Acute Cocaine Differentially Induces PKA Phosphorylation Substrates in Male and Female Rats

Background: Sex differences in intracellular dopamine pathways may contribute to the known sex differences in psychomotor responses to cocaine and differential development of dependence. This study aimed to determine whether there are sex differences in the activation of the extracellular signal-regulated kinases (ERK1/2, or p44/p42 MAPK) and PKA phosphorylation-dependent substrates in the nucleus accumbens (NAcc) of male and female rats at baseline or after acute cocaine administration. Methods: 60-day-old male and female Fischer rats were injected with saline or cocaine (30 mg/kg) and sacrificed 5, 15, 30, 45 or 90 minutes later. Total locomotor activity, Stereotypic, rearing, and ambulatory behaviors was measured for 90 minutes using a two-frame automated Photobeam Activity Results: Similar to our previous findings, total locomotor activities were higher in female rats after this single cocaine administration. Females had higher levels of phosphorylated PKA substrates after cocaine administration, and this change lasted longer and had a greater magnitude than in cocaine treated male rats. Furthermore, although cocaine administration increased the phosphorylation of ERK proteins, there were no sex differences in p-ERK protein levels either at baseline or after acute cocaine administration. Conclusion: Taken together, these findings suggest that sex differences in basal and cocaine-induced alterations in PKA signaling activity in the NAcc may contribute to sex differences in psychomotor responses to cocaine. However, not all the components of the DA-intracellular signaling pathway maybe heightened in female rats as ERK phosphorylation patterns did not differ between the sexes

A single brain-derived neurotrophic factor infusion into the dorsomedial prefrontal cortex attenuates cocaine self-administration-induced phosphorylation of synapsin in the nucleus accumbens during early withdrawal

BACKGROUND: Dysregulation in the prefrontal cortex-nucleus accumbens pathway has been implicated in cocaine addiction. We have previously demonstrated that one intra-dorsomedial prefrontal cortex brain-derived neurotrophic factor (BDNF) infusion immediately following the last cocaine self-administration session caused a long-lasting inhibition of cocaine-seeking and normalized the cocaine-induced disturbance of glutamate transmission in the nucleus accumbens after extinction and a cocaine prime. However, the molecular mechanism mediating the brain-derived neurotrophic factor effect on cocaine-induced alterations in extracellular glutamate levels is unknown. METHODS: In the present study, we determined the effects of brain-derived neurotrophic factor on cocaine-induced changes in the phosphorylation of synapsin (p-synapsin), a family of presynaptic proteins that mediate synaptic vesicle mobilization, in the nucleus accumbens during early withdrawal. RESULTS: Two hours after cocaine self-administration, p-synapsin Ser9 and p-synapsin Ser62/67, but not p-synapsin Ser603, were increased in the nucleus accumbens. At 22 hours, only p-synapsin Ser9 was still elevated. Elevations at both time points were attenuated by an intra-dorsomedial prefrontal cortex brain-derived neurotrophic factor infusion immediately after the end of cocaine self-administration. Brain-derived neurotrophic factor also reduced cocaine self-administration withdrawal-induced phosphorylation of the protein phosphatase 2A C-subunit, suggesting that brain-derived neurotrophic factor disinhibits protein phosphatase 2A C-subunit, consistent with p-synapsin Ser9 dephosphorylation. Further, co-immunoprecipitation demonstrated that protein phosphatase 2A C-subunit and synapsin are associated in a protein-protein complex that was reduced after 2 hours of withdrawal from cocaine self-administration and reversed by brain-derived neurotrophic factor. CONCLUSIONS: Taken together, these findings demonstrate that brain-derived neurotrophic factor normalizes the cocaine self-administration–induced elevation of p-synapsin in nucleus accumbens that may underlie a disturbance in the probability of neurotransmitter release or represent a compensatory neuroadaptation in response to the hypofunction within the prefrontal cortex-nucleus accumbens pathway during cocaine withdrawal

The Role of BDNF/TrkB Signaling in Acute Amphetamine-Induced Locomotor Activity and Opioid Peptide Gene Expression in the Rat Dorsal Striatum

Exposure to psychostimulants increases brain-derived neurotrophic factor (BDNF) mRNA and protein levels in the cerebral cortex and subcortical structures. Because BDNF is co-localized with dopamine and glutamate in afferents to the striatum of rats, it may be co-released with those neurotransmitters upon stimulation. Further, there may be an interaction between the intracellular signaling cascades activated by dopamine, glutamate, and TrkB receptors in medium spiny striatal neurons. In the present study, the effect of acute amphetamine administration on TrkB phosphorylation, as an indirect indicator of activation, and striatal gene expression, was evaluated. In Experiment 1, 15 min or 2 h after a single saline or amphetamine (2.5 mg/kg, i.p.) injection, the caudate–putamen (CPu), nucleus accumbens (NAc), and dorsomedial prefrontal cortex (dmPFC) were extracted and processed for phospho (p)-TrkB immunoreactivity. Immunoprecipitation analyses indicated that neither the tyrosine phosphorylation (p-Tyr) or autophosphorylation sites of TrkB (706) were changed in NAc, CPu, or dmPFC 15 min after amphetamine administration. In contrast, p-Tyr and the PLCγ phosphorylation site of TrkB (816) were increased in the NAc and CPu 2 h after amphetamine. In Experiment 2, intra-striatal infusion of the tyrosine kinase inhibitor, K252a, increased amphetamine-induced vertical activity but not total distance traveled. In addition, K252a inhibited amphetamine-induced preprodynorphin, but not preproenkephalin, mRNA expression in the striatum. These data indicate that acute amphetamine administration induces p-TrkB activation and signaling in a time- and brain region-dependent manner and that TrkB/BDNF signaling plays an important role in amphetamine-induced behavior and striatal gene expression

Templated fabrication of large area subwavelength antireflection gratings on silicon

We report a cheap and scalable bottom-up technique for fabricating wafer-scale, subwavelength-structured antireflection coatings on single-crystalline silicon substrates. Spin-coated monolayer colloidal crystals are utilized as shadow masks to generate metallic nanohole arrays. Inverted pyramid arrays in silicon can then be templated against nanoholes by anisotropic wet etching. The resulting subwavelength gratings greatly suppress specular reflection at normal incidence. The reflection spectra for flat silicon and the templated gratings at long wavelengths agree well with the simulated results using a rigorous coupled wave analysis model. These subwavelength gratings are of great technological importance in crystalline silicon solar cells

Allosteric Modulatory Effects of SRI-20041 and SRI-30827 on Cocaine and HIV-1 Tat Protein Binding to Human Dopamine Transporter

Dopamine transporter (DAT) is the target of cocaine and HIV-1 transactivator of transcription (Tat) protein. Identifying allosteric modulatory molecules with potential attenuation of cocaine and Tat binding to DAT are of great scientific and clinical interest. We demonstrated that tyrosine 470 and 88 act as functional recognition residues in human DAT (hDAT) for Tat-induced inhibition of DA transport and transporter conformational transitions. Here we investigated the allosteric modulatory effects of two allosteric ligands, SRI-20041 and SRI-30827 on cocaine binding on wild type (WT) hDAT, Y470 H and Y88 F mutants. Effect of SRI-30827 on Tat-induced inhibition of [3H]WIN35,428 binding was also determined. Compared to a competitive DAT inhibitor indatraline, both SRI-compounds displayed a similar decrease (30%) in IC50 for inhibition of [3H]DA uptake by cocaine in WT hDAT. The addition of SRI-20041 or SRI-30827 following cocaine slowed the dissociation rate of [3H]WIN35,428 binding in WT hDAT relative to cocaine alone. Moreover, Y470H and Y88F hDAT potentiate the inhibitory effect of cocaine on DA uptake and attenuate the effects of SRI-compounds on cocaine-mediated dissociation rate. SRI-30827 attenuated Tat-induced inhibition of [3H]WIN35,428 binding. These observations demonstrate that tyrosine 470 and 88 are critical for allosteric modulatory effects of SRI-compounds on the interaction of cocaine with hDAT

Role of Histidine 547 of Human Dopamine Transporter in Molecular Interaction with HIV-1 Tat and Dopamine Uptake

HIV-1 Tat plays an important role in HIV-associated neurocognitive disorders (HAND) by disrupting neurotransmission including dopamine uptake by human dopamine transporter (hDAT). Previous studies have demonstrated that HIV-1 Tat directly binds to hDAT and some amino-acid mutations that attenuate the hDAT-Tat binding also significantly decreased dopamine uptake activity of hDAT. This combined computational-experimental study demonstrates that histidine-547 (H547) of hDAT plays a crucial role in the hDAT-Tat binding and dopamine uptake by hDAT, and that the H547A mutation can not only considerably attenuate Tat-induced inhibition of dopamine uptake, but also significantly increase the Vmax of hDAT for dopamine uptake. The finding of such an unusual hDAT mutant capable of both increasing the Vmax of hDAT for dopamine uptake and disrupting the hDAT-Tat binding may provide an exciting knowledge basis for development of novel concepts for therapeutic treatment of the HAND

Molecular Mechanism: The Human Dopamine Transporter Histidine 547 Regulates Basal and HIV-1 Tat Protein-Inhibited Dopamine Transport

Abnormal dopaminergic transmission has been implicated as a risk determinant of HIV-1-associated neurocognitive disorders. HIV-1 Tat protein increases synaptic dopamine (DA) levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Through integrated computational modeling prediction and experimental validation, we identified that histidine547 on human DAT (hDAT) is critical for regulation of basal DA uptake and Tat-induced inhibition of DA transport. Compared to wild type hDAT (WT hDAT), mutation of histidine547 (H547A) displayed a 196% increase in DA uptake. Other substitutions of histidine547 showed that DA uptake was not altered in H547R but decreased by 99% in H547P and 60% in H547D, respectively. These mutants did not alter DAT surface expression or surface DAT binding sites. H547 mutants attenuated Tat-induced inhibition of DA transport observed in WT hDAT. H547A displays a differential sensitivity to PMA- or BIM-induced activation or inhibition of DAT function relative to WT hDAT, indicating a change in basal PKC activity in H547A. These findings demonstrate that histidine547 on hDAT plays a crucial role in stabilizing basal DA transport and Tat-DAT interaction. This study provides mechanistic insights into identifying targets on DAT for Tat binding and improving DAT-mediated dysfunction of DA transmission

Mutational Effects of Human Dopamine Transporter at Tyrosine88, Lysine92, and Histidine547 on Basal and HIV-1 Tat-Inhibited Dopamine Transport

Dysregulation of dopaminergic system induced by HIV-1 Tat protein-mediated direct inhibition of the dopamine transporter (DAT) has been implicated as a mediating factor of HIV-1 associated neurocognitive disorders. We have reported that single point mutations on human DAT (hDAT) at tyrosine88 (Y88F), lysine92 (K92M), and histidine547 (H547A) differentially regulate basal dopamine uptake but diminish Tat-induced inhibition of dopamine uptake by changing dopamine transport process. This study evaluated the effects of double (Y88F/H547A) and triple (Y88F/K92M/H547A) mutations on basal dopamine uptake, Tat-induced inhibition of DAT function, and dynamic transport process. Compared to wild-type hDAT, the Vmax values of [3H]Dopamine uptake were increased by 96% in Y88F/H547A but decreased by 97% in Y88F/K92M/H547A. [3H]WIN35,428 binding sites were not altered in Y88F/H547A but decreased in Y88F/K92M/H547A. Y88F/H547A mutant attenuated Tat-induced inhibition of dopamine uptake observed in wild-type hDAT. Y88F/H547A displayed an attenuation of zinc-augmented [3H]WIN35,428 binding, increased basal dopamine efflux, and reduced amphetamine-induced dopamine efflux, indicating this mutant alters transporter conformational transitions. These findings further demonstrate that both tyrosine88 and histidine547 on hDAT play a key role in stabilizing basal dopamine transport and Tat-DAT integration. This study provides mechanistic insights into developing small molecules to block multiple sites in DAT for Tat binding