Conditioned inhibition of amphetamine sensitization.

Repeated intermittent exposure to psychostimulants, such as amphetamine, leads to a progressive enhancement of the drug's ability to increase both behavioral and brain neurochemical responses. The expression of these enhancements, known as sensitization, can be regulated by Pavlovian conditioned stimuli. Cues that are associated with drug experience can facilitate sensitization so that it only occurs in the presence of these stimuli (context-specific sensitization). In contrast, cues that are explicitly related to the absence of drugs (conditioned inhibitors) can prevent the expression of sensitization. We hypothesized that disrupting conditioned inhibition would enable amphetamine sensitization in new contexts. Using male Sprague Dawley rats and a two-context amphetamine conditioning procedure, we found that extinguishing amphetamine experience in one environment led to the loss of conditioned inhibition in a separate context. Thus, amphetamine-induced sensitized locomotion, as well as both enhanced dopamine and glutamate neurotransmission in the nucleus accumbens, were observed in a context where the drug was never experienced before. A similar loss of contextual control of sensitization was seen after using baclofen/muscimol microinjections to transiently inhibit the medial prefrontal cortex, basolateral amygdala, or ventral subiculum of the hippocampus. In other words, compared to control infusions, these intracranial injections of GABA-receptor agonists were able to block conditioned inhibitors from preventing the expression of sensitized locomotion. Together, these findings reveal the importance of conditioned inhibitors for regulating addiction-like behavior. The results suggest that dopaminergic and glutamatergic brain circuitry controls the context-specific expression of amphetamine sensitization.

Transient overexpression of alpha-Ca2+/calmodulin-dependent protein kinase II in the nucleus accumbens shell enhances behavioral responding to amphetamine.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is known to contribute to the expression of psychostimulant sensitization by regulating dopamine (DA) overflow from DA neuron terminals in the nucleus accumbens (NAcc). The present experiments explored the contribution of CaMKII in NAcc neurons postsynaptic to these terminals where it is known to participate in a number of signaling pathways that regulate responding to psychostimulant drugs. Exposure to amphetamine transiently increased alphaCaMKII levels in the shell but not the core of the NAcc. Thus, HSV (herpes simplex viral) vectors were used to transiently overexpress alphaCaMKII in NAcc neurons in drug-naive rats, and behavioral responding to amphetamine was assessed. Transiently overexpressing alphaCaMKII in the NAcc shell led to long-lasting enhancement of amphetamine-induced locomotion and self-administration manifested when alphaCaMKII levels were elevated and persisting long after they had returned to baseline. Enhanced locomotion was not observed after infection in the NAcc core or sites adjacent to the NAcc. Transient elevation of NAcc shell alphaCaMKII levels also enhanced locomotor responding to NAcc AMPA and increased phosphorylation levels of GluR1 (Ser831), a CaMKII site, both soon and long after infection. Similar increases in pGluR1 (Ser831) were observed both soon and long after exposure to amphetamine. These results indicate that the transient increase in alphaCaMKII observed in neurons of the NAcc shell after viral-mediated gene transfer and likely exposure to amphetamine leads to neuroadaptations in AMPA receptor signaling in this site that may contribute to the long-lasting maintenance of behavioral and incentive sensitization by psychostimulant drugs like amphetamine.

Casein kinase 1 enables nucleus accumbens amphetamine-induced locomotion by regulating AMPA receptor phosphorylation.

The closely related δ and ε isoforms of the serine/threonine protein kinase casein kinase 1 (Csnk1) have been implicated in the generation of psychostimulant-induced behaviors. In this study, we show that Csnk1δ/ε produces its effects on behavior by acting on the Darpp-32-PP1 signaling pathway to regulate AMPA receptor phosphorylation in the nucleus accumbens (NAcc). Inhibiting Csnk1δ/ε in the NAcc with the selective inhibitor PF-670462 blocks amphetamine induced locomotion and its ability to increase phosphorylation of Darpp-32 at S137 and T34, decrease PP1 activity and increase phosphorylation of the AMPA receptor subunit at S845. Consistent with these findings, preventing GluR1 phosphorylation with the alanine mutant GluR1(S845A) reduces glutamate-evoked currents in cultured medium spiny neurons and blocks the locomotor activity produced by NAcc amphetamine. Thus, Csnk1 enables the locomotor and likely the incentive motivational effects of amphetamine by regulating Darrp-32-PP1-GlurR1(S845) signaling in the NAcc. As such, Csnk1 may be a critical target for intervention in the treatment of drug use disorders.

Exposure to conditions of uncertainty promotes the pursuit of amphetamine.

Prior exposure to abused drugs leads to long-lasting neuroadaptations culminating in excessive drug intake. Given the comorbidity between substance use and gambling disorders, surprisingly little is known about the effects of exposure to reinforcement contingencies experienced during games of chance. As it is a central feature of these games, we characterized the effects of exposure to uncertainty on biochemical and behavioral effects normally observed in rats exposed to amphetamine. Rats in different groups were trained to nose-poke for saccharin under certain [fixed-ratio (FR)] or uncertain conditions [variable-ratio (VR)] for 55 1-h sessions. Ratios were escalated on successive sessions and rats maintained on the last ratio (FR/VR 20) for 20-25 days. Two to three weeks later, rats were tested for their locomotor or nucleus accumbens dopamine (NAcc DA) response to amphetamine or self-administration of the drug using a lever press operant. NAcc DA overflow was also assessed in additional rats during the saccharin sessions. Rats exposed to uncertainty subsequently showed a higher locomotor and NAcc DA response to amphetamine and self-administered more drug infusions relative to rats exposed to predictable reinforcement. NAcc DA levels during the saccharin sessions tracked the variance of the scheduled ratios (a measure of uncertainty). VR rats showed escalating DA overflow with increasing ratios. Exposure to uncertainty triggered neuroadaptations similar to those produced by exposure to abused drugs. As these were produced in drug naive rats both during and after exposure to uncertainty, they provide a novel common pathway to drug and behavioral addictions.

Sensitizing exposure to amphetamine increases AMPA receptor phosphorylation without increasing cell surface expression in the rat nucleus accumbens.

Exposure to psychostimulants like cocaine or amphetamine leads to long-lasting sensitization of their behavioral and neurochemical effects. Here we characterized changes in AMPA receptor distribution and phosphorylation state in the rat nucleus accumbens (NAcc) weeks after amphetamine exposure to assess their potential contribution to sensitization by this drug. Using protein cross-linking, biochemical, subcellular fractionation, and slice electrophysiological approaches in the NAcc, we found that, unlike cocaine, previous exposure to amphetamine did not increase cell surface levels of either GluA1 or GluA2 AMPA receptor subunits, redistribution of these subunits to the synaptic or perisynaptic cellular membrane domains, protein-protein associations required to support the accumulation and retention of AMPA receptors in the PSD, or the peak amplitude of AMPA receptor mediated mEPSCs recorded in NAcc slices. On the other hand, exposure to amphetamine significantly slowed mEPSC decay times and increased levels in the PSD of PKA and CaMKII as well as phosphorylation by these kinases of the GluA1 S845 and S831 residues selectively in this cellular compartment. As the latter effects are known to respectively regulate channel open probability and duration as well as conductance, they provide a novel mechanism that could contribute to the long-lasting AMPA receptor dependent expression of sensitization by amphetamine. Rather than increase the number of surface and synaptic AMPA receptors as with cocaine, this mechanism could increase NAcc medium spiny neuron reactivity to glutamate afferents by increasing the phosphorylation state of critical regulatory sites in the AMPA receptor GluA1 subunit in the PSD.

Operant responding for optogenetic excitation of LDTg inputs to the VTA requires D1 and D2 dopamine receptor activation in the NAcc.

Behavioral studies in rats and mice indicate that laterodorsal tegmental nucleus (LDTg) inputs to the ventral tegmental area (VTA) importantly contribute to reward function. Further evidence from anesthetized rat and mouse preparations suggests that these LTDg inputs may exert this effect by regulating mesolimbic dopamine (DA) signaling. Direct evidence supporting this possibility remains lacking however. To address this lack, rat LDTg neurons were transfected with adeno-associated viral vectors encoding channelrhodopsin2 and eYFP (ChR2) or eYFP alone (eYFP) and rats were subsequently trained to lever press for intracranial self-stimulation (ICSS) of the inputs of these neurons to the VTA. First, we found that DA overflow in the forebrain nucleus accumbens (NAcc) increased maximally during ICSS to approximately 240% of baseline levels in ChR2, but not in eYFP, rats. Based on these findings, we next tested the contribution of NAcc D1 and D2 DA receptors to the reinforcing effects of optogenetic excitation of LDTg inputs to the VTA. Microinjecting SCH23390 or raclopride, D1 and D2 DA receptor antagonists respectively, into the NAcc significantly reduced operant responding for this stimulation. Together these results demonstrate for the first time that optogenetic ICSS of LDTg inputs to the VTA increases DA overflow in the NAcc and requires activation of D1 and D2 DA receptors in this site.

PKC phosphorylates residues in the N-terminal of the DA transporter to regulate amphetamine-induced DA efflux.

The DA transporter (DAT), a phosphoprotein, controls extracellular dopamine (DA) levels in the central nervous system through transport or reverse transport (efflux). Multiple lines of evidence support the claim that PKC significantly contributes to amphetamine-induced DA efflux. Other signaling pathways, involving CaMKII and ERK, have also been shown to regulate DAT mediated efflux. Here we assessed the contribution of putative PKC residues (S4, S7, S13) in the N-terminal of the DAT to amphetamine-induced DA efflux by transfecting DATs containing different serine to alanine (S-A) point mutations into DA pre-loaded HEK-293 cells and incubating these cells in amphetamine (2µM). The effects of a S-A mutation at the non-PKC residue S12 and a threonine to alanine (T-A) mutation at the ERK T53 residue were also assessed for comparison. WT-DATs were used as controls. In an initial experiment, we confirmed that inhibiting PKC with Go6976 (130nM) significantly reduced amphetamine-induced DA efflux. In subsequent experiments, cells transfected with the S4A, S12A, S13A, T53A and S4,7,13A mutants showed a reduction in amphetamine-induced DA efflux similar to that observed with Go6976. Interestingly, cells transfected with the S7A mutant, identified by some as a PKC-PKA residue, showed unperturbed WT-DAT levels of amphetamine-induced DA efflux. These results indicate that phosphorylation by PKC of select residues in the DAT N-terminal can regulate amphetamine-induced efflux. PKC can act either independently or in concert with other kinases such as ERK to produce this effect.

Stimuli associated with the presence or absence of amphetamine regulate cytoskeletal signaling and behavior.

Drug-paired stimuli rapidly enlarge dendritic spines in the nucleus accumbens (NAcc). While increases in spine size and shape are supported by rearrangement of the actin cytoskeleton and facilitate the synaptic expression of AMPA-type glutamate receptors, it remains unclear whether drug-related stimuli can influence signaling pathways known to regulate these changes in spine morphology. These pathways were studied in rats trained on a discrimination learning paradigm using subcellular fractionation and protein immunoblotting to isolate proteins within dendritic spine compartments in the NAcc shell. An open field chamber was repeatedly associated with amphetamine in one group (Paired) and explicitly unpaired with amphetamine in another (Unpaired). Rats in a third group were exposed to the open field but never administered amphetamine (Control). When administered saline and returned to the open field one week later, Paired rats as expected displayed a conditioned locomotor response relative to rats in the other two groups. NAcc shell tissues were harvested immediately after this 30-minute test. Re-exposing Paired rats to the drug-paired excitatory context significantly decreased p-GluA2(S880), an effect consistent with reduced internalization of this subunit and increased spine proliferation in these rats. In contrast, re-exposing Unpaired rats to the drug-unpaired context, capable of inhibiting conditioned responding in these animals, significantly decreased levels of both actin binding protein Arp2/3 and p-cofilin, consistent with spine volatility, shrinkage, and inhibition of spine proliferation in these rats. These findings show that contextual stimuli previously associated with either the presence or absence of amphetamine differentially regulate cytoskeletal signaling pathways in the NAcc.

Drug-Paired Contextual Stimuli Increase Dendritic Spine Dynamics in Select Nucleus Accumbens Neurons.

Repeated exposure to amphetamine leads to both associative conditioning and nonassociative sensitization. Here we assessed the contribution of neuronal ensembles in the nucleus accumbens (NAcc) to these behaviors. Animals exposed to amphetamine IP or in the ventral tegmental area (VTA) showed a sensitized locomotor response when challenged with amphetamine weeks later. Both exposure routes also increased ΔFosB levels in the NAcc. Further characterization of these ΔFosB+ neurons, however, revealed that amphetamine had no effect on dendritic spine density or size, indicating that these neurons do not undergo changes in dendritic spine morphology that accompany the expression of nonassociative sensitization. Additional experiments determined how neurons in the NAcc contribute to the expression of associative conditioning. A discrimination learning procedure was used to expose rats to IP or VTA amphetamine either Paired or Unpaired with an open field. As expected, compared with Controls, Paired rats administered IP amphetamine subsequently showed a conditioned locomotor response when challenged with saline in the open field, an effect accompanied by an increase in c-Fos+ neurons in the medial NAcc. Further characterization of these c-Fos+ cells revealed that Paired rats showed an increase in the density of dendritic spines and the frequency of medium-sized spines in the NAcc. In contrast, Paired rats previously exposed to VTA amphetamine showed neither conditioned locomotion nor conditioned c-Fos+ expression. Together, these results suggest a role for c-Fos+ neurons in the medial NAcc and rapid changes in the morphology of their dendritic spines in the expression of conditioning evoked by amphetamine-paired contextual stimuli.

Long-chain fatty acids increase cellular dopamine in an immortalized cell line (MN9D) derived from mouse mesencephalon.

The lysate of an immortalized monoclonal cell line derived from the striatum (X61) contains a dopaminergic stimulatory activity that is capable of increasing the dopamine content of an immortalized mouse mesencephalic cell line (MN9D) which expresses a dopaminergic phenotype. Purification of an isoamyl alcohol extract of this lysate and subsequent identification by NMR spectroscopic analysis demonstrated that the dopaminergic stimulatory activity contained within the lysate was a mixture of 80-90% cis-9-octadecenoic acid (oleic acid) and 10-20% cis-11-octadecenoic acid (cis-vaccenic acid). The effect of oleic acid on MN9D dopamine is a prolonged event. MN9D dopamine increases linearly over a 48 h period suggesting the induction of an increased dopaminergic phenotype in these dividing cells. The ability to increase MN9D dopamine by oleic and cis-vaccenic acids is shared by a number of other long-chain fatty acids including arachidonic, linoleic, linolenic, palmitoleic, and cis-13-octadecenoic acid. The possibility that oleic or other relatively innocuous fatty acids might affect dopaminergic function in primary neurons is intriguing with respect to possible therapeutic approaches to the treatment of dopaminergic cell loss and the motor sequelae of Parkinson's disease.

Fetal exposure to (+/-)-methylenedioxymethamphetamine in utero enhances the development and metabolism of serotonergic neurons in three-dimensional reaggregate tissue culture.

Methylenedioxymethamphetamine (MDMA, Ecstasy) is a potent psychomotor stimulant with neurotoxic potential which is widely abused by females of childbearing age raising serious public health concerns in terms of exposure of the fetus to the drug. The current study was conducted using the three-dimensional reaggregate tissue culture system as an approach to the assessment of risk to fetal brain cells following exposure to MDMA during early to mid-gestation. In this culture system, the serotonergic and dopaminergic mesencephalic-striatal projections are reconstructed and develop with a time course similar to that observed in vivo. Pregnant C57Bl/6J mice were injected twice daily with 40 mg/kg MDMA or saline from gestational day 6 to 13. On gestational day 14, mesencephalic and striatal cells from MDMA- and saline-exposed embryos were used to prepare reaggregate cultures. Levels of neurotransmitters and their metabolites in the reaggregates and culture medium were assessed at 22 and 36 days of culture. There was a long-term enhancement of serotonergic development and metabolism by fetal exposure to MDMA as evidenced by increased reaggregate serotonin levels as well as the elevated production and release of 5-hydroxyindoleacetic acid in cultures prepared from MDMA-exposed embryos which persisted for up to 36 days of culture. Dopaminergic neurons in such cultures also exhibited increased metabolism as indicated by elevated levels of dihydroxyphenylacetic acid in reaggregate tissue and culture medium. The data obtained suggest that exposure to MDMA in utero during early to mid-gestation may result in more active serotonergic and dopaminergic neurons.

Enhanced survival of primary murine dopaminergic neurons induced by a partially purified cell lysate fraction from mouse-derived striatal hybrid monoclonal cells.

Lysates of X61, a striatal-derived cell line, and a partially purified preparation from the lysate (UF4) contain a factor(s) capable of increasing the dopamine content of a mesencephalic-derived dopaminergic cell line (MN9D) and of cultures containing primary dopaminergic neurons. Treatment of cultures containing dopaminergic primary neurons grown in the absence of target cells over a 2 week period with X61 lysate or UF4 resulted in an elevation of dopamine levels of the cultures and of media homovanillic acid as well as a 2.0-fold (UF4) to 2.9-fold (X61 lysate) increase in the density of dopaminergic neurons in treated cultures. The results suggest that the activity factor derived from X61 is capable of preventing dopaminergic cell loss which occurs in the absence of dopaminergic target cells of the corpus striatum.

Locomotor conditioning by amphetamine requires cyclin-dependent kinase 5 signaling in the nucleus accumbens.

Intermittent systemic exposure to psychostimulants leads to several forms of long-lasting behavioral plasticity including nonassociative sensitization and associative conditioning. In the nucleus accumbens (NAcc), the protein serine/threonine kinase cyclin-dependent kinase 5 (Cdk5) and its phosphorylation target, the guanine-nucleotide exchange factor kalirin-7 (Kal7), may contribute to the neuroadaptations underlying the formation of conditioned associations. Pharmacological inhibition of Cdk5 in the NAcc prevents the increases in dendritic spine density normally observed in this site following repeated cocaine. Mice lacking the Kal7 gene display similar effects. As increases in spine density may relate to the formation of associative memories and both Cdk5 and Kal7 regulate the generation of spines following repeated drug exposure, we hypothesized that either inhibiting Cdk5 or preventing its phosphorylation of Kal7 in the NAcc may prevent the induction of drug conditioning. In the present experiments, blockade in rats of NAcc Cdk5 activity with roscovitine (40 nmol/0.5 µl/side) prior to each of 4 injections of amphetamine (1.5 mg/kg; i.p.) prevented the accrual of contextual locomotor conditioning but spared the induction of locomotor sensitization as revealed on tests conducted one week later. Similarly, transient viral expression in the NAcc exclusively during amphetamine exposure of a threonine-alanine mutant form of Kal7 [mKal7(T1590A)] that is not phosphorylated by Cdk5 also prevented the accrual of contextual conditioning and spared the induction of sensitization. These results indicate that signaling via Cdk5 and Kal7 in the NAcc is necessary for the formation of context-drug associations, potentially through the modulation of dendritic spine dynamics in this site.

α-1 Adrenergic receptors are localized on presynaptic elements in the nucleus accumbens and regulate mesolimbic dopamine transmission.

Brainstem noradrenergic neurons innervate the mesocorticolimbic reward pathway both directly and indirectly, with norepinephrine facilitating dopamine (DA) neurotransmission via α1-adrenergic receptors (α1ARs). Although α1AR signaling in the prefrontal cortex (PFC) promotes mesolimbic transmission and drug-induced behaviors, the potential contribution of α1ARs in other parts of the pathway, such as the ventral tegmental area (VTA) and nucleus accumbens (NAc), has not been investigated before. We found that local blockade of α1ARs in the medial NAc shell, but not the VTA, attenuates cocaine- and morphine-induced locomotion. To determine the neuronal substrates that could mediate these effects, we analyzed the cellular, subcellular, and subsynaptic localization of α1ARs and characterized the chemical phenotypes of α1AR-containing elements within the mesocorticolimbic system using single and double immunocytochemical methods at the electron microscopic (EM) level. We found that α1ARs are found mainly extra-synaptically in axons and axon terminals in the NAc and are enriched in glutamatergic and dopaminergic elements. α1ARs are also abundant in glutamatergic terminals in the PFC, and in GABA-positive terminals in the VTA. In line with these observations, microdialysis experiments revealed that local blockade of α1ARs attenuated the increase in extracellular DA in the medial NAc shell following administration of cocaine. These data indicate that local α1ARs control DA transmission in the medial NAc shell and behavioral responses to drugs of abuse.

Fetal exposure to methamphetamine in utero stimulates development of serotonergic neurons in three-dimensional reaggregate tissue culture.

Methamphetamine is a potent psychomotor stimulant with neurotoxic potential which is widely abused by females of childbearing age, raising serious public health concerns in terms of exposure of the fetus to the drug. The current study was conducted to determine the effect of maternal administration of methamphetamine on developing monoaminergic neurons using three-dimensional reaggregate tissue cultures prepared from fetal mesencephalic and striatal cells. In this culture system, the dopaminergic and serotonergic mesencephalic-striatal projections are reconstructed and develop with a time course similar to that observed in vivo. Pregnant C57Bl/6J mice were injected twice daily with 40 mg/kg methamphetamine or saline from gestational days 6-13. On gestational day 14, cells from methamphetamine and saline-exposed embryos were used to prepare reaggregate cultures. Levels of neurotransmitters and their metabolites in the reaggregates and culture medium were monitored at 14, 29, 43, and 64 days of culture. Reaggregates prepared from methamphetamine-exposed embryos showed a significant elevation in serotonin levels at all culture ages compared to reaggregates prepared from saline-treated embryos. Levels of 5-HIAA in reaggregates and culture medium were also elevated in 14- and 29-day-old cultures derived from drug-exposed embryos. The development of the dopaminergic nigrostriatal projection was resistant to repeated in utero exposure to methamphetamine. In contrast, exposure of the fetus to methamphetamine, during early to midgestation, produced a long-lasting stimulatory effect on serotonergic development in culture.