Nancy Bubula

Alpha-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.

Methamphetamine concentrations in fetal and maternal brain following prenatal exposure

Levels of methamphetamine in maternal striatum and whole fetal mouse brain were assessed at 0.5, 1, 2, and 4 h postinjection on gestational day 14 (GD14) following a single, subcutaneous injection of 40 mg/kg (+)-methamphetamine hydrochloride to pregnant mice. In the dams, striatal concentrations of methamphetamine peaked at 1 h postinjection, reaching levels of approximately 510 ng/mg protein. Amphetamine, the primary metabolite of methamphetamine, increased to 77 ng/mg protein at 2 h and remained elevated by 4 h postinjection. In the fetal brain, peak methamphetamine concentrations of approximately 122 ng/mg protein were attained at 1 h. Amphetamine was only detectable in fetal brain at 2 and 4 h postinjection. Regional analysis of methamphetamine levels in fetal striatum, cortex, and brainstem revealed that the drug was not uniformly distributed. Maternal administration of methamphetamine results in fetal brain drug concentrations, which approximate those reported in human infants whose mother abused methamphetamine. This dosage regimen, therefore, serves as an appropriate animal model for assessing the potential risks to human offspring exposed to methamphetamine in utero.

Transient Overexpression of α-Ca2+/Calmodulin-Dependent Protein Kinase II in the Nucleus Accumbens Shell Enhances Behavioral Responding to Amphetamine

Ca2+/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 αCaMKII levels in the shell but not the core of the NAcc. Thus, HSV (herpes simplex viral) vectors were used to transiently overexpress αCaMKII in NAcc neurons in drug-naive rats, and behavioral responding to amphetamine was assessed. Transiently overexpressing αCaMKII in the NAcc shell led to long-lasting enhancement of amphetamine-induced locomotion and self-administration manifested when αCaMKII 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 αCaMKII 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 αCaMKII 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

J. Neurochem. (2011) 118, 237–247.

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.

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 Parkinsons disease.

Elevation of fetal dopamine following exposure to methamphetamine in utero

The effect of methamphetamine on fetal dopaminergic function was examined following treatment of pregnant mice twice daily with 40 mg/kg methamphetamine from either gestational day (GD) 7-13 or from GD 7-15. Dopamine levels were elevated in fetal GD 16 corpus striatum and rostral mesencephalon following both treatment regimens. This increase in fetal dopamine is consistent with our findings that exposure to methamphetamine in utero results in adult dopaminergic neurons which are more responsive in terms of methamphetamine induced release of the neurotransmitter and more sensitive to the neurotoxic effects of the drug.

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.

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

HighlightsRats acquire VTA optogenetic intracranial self‐stimulation (ICSS) of LDTg inputs.This ICSS increases DA overflow in the forebrain NAcc.D1 or D2 receptor blockade in the NAcc reduces optogenetic ICSS of LDTg inputs to the VTA. Abstract 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.

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.