Devon L. Graham

Developmental Consequences of Fetal Exposure to Drugs: What We Know and What We Still Must Learn

Most drugs of abuse easily cross the placenta and can affect fetal brain development. In utero exposures to drugs thus can have long-lasting implications for brain structure and function. These effects on the developing nervous system, before homeostatic regulatory mechanisms are properly calibrated, often differ from their effects on mature systems. In this review, we describe current knowledge on how alcohol, nicotine, cocaine, amphetamine, Ecstasy, and opiates (among other drugs) produce alterations in neurodevelopmental trajectory. We focus both on animal models and available clinical and imaging data from cross-sectional and longitudinal human studies. Early studies of fetal exposures focused on classic teratological methods that are insufficient for revealing more subtle effects that are nevertheless very behaviorally relevant. Modern mechanistic approaches have informed us greatly as to how to potentially ameliorate the induced deficits in brain formation and function, but conclude that better delineation of sensitive periods, dose–response relationships, and long-term longitudinal studies assessing future risk of offspring to exhibit learning disabilities, mental health disorders, and limited neural adaptations are crucial to limit the societal impact of these exposures.

Evidence against dopamine D1/D2 receptor heteromers

Hetero-oligomers of G-protein-coupled receptors have become the subject of intense investigation, because their purported potential to manifest signaling and pharmacological properties that differ from the component receptors makes them highly attractive for the development of more selective pharmacological treatments. In particular, dopamine D1 and D2 receptors have been proposed to form hetero-oligomers that couple to Gαq proteins, and SKF83959 has been proposed to act as a biased agonist that selectively engages these receptor complexes to activate Gαq and thus phospholipase C. D1/D2 heteromers have been proposed as relevant to the pathophysiology and treatment of depression and schizophrenia. We used in vitro bioluminescence resonance energy transfer, ex vivo analyses of receptor localization and proximity in brain slices, and behavioral assays in mice to characterize signaling from these putative dimers/oligomers. We were unable to detect Gαq or Gα11 protein coupling to homomers or heteromers of D1 or D2 receptors using a variety of biosensors. SKF83959-induced locomotor and grooming behaviors were eliminated in D1 receptor knockout (KO) mice, verifying a key role for D1-like receptor activation. In contrast, SKF83959-induced motor responses were intact in D2 receptor and Gαq KO mice, as well as in knock-in mice expressing a mutant Ala286-CaMKIIα that cannot autophosphorylate to become active. Moreover, we found that, in the shell of the nucleus accumbens, even in neurons in which D1 and D2 receptor promoters are both active, the receptor proteins are segregated and do not form complexes. These data are not compatible with SKF83959 signaling through Gαq or through a D1/D2 heteromer and challenge the existence of such a signaling complex in the adult animals that we used for our studies.

GLP-1 analog attenuates cocaine reward

Letter to the Editor Glucagon-like peptide-1 (GLP-1) is released in response to food intake and acts through both peripheral and central mechanisms to regulate energy homeostasis and feeding behavior. Exendin-4 (Ex-4) is an analog of GLP-1 with a significantly longer half-life and greater potency (1) and is marketed synthetically as exenatide (Byetta™ and Bydureon™) as a treatment for type 2 diabetes mellitus. We show that administration of exendin-4 attenuates the rewarding effects of cocaine in mice, thus highlighting the therapeutic potential of GLP-1 receptor (GLP-1R) agonists for the treatment of psychostimulant addiction. Emerging data suggest that hormones and peptides that play a role in feeding behavior, such as insulin, GLP-1, leptin, orexin, and ghrelin, may also be involved in drug reward (2, 3) and could thereby be targeted as therapies for the treatment of addiction. GLP1-Rs are expressed within the brain, including in the ventral tegmental area (VTA) and the nucleus accumbens (NAc) (4, 5). These brain regions are part of the mesolimbic reward circuit considered to play a major role in the rewarding properties of drugs, including the cocaine (6). Activation of mesolimbic GLP-1Rs decreases the intake of highly palatable foods, suggesting that GLP-1R activation contributes to the hedonic components of food intake (4). Drug reward and feeding behavior utilize overlapping brain circuitry and mechanisms, thus we hypothesized that the GLP-1R agonist Ex-4 would reduce the rewarding effects of cocaine. In a test of conditioned place preference (CPP), a significant cocaine-induced CPP was achieved (SAL/Cocaine group, Fig. 1). However, the rewarding effects of cocaine were attenuated in mice pretreated with Ex-4 (Ex-4/Cocaine group, regardless of pretreatment dose), suggesting that Ex-4 reduces the hedonic effects of cocaine. This response was not influenced by Ex-4-induced hypoactivity, as Ex-4 pretreatment did not significantly lessen cocaine’s locomotor-stimulating properties (Supplementary Fig. 1C). As hypothesized, Ex-4-pretreated mice spent more time in the treatment chamber during the test phase following cocaine treatment but not to the same extent as cocaine-treated mice without the Ex-4 pretreatment, and this finding was not contingent upon the Ex-4 dose used. Notably, Ex-4 treatment alone at any of the doses investigated did not condition an aversion (or a preference) to the treatment-associated chamber as there was no significant change in the amount or percentage of time spent in the treatment chamber (Fig. 1, all Ex-4/SAL groups). Figure 1 Percent time spent on the treatment-affiliated compartment during the CPP test phase. A main effect of Treatment was found (F(6,117)=31.66, p<0.0001). Data are normalized against the SAL/cocaine group. Sample size for each group is indicated in ... How Ex-4 exerts this decrease in cocaine reward is unknown. Ex-4 readily crosses the blood brain barrier (7), GLP-1Rs are present in brain reward circuitry (4, 5), and local administration of Ex-4 into the mesolimbic dopamine system alters hedonic responses to food (4). It is thus likely that Ex-4 blunts cocaine CPP by targeting GLP-1Rs within the NAc or ventral midbrain. A previous study has shown that GLP-1Rs can specifically interact with the G-protein coupled receptor sorting protein (GASP-1) (8), and genetic knockout loss of GASP-1, in turn, alters cocaine-induced behavioral responses (9). Therefore, GLP-1Rs in the hypothalamus may mediate homeostatic regulation of food intake (10), whereas GLP-1Rs in the VTA and NAc might instead represent hedonic value, perhaps through alterations in dopamine transporter and/or receptor function (9, 11). However, other mechanisms, including a role of peripheral GLP-1Rs, cannot be ruled out at this time. Our study extends the potential therapeutic value of GLP-1R stimulation beyond metabolic disorders and identifies a new molecular target for the treatment of psychostimulant abuse. Moreover, even the lowest dose of Ex-4 used (10 µg/kg) produced a blunted cocaine CPP response, suggesting a GLP-1R-dependent mechanism involved in drug reward in addition to those already known involving neurotransmitters (e.g., dopamine, serotonin, norepinephrine, glutamate, and the opioids). Importantly, these data and that of others (5) indicate that Ex-4 treatment alone appears to be neither aversive nor pleasurable, suggesting that patient compliance due to negative side-effects or the potential for addiction to Ex-4, respectively, is of little concern. Ex-4, marketed in its synthetic form as exenatide, is already approved for human use as a treatment for type 2 diabetes. Whether these findings extend to other psychostimulants (e.g. amphetamines) and other drug classes has yet to be determined. Additional studies will also be required to examine full doseresponse functions, as it appears that even the 10 µg/kg dose was sufficient to attenuate cocaine-induced CPP; the duration of effectiveness; and the blockade or reversal of the cellular and molecular neuroadaptations that accompany chronic drug use and addiction. Self-administration studies will also be needed to fully explore the ability of GLP-1 signaling to regulate addictive processes. Finally, it is also worth noting that, although effective, even the highest dose of Ex-4 (100 µg/kg) did not completely eliminate cocaine-induced CPP. Thus our data also demonstrate the presence of a GLP-1 receptor-independent component to cocaine reward, suggesting exenatide treatment as a possible complementary therapy of drug abuse.

Creatine Transporter (CrT; Slc6a8) Knockout Mice as a Model of Human CrT Deficiency

Mutations in the creatine (Cr) transporter (CrT; Slc6a8) gene lead to absence of brain Cr and intellectual disabilities, loss of speech, and behavioral abnormalities. To date, no mouse model of CrT deficiency exists in which to understand and develop treatments for this condition. The purpose of this study was to generate a mouse model of human CrT deficiency. We created mice with exons 2–4 of Slc6a8 flanked by loxP sites and crossed these to Cre:CMV mice to create a line of ubiquitous CrT knockout expressing mice. Mice were tested for learning and memory deficits and assayed for Cr and neurotransmitter levels. Male CrT−/y (affected) mice lack Cr in the brain and muscle with significant reductions of Cr in other tissues including heart and testes. CrT−/y mice showed increased path length during acquisition and reversal learning in the Morris water maze. During probe trials, CrT−/y mice showed increased average distance from the platform site. CrT−/y mice showed reduced novel object recognition and conditioned fear memory compared to CrT+/y. CrT−/y mice had increased serotonin and 5-hydroxyindole acetic acid in the hippocampus and prefrontal cortex. Ubiquitous CrT knockout mice have learning and memory deficits resembling human CrT deficiency and this model should be useful in understanding this disorder.

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). Synapse 2012.

Effects of (+)-methamphetamine on path integration and spatial learning, but not locomotor activity or acoustic startle, align with the stress hyporesponsive period in rats

Rats treated with (+)‐methamphetamine (MA) on postnatal days (P) 11–20 exhibit long‐term spatial and path integration (Morris water maze (MWM) and Cincinnati water maze (CWM)) learning deficits whereas those treated on P1–10 do not. MA treatment increases corticosterone release in an age‐dependent U‐shaped pattern that corresponds to the stress hyporesponsive period (SHRP; P4–15). Here we tested the hypothesis that the cognitive effects induced by MA are associated with treatment that begins within the SHRP. Three treatment regimens were compared, P1–10, P6–15, and P11–20. One male/female pair/litter received 0, 10, or 25 mg/kg MA/dose (four doses/day at 2 h intervals given s.c. with 19–21 litters/regimen). Locomotor activity and acoustic startle were tested as behaviors not predicted to be associated with the SHRP. Cincinnati and Morris water maze findings were consistent with the hypothesis in that MA‐treated animals exposed from P6–15 or P11–20 showed impaired learning compared to those exposed from P1–10; however, on probe trials in the Morris water maze, MA‐induced memory impairments were not regimen‐specific and were contributed to by all treatment regimens. All MA treatment regimens induced reductions in locomotor activity and acoustic startle facilitation as expected. No differential effect on prepulse trials was seen suggesting no impairment in sensory gating. Cognitive deficits from neonatal MA treatment are associated with the SHRP and may be the product of hypothalamic–pituitary–adrenal (HPA) axis dysregulation during critical periods of brain development.

Effect of a neurotoxic dose regimen of (+)-methamphetamine on behavior, plasma corticosterone, and brain monoamines in adult C57BL/6 mice

RATIONALE In rats, neurotoxic doses of methamphetamine (MA) induce astrogliosis, long lasting monoamine reductions, reuptake transporter down-regulation, and learning impairments. OBJECTIVE We tested whether comparable effects occur in C57BL/6 mice. METHOD C57BL/6 mice were treated with 10mg/kgs.c.x4 MA on a single day and evaluated at various intervals thereafter. RESULTS The neurotoxic dose regimen of MA caused the predicted acute hyperthermia and increased striatal glial fibrillary acidic protein and reduced neostriatal dopamine. The MA-treated mice were hypoactive 24h later but not 48h later. MA-treated mice also showed exaggerated initial hyperactivity after a pharmacological dose of MA used to stimulate locomotion followed by a later phase of hypoactivity compared to saline-treated mice. No differences were observed on learning or memory tests (novel object recognition, egocentric, or spatial learning/memory). MA-treated mice showed a trend toward increased prepulse inhibition but not baseline acoustic startle reactivity. After testing, MA-treated mice showed reduced neostriatal dopamine and increased basal plasma corticosterone. CONCLUSIONS A neurotoxic/binge regimen of MA in mice that produces the typical pattern of neurotoxic changes to those seen in rats, results in few behavioral changes. This may limit the utility of C57BL/6 mice for modeling the cognitive and behavioral effects described in human MA users who show such changes even after prolonged abstinence.

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.

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.

The glucagon-like peptide 1 (GLP-1) receptor agonist exendin-4 reduces cocaine self-administration in mice

Glucagon-like peptide 1 (GLP-1) analogues are used for the treatment of type 2 diabetes. The ability of the GLP-1 system to decrease food intake in rodents has been well described and parallels results from clinical trials. GLP-1 receptors are expressed in the brain, including within the ventral tegmental area (VTA) and the nucleus accumbens (NAc). Dopaminergic neurons in the VTA project to the NAc, and these neurons play a pivotal role in the rewarding effects of drugs of abuse. Based on the anatomical distribution of GLP-1 receptors in the brain and the well-established effects of GLP-1 on food reward, we decided to investigate the effect of the GLP-1 analogue exendin-4 on cocaine- and dopamine D1-receptor agonist-induced hyperlocomotion, on acute and chronic cocaine self-administration, on cocaine-induced striatal dopamine release in mice and on cocaine-induced c-fos activation. Here, we report that GLP-1 receptor stimulation reduces acute and chronic cocaine self-administration and attenuates cocaine-induced hyperlocomotion. In addition, we show that peripheral administration of exendin-4 reduces cocaine-induced elevation of striatal dopamine levels and striatal c-fos expression implicating central GLP-1 receptors in these responses. The present results demonstrate that the GLP-1 system modulates cocaines effects on behavior and dopamine homeostasis, indicating that the GLP-1 receptor may be a novel target for the pharmacological treatment of drug addiction.