechanism involving 5‐HT1A receptors. Finally, the effect of low doses of cyanopindolol and fluvoxamine to inhibit clearance of 5‐HT from ECF was additive. These data are consistent with the hypothesis that activation of terminal 5‐HT1B autoreceptors increases 5‐HTT activity.

Ethanol Inhibits Clearance of Brain Serotonin by a Serotonin Transporter-Independent Mechanism

Brain serotonin (5-HT) modulates the neural and behavioral effects of ethanol in a manner that remains poorly understood. Here we show that treatment with physiologically relevant (i.e., moderately intoxicating) doses of ethanol inhibits clearance of 5-HT from extracellular fluid in the mouse hippocampus. This finding demonstrates, in vivo, a key molecular mechanism by which ethanol modulates serotonergic neurotransmission. The 5-HT transporter (5-HTT) is the principle means of 5-HT reuptake in the brain and an obvious candidate mechanism for the effect of ethanol to inhibit 5-HT clearance. However, our second major finding was that genetic inactivation of the 5-HTT in a knock-out mouse not only failed to prevent ethanol-induced inhibition of 5-HT clearance, but actually potentiated this effect. Ethanol-induced inhibition of 5-HT clearance was also potentiated in nonmutant mice by cotreatment with a 5-HTT antagonist. Providing a link with potential behavioral manifestations of this neural phenotype, 5-HTT knock-out mice also exhibited exaggerated sensitivity to behavioral intoxication, as assayed by the sedative/hypnotic effects of ethanol. This clearly demonstrates that the 5-HTT is not necessary for the neural and behavioral effects of ethanol observed herein and that genetic or pharmacological inactivation of the 5-HTT unmasks involvement of other principle mechanisms. These data are intriguing given growing evidence implicating the 5-HTT in the pathophysiology and treatment of alcoholism and neuropsychiatric conditions frequently comorbid with alcoholism, such as depression. The present findings provide new insights into the actions of ethanol on brain function and behavior.