Basolateral amygdala and stress-induced hyperexcitability affect motivated behaviors and addiction.

The amygdala integrates and processes incoming information pertinent to reward and to emotions such as fear and anxiety that promote survival by warning of potential danger. Basolateral amygdala (BLA) communicates bi-directionally with brain regions affecting cognition, motivation and stress responses including prefrontal cortex, hippocampus, nucleus accumbens and hindbrain regions that trigger norepinephrine-mediated stress responses. Disruption of intrinsic amygdala and BLA regulatory neurocircuits is often caused by dysfunctional neuroplasticity frequently due to molecular alterations in local GABAergic circuits and principal glutamatergic output neurons. Changes in local regulation of BLA excitability underlie behavioral disturbances characteristic of disorders including post-traumatic stress syndrome (PTSD), autism, attention-deficit hyperactivity disorder (ADHD) and stress-induced relapse to drug use. In this Review, we discuss molecular mechanisms and neural circuits that regulate physiological and stress-induced dysfunction of BLA/amygdala and its principal output neurons. We consider effects of stress on motivated behaviors that depend on BLA; these include drug taking and drug seeking, with emphasis on nicotine-dependent behaviors. Throughout, we take a translational approach by integrating decades of addiction research on animal models and human trials. We show that changes in BLA function identified in animal addiction models illuminate human brain imaging and behavioral studies by more precisely delineating BLA mechanisms. In summary, BLA is required to promote responding for natural reward and respond to second-order drug-conditioned cues; reinstate cue-dependent drug seeking; express stress-enhanced reacquisition of nicotine intake; and drive anxiety and fear. Converging evidence indicates that chronic stress causes BLA principal output neurons to become hyperexcitable.

Gene expression in accumbens GABA neurons from inbred rats with different drug-taking behavior.

Inbred Lewis and Fisher 344 rat strains differ greatly in drug self-administration; Lewis rats operantly self-administer drugs of abuse including nicotine, whereas Fisher self-administer poorly. As shown herein, operant food self-administration is similar. On the basis of their pivotal role in drug reward, we hypothesized that differences in basal gene expression in GABAergic neurons projecting from nucleus accumbens (NAcc) to ventral pallidum (VP) play a role in vulnerability to drug-taking behavior. The transcriptomes of NAcc shell-VP GABAergic neurons from these two strains were analyzed in adolescents, using a multidisciplinary approach that combined stereotaxic ionotophoretic brain microinjections, laser-capture microdissection (LCM) and microarray measurement of transcripts. Laser-capture microdissection enriched the gene transcripts detected in gamma-aminobutyric acid (GABA) neurons compared to the residual NAcc tissue: a ratio of neuron/residual >1 and false discovery rate (FDR) <5% yielded 6623 transcripts, whereas a ratio of >3 yielded 3514. Strain-dependent differences in gene expression within GABA neurons were identified; 322 vs. 60 transcripts showed 1.5-fold vs. 2-fold differences in expression (FDR < 5%). Classification by gene ontology showed that these 322 transcripts were widely distributed, without categorical enrichment. This is most consistent with a global change in GABA neuron function. Literature mining by Chilibot found 38 genes related to synaptic plasticity, signaling and gene transcription, all of which determine drug abuse; 33 genes have no known association with addiction or nicotine. In Lewis rats, upregulation of Mint-1, Cask, CamkII , Ncam1, Vsnl1, Hpcal1 and Car8 indicates that these transcripts likely contribute to altered signaling and synaptic function in NAcc GABA projection neurons to VP.

Phosphorylation of activating transcription factor in murine splenocytes through delta opioid receptors.

Delta opioid receptors (DORs) modulate TCR signaling through the mitogen-activated protein kinases (MAPKs), ERKs 1 and 2. These studies determined whether a DOR agonist alone ([D-Ala(2)-D-Leu(5)]enkephalin; DADLE) affects phosphorylation of the activating transcription factor (ATF-2) and its interaction with the MAPK, c-Jun NH(2)-terminal kinase (JNK). DOR expression was induced on murine splenocytes by anti-CD3 and then quiescent cells were treated with DADLE. DADLE, itself, dose-dependently induced maximal phosphorylation of ATF-2 within 5-10min; naltrindole, a specific antagonist, abolished this. Anti-ATF-2 immunoprecipitates from control and DADLE-treated splenocytes showed a dominant 59kDa phosphorylated band and a 71kDa band. DADLE stimulated phosphorylation of both bands, although the 71kDa band was selectively immunoprecipitated by anti-JNK. Thus, DADLE stimulated phosphorylation of 71kDa ATF-2 and its association with JNK, suggesting that JNK is activated through DORs. Along with previous observations, these studies suggest that lymphocyte DORs can affect the activation of MAPKs by TCR-independent stimulation (e.g., JNK) or indirectly by modulating TCR-dependent stimulation (e.g., ERK).

Norepinephrine secretion in the hypothalamic paraventricular nucleus of rats during unlimited access to self-administered nicotine: An in vivo microdialysis study.

Norepinephrine (NE) secretion within the hypothalamic paraventricular nucleus (PVN) is pivotal to endocrine and behavioral responses. Activation of NE afferents to PVN also is necessary for the hypothalamo-pituitary-adrenal axis response to passively administered nicotine. The mode of drug delivery is a critical determinant of the dynamics of neurotransmitter secretion, yet the PVN NE response to nicotine self-administration (SA) is unknown. Herein, rats housed in operant chambers had unlimited 23 hr access to self-administered nicotine. In vivo microdialysis of PVN NE was performed, collecting consecutive 7 min samples over 9 hr sessions during three phases of nicotine SA: acquisition (day 1); early maintenance, once stable rates of SA were achieved (day 9.2 +/- 0.6); later maintenance (day 18.6 +/- 0.8). On d1, nicotine animals had an increased percentage of SA episodes (SAEs) in which NE levels were elevated (80 vs 30% with saline; p < 0.01). By early maintenance, a fourfold increase in such episodes was observed in nicotine animals (p < 0.01), and the overall NE level was greater (1.30 +/- 0.24 vs 0.63 +/- 0.07 pg/10 microl in saline; p < 0.05); NE increased during the first, but not the last, SAE. The pattern was similar during later maintenance, although NE responsiveness declined (overall NE level, 0.96 +/- 0.19 in nicotine vs 0.52 +/- 0.08 pg/10 microl in saline; p < 0.05). Therefore, nicotine SAEs were associated with sustained increases in NE secretion during all three phases of SA. However, the reduced NE responsiveness observed both within the dialysis session in each phase and by later versus early maintenance is consistent with progression of partial daily desensitization of PVN NE secretion to nicotine SA. Therefore, in rats chronically self-administering nicotine, the drug stimulates sustained PVN NE secretion that may alter neuroendocrine and behavioral responses mediated by the PVN. Compared with studies of chronic human smokers, our nicotine SA model may reflect the CNS noradrenergic responses that occur during human cigarette smoking.

Immunofluorescence detection of delta opioid receptors (DOR) on human peripheral blood CD4+ T cells and DOR-dependent suppression of HIV-1 expression.

The delta opioid receptors (DORs) modulate T cell proliferation, IL-2 production, chemotaxis, and intracellular signaling. Moreover, in DOR-transfected Jurkat cells, delta opioids have been shown to suppress HIV-1 p24 Ag expression. These observations led us to characterize the expression of DORs by human peripheral blood T cells and to determine whether a specific DOR agonist, benzamide,4-([2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-methoxyphenyl)methyl]-N,-,(2S[1(S*),2alpha,5beta])-(9Cl) (SNC-80), can suppress p24 Ag expression by HIV-1-infected CD4+ T cells obtained from normal donors. By immunofluorescence flow cytometry, PHA stimulated the expression of DOR from 1.94 +/- 0.70 (mean +/- SEM) to 20.70 +/- 1.88% of the PBMC population by 48 h (p < 0.0001). DOR expression was approximately 40% of both the PHA-stimulated CD4+ and CD8+ T cell subsets, and virtually all DORs were found on these subsets. To determine whether activated DORs suppress HIV-1 expression, PBMC were prestimulated with PHA, and then CD4+ T cells were purified, pretreated with SNC-80, and infected with HIV-1. In a concentration-dependent manner, SNC-80 inhibited production of p24 Ag. SNC-80 10(-10) M maximally suppressed (approximately 50%) both lymphocytotropic (HIV-1 MN) and monocytotropic (SF162) strains; higher concentrations were less effective. Naltrindole, a selective DOR antagonist, abolished the inhibitory effects of SNC-80. Kinetic studies indicated that 24-h pre- or postincubation with SNC-80, relative to infection with HIV-1, eliminated its suppressive effects. Thus, stimulating the DORs expressed by activated CD4+ T cells significantly suppressed the expression of HIV-1. These findings suggest that opioid immunomodulation directed at host T cells may be adjunctive to standard antiviral approaches to HIV-1 infection.

Expression of delta opioid receptors by splenocytes from SEB-treated mice and effects on phosphorylation of MAP kinase.

Delta opioid receptors (DORs) are known to modulate multiple T-cell responses. However, little is known about the expression of these receptors. These studies evaluated the expression of DOR mRNA and protein after a single in vivo exposure to staphylococcal enterotoxin B (SEB). SEB (20 microg, ip) significantly enhanced splenocyte DOR mRNA expression 8 and 24 h after injection. SEB also increased the fractions of the total splenocyte (5 to 20%) and T-cell (8 to 50%) populations expressing DOR protein. In saline-treated animals, DOR relative fluorescence intensity per cell was 11.1 +/- 0.62 units (mean +/- SEM), increasing to 16.1 +/- 1.7 after exposure to SEB. DOR fluorescence intensity significantly increased to 33.5 +/- 2.0 units in a subpopulation of T-cells. Thus, SEB significantly increased DOR expression in vivo, affecting both mRNA and protein levels primarily within the T-cell population. To determine whether T-cell DORs modulate the activity of extracellular-regulated kinases (ERKs), the phosphorylation of ERKs 1 and 2 was studied using splenocytes from SEB-treated mice. At concentrations from 10(-8) to 10(-6) M, [d-Ala(2)-d-Leu(5)]-enkephalin, a selective DOR agonist, significantly inhibited anti-CD3-epsilon-induced phosphorylation of the ERKs. Therefore, the DORs expressed by activated T-cells are capable of attenuating T-cell activation that depends on ERK phosphorylation.

Local alpha-bungarotoxin-sensitive nicotinic receptors in the nucleus accumbens modulate nicotine-stimulated dopamine secretion in vivo.

Nicotinic cholinergic receptors in the ventral tegmental area are required for the accumbal dopamine response to systemic nicotine. In contrast, the role of nicotinic receptors located within the nucleus accumbens itself has not been clarified for systemically administered nicotine. In the present study, in vivo microdialysis of accumbal dopamine secretion and receptor antagonist blockade in both the ventral striatal nucleus accumbens and the midbrain ventral tegmental area were used to evaluate this question. The nicotinic receptor antagonists methyllycaconitine or mecamylamine were delivered through the accumbal dialysis probe, followed by 0.09mg/kg nicotine (i.v.). The alpha7 subunit antagonist methyllycaconitine inhibited 71% of the dopamine response (P<0.01), whereas mecamylamine was completely ineffective. In addition, the classical alpha7 subunit antagonist alpha-bungarotoxin infused into the nucleus accumbens adjacent to the microdialysis probe, significantly reduced dopamine release by 0.065 or 0.09mg/kg nicotine (i.v.; P<0. 05). Combined, these data indicate the involvement of alpha7 subunit-containing nicotinic receptors in the nucleus accumbens. In contrast, local infusion of mecamylamine into the ventral tegmental area effectively blocked nicotine-induced accumbal dopamine release. Simultaneous infusions of methyllycaconitine into the accumbens and mecamylamine into the ventral tegmental area induced greater blockade of nicotine-stimulated dopamine secretion than methyllycaconitine or mecamylamine alone. In conclusion, the present study demonstrates that different types of nicotinic cholinergic receptors, located in the ventral striatal nucleus accumbens (alpha-bungarotoxin sensitive and mecamylamine insensitive) and the midbrain ventral tegmental area (mecamylamine sensitive), may be required for the full effects of nicotine on the mesostriatal dopaminergic pathway. While activation of nicotinic cholinergic receptors in the ventral tegmentum is required for the accumbal dopamine response to systemic nicotine, accumbal nicotinic receptors themselves act as modulators of this response. This fine tuning of the dopamine reward pathway through alpha7 nicotinic cholinergic receptors in the nucleus accumbens may amplify the secretion of dopamine, allowing a subthreshold brain concentration of nicotine to become an effective stimulus for dopamine secretion.

Nicotine up-regulates expression of orexin and its receptors in rat brain.

Orexins are two recently discovered neuropeptides that can stimulate food intake. As the chronic use of tobacco typically leads to a reduction in body weight, it is of interest to determine whether nicotine, the major biologically active tobacco ingredient, has an effect on orexin metabolism in the brain. Using a semiquantitative RT-PCR technique, the levels of messenger RNA (mRNA) for prepro-orexin, orexin A (OX1-R) and orexin B (OX2-R) receptors were 20-50% higher in rats receiving nicotine for 14 days at the level of 2-4 mg/kg day compared with rats receiving saline solvent alone. In animals treated with nicotine at 4 mg/kg x day, the expression levels of mRNA for prepro-orexin, OX1-R, and OX2-R were significantly higher compared with those in either the free-feeding control or pair-fed saline control rats. RIA data indicated that both orexin A and orexin B peptide levels were significantly elevated (45-54%; P < 0.01) in the dorsomedial nucleus (DMH) of the nicotine-treated rats compared with either solvent-only or pair-fed controls. Additionally, orexin B was significantly elevated (83%; P < 0.01), over levels in both types of the control animals, in the paraventricular nucleus (PVN) region. In summary, we demonstrated that an inverse association between nicotine and food intake as well as body weight held with doses comparable to those consumed by average human smokers. Moreover, our data indicated that chronic exposure to nicotine can induce a long-term increase in the expression levels of prepro-orexin and their receptor mRNA in the rat hypothalamus and in the levels of orexin A in the DMH and orexin B in the DMH and PVN among the six hypothalamic regions that we examined.

Systemic nicotine stimulates dopamine release in nucleus accumbens: re-evaluation of the role of N-methyl-D-aspartate receptors in the ventral tegmental area.

Systemic nicotine stimulates dopamine (DA) release in the nucleus accumbens (NAcc), and N-methyl-D-aspartate (NMDA) receptors in the ventral tegmental area (VTA) appear to be involved. However, it is not known whether the secretion of DA elicited by nicotine depends on the tonic and/or phasic activation of NMDA receptors by glutamate (Glu). To clarify this, in vivo microdialysis was conducted in freely moving, alert rats to measure DA and Glu overflows in the NAcc and Glu in the VTA. Nicotine (0.065, 0.09, or 0.135 mg/kg delivered i.v. at 0.09 mg/kg/60 s via a jugular cannula) dose dependently stimulated NAcc DA secretion (P <.05). However, 0.065 mg/kg nicotine failed to stimulate Glu release in the VTA, whereas higher doses of nicotine (> or =0.09 mg/kg) were effective (P <.05). Administering the competitive NMDA receptor antagonists, 2-amino-5-phosphonopentanoic acid (AP-5; 1 mM) or 0.2 mM cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS 19755) through the VTA probe, abolished NAcc DA release after 0.065 mg/kg nicotine (P <.01) and reduced the response to 0.09 mg/kg nicotine. Therefore, the NAcc DA response to a relatively low dose of nicotine depends on the tonic activation of NMDA receptors in the VTA. In contrast, infusing 1 mM 2-amino-5-phosphonopentanoic acid or 1 mM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist, into the NAcc through the microdialysis probe had no effect on NAcc DA secretion in response to 0.09 mg/kg nicotine. These findings, coupled with data showing that Glu secretion in the VTA was stimulated only by higher doses of nicotine, indicate that the phasic release of VTA Glu is involved in the NAcc DA response to higher doses of nicotine (> or =0.09 mg/kg).

Nicotine administration enhances NPY expression in the rat hypothalamus.

Epidemiological studies have shown an inverse relationship between cigarette smoking and body weight. In rodents, a negative correlation between nicotine and body weight has been reported, but this observation was largely derived from studies where relatively high doses of nicotine ( approximately 12 mg/kg/day) were used. In the current study, we showed that a negative relationship also holds for low doses of nicotine that are comparable to that consumed by average human smokers (<6 mg/kg/day). We also demonstrated that 14 days of nicotine administration (4 mg/kg/day) reduced average daily food intake by 19.5% (P<0.01) in the free-feeding nicotine-treated group compared to saline controls. No significant differences in body weight were detected between the nicotine-treated and pair-fed groups. To determine whether the effects of nicotine on food intake and body weight were related to neuropeptide Y (NPY) expression, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and radioimmunoassay were utilized to measure NPY mRNA and peptide levels in various regions of the hypothalamus. Significantly higher levels of NPY mRNA (ca. 20-50%) and peptide (ca. 24-69%) were only detected in the nicotine-treated groups. In addition, significantly higher NPY contents were also obtained in two hypothalamic areas of pair-fed control animals. In summary, our data suggest that the pharmacological effects of nicotine on food intake and body weight may be mediated by changes in hypothalamic NPY levels, a neuropeptide that is pivotal to the hypothalamic regulation of food intake.

Nicotine enhances the biosynthesis and secretion of transthyretin from the choroid plexus in rats: implications for beta-amyloid formation.

Epidemiological studies indicated that cigarette smoking protects against the development of several neurodegenerative disorders, including Alzheimer's disease (AD). However, the molecular mechanism(s) underlying this is poorly understood. To gain insight into these protective effects, we used differential display PCR (DD-PCR) to amplify RNA from various brain regions of rats self-administering (SA) nicotine compared with yoked-saline controls. We found that the transthyretin (TTR) gene, whose product has been shown to bind to amyloid beta (Abeta) protein and prevent Abeta aggregation, was more abundantly expressed ( approximately 1.5- to 2.0-fold) in the brainstem and hippocampus (areas containing choroid plexus) of nicotine SA rats. Subsequently, quantitative reverse transcription-PCR analysis confirmed these DD-PCR findings and demonstrated that nicotine increased TTR mRNA levels in these regions in a time- and dose-dependent manner. Significantly higher TTR protein concentrations were also detected in the ventricular CSF of nicotine-treated rats. In contrast, no differences either in plasma TTR or in CSF and plasma retinol-binding protein were detected. Immunohistochemical analysis showed that immunoreactive TTR was 41.5% lower in the choroid plexus of nicotine-treated rats compared with the saline controls. On the basis of these data, we speculate that the protective effects of nicotine on the development of AD may be attributable, in part, to the increased biosynthesis and secretion of TTR from the choroid plexus. These findings also point toward new approaches that may take advantage of the potentially novel therapeutic effects of nicotinic agonists in patients with AD.

Expression of delta opioid receptors and transcripts by splenic T cells.

Delta opioid receptors (DORs) and preproenkephalin-A-derived opiate peptides are expressed by mononuclear cells in various lymphoid organs. DOR ligands modulate a variety of immune functions, such as T-cell proliferation, calcium mobilization, and cytokine production. Recently, quiescent T cells were found to express low levels of DOR transcripts, which increased due to the following: cell culture of unstimulated murine splenocytes (depending on cell density); cross-linking the T-cell receptor (TCR) with anti-CD3-epsilon; and a single in vivo exposure to staphylococcal enterotoxin B (SEB). Enhanced expression of DOR mRNA was mediated transcriptionally. Moreover, PMA + ionomycin, which mimic the proliferative signal of anti-CD3, inhibited the expression of DOR mRNA. Using semiquantitative immunofluorescence to detect DORs, SEB was found to increase the fraction of T cells that expressed DOR and to enhance the relative level of DOR expression per T cell. Previous studies have shown that DOR agonists inhibited the anti-CD3-stimulated production of interleukin-2 and T-cell proliferation. Therefore, the enhanced expression of DORs by activated T cells may be capable of downregulating the T-cell activation program.

Inhibition of nicotine-induced hippocampal norepinephrine release in rats by alpha-conotoxins MII and AuIB microinjected into the locus coeruleus.

Hippocampal norepinephrine (NE) is secreted by neurons projecting from the locus coeruleus (LC) to the hippocampus; LC nicotinic receptors (NAchRs) are involved in the effects of systemic nicotine on this pathway. To clarify the NAchR subtypes, NAchR antagonists, termed alpha-conotoxins, were microinjected into the LC before nicotine; MII and AuIB were used to assess the potential involvement of alpha3beta2 and alpha3beta4 subunit-containing NAchRs, respectively. Nicotine dose-dependently stimulated hippocampal NE release (P < 0.01). MII (>0.25 pmol) reduced the NE response to nicotine (67% decrease; P < 0.05), as did AuIB (44% reduction by 25 pmol; P < 0.05). Administered together, however, MII and AuIB were no more effective than MII. Thus, MII and AuIB are capable of interacting with NAchR subtypes other than those previously defined as alpha3beta2 and alpha3beta4, respectively. NAchRs containing both beta2 and beta4-subunits may be involved.

Delta opioid receptors expressed by stably transfected jurkat cells signal through the map kinase pathway in a ras-independent manner.

Delta opioid receptors (DOR) are G-protein coupled 7-transmembrane receptors (GPCR), expressed by thymic and splenic T cells, that modulate interleukin (IL)-2 production and proliferation in response to concanavalin A or crosslinking the TCR. Mitogen-activated protein kinases (MAPKs) are involved in mediating intracellular responses to TCR crosslinking. In addition, MAPKs can be activated by signaling cascades that are initiated by the release of G-proteins from GPCRs. To determine whether DORs expressed by T cells signal through the MAPKs, extracellular-regulated kinases (ERKs) 1 and 2, two delta opioid peptides, deltorphin and [D-Ala2,D-Leu5]-enkephalin (DADLE), were studied in Jurkat cells that had been stably transfected with DOR (DOR-Ju.1). These peptides rapidly and dose-dependently induced ERK phosphorylation; pretreatment with naltrindole (NTI), a selective DOR antagonist, abolished this. Pertussis toxin (PTX) also inhibited phosphorylation, indicating the involvement of the Gi/o proteins. Herbimycin A, a protein tyrosine kinase (PTK) inhibitor, reduced the DADLE-induced ERK phosphorylation by 68%. ERK phosphorylation was inhibited by Bisindolylmaleimide 1 (GF109203X), an inhibitor of PKC, and by pretreatment with PMA prior to DADLE. A GTP/GDP exchange assay was used to assess the potential role of Ras in the pathway leading to ERK phosphorylation; DADLE failed to stimulate GTP/GDP exchange in comparison to PMA. Additional studies showed that DADLE stimulated an increase in cfos mRNA; this was reduced by the inhibitor of MAPK/ERK kinase (MEK), PD98059. Therefore, in DOR-Ju.1 cells, DOR agonists stimulate ERK phosphorylation in a Ras independent and PKC-dependent manner; PTKs appear to be involved. MAPKs mediate the increase in cfos mRNA induced by DOR agonists.

Regulation of delta opioid receptor expression by anti-CD3-epsilon, PMA, and ionomycin in murine splenocytes and T cells.

Previous studies have shown that low levels of delta opioid receptor (DOR) mRNA were detectable by reverse transcription polymerase chain reaction (RT-PCR) in unstimulated splenocytes from BALB/c female mice. This study demonstrates that DOR transcripts can be detected in freshly obtained splenocytes froin CD 1 female mice as well. The results of studies using quantitative competitive RT-PCR showed that DOR transcripts in splenic T cells increased from < 1 copy/cell to 22 and 42 copies/cell, respectively, after stimulation with anti-CD3-epsilon for 24 and 48 h compared to the level in freshly obtained T cells. In the presence of actinomycin D, anti-CD3-epsilon did not affect the rate of degradation of DOR mRNA, suggesting that its stability is not altered by anti-CD3-epsilon. After incubation with phorbol myristate acetate (PMA) and ionomycin, the expression of DOR mRNA in splenocytes and T cells was significantly reduced compared with unstimulated cells in culture. In addition, the inhibitory effect of PMA prevented anti-CD3-epsilon-stimulated DOR expression. These data suggest that signaling through the T cell receptor complex by anti-CD3-epsilon regulates DOR expression differently than PMA and ionomycin.

Local alpha-bungarotoxin-sensitive nicotinic receptors modulate hippocampal norepinephrine release by systemic nicotine.

Previous studies have shown that nicotinic receptors (NAChRs) accessible from the cerebral aqueduct of the brainstem mediate the hippocampal norepinephrine (NE) release induced by i.v. nicotine. The present study was designed to investigate the role of hippocampal NAChRs in this process. Nicotinic antagonists were microinjected or microdialyzed into the hippocampus (HP) before administering nicotine (0.09 mg/kg over 60 s, i.v.) to freely moving rats. alpha-Bungarotoxin (0.3 nmol by microinjection) blocked nicotine-induced hippocampal NE release by 47% (p <.05) and abolished the effect of 0.065 mg/kg nicotine. Methyllycaconitine (1.4-5.6 mM in the dialysate) inhibited the stimulatory effect of nicotine 0.09 mg/kg by 48 to 75% (p <.05). In contrast, mecamylamine (2.9-5.8 mM) and dihydro-beta-erythroidine (7-14 mM) were completely ineffective. The role of hippocampal NAChRs was demonstrated further by selectively desensitizing these receptors before the systemic infusion of nicotine. To do so, the HP was pretreated with nicotine (0.1 mM) delivered through the microdialysis probe; this concentration was calculated to yield tissue concentrations similar to those produced by the systemic infusions of nicotine. Dialyzing this concentration of nicotine into the HP inhibited the NE response to i.v. nicotine by 34% (p <.05), and 1.0 mM nicotine reduced the response by 40%. These studies indicate that alpha-bungarotoxin-sensitive hippocampal NAChRs, probably containing alpha7 subunits, modulate hippocampal NE release because of systemic nicotine.

Delta-opioid suppression of human immunodeficiency virus-1 expression in T cells (Jurkat).

Delta-opioid receptor (DOR) transcripts and binding sites are expressed by lymphocytes and lymphoid cell lines from several species. Direct modulation of lymphocyte function through DORs affects T cell proliferation, interleukin-2 production, chemotaxis, and intracellular signaling. Moreover, in human DOR-transfected T cells (DOR-Ju.1), delta-opioids have been shown previously to mobilize intracellular calcium rapidly, to inhibit forskolin-stimulated cyclic AMP production, and to activate the mitogen-activated protein kinases ERKs 1 and 2. These observations led us to consider whether delta agonists modify T cell functions, thus affecting the expression of human immunodeficiency virus-1 (HIV-1) by CD4+ T cells. To test this hypothesis, DOR-Ju.1 cells, derived from Jurkat cells stably transfected with a cDNA encoding the neuronal DOR, were stimulated with deltorphin or benzamide, 4-[[2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-methoxyphenyl)methyl]N- ,[2S[(S*),2alpha,5beta]]-(9Cl) (SNC-80) prior to the addition of HIV-1. Both deltorphin and SNC-80 concentration-dependently inhibited the production of p24 antigen, an index of HIV-1 expression. Inhibition was maximal with 10(-13)-10(-9) M SNC-80 (>60% reduction) or 10(-15)-10(-11) M deltorphin (>50% reduction). At higher concentrations, less inhibition of p24 antigen production was found. Naltrindole (NTI, 10(-11) M), a selective DOR antagonist, abolished the inhibitory effects of 10(-9) M SNC-80, whereas 10(-13) M NTI partially reversed the effect of SNC-80. Thus, activation of DORs expressed by CD4+ T cells significantly (P < 0.05) reduced the expression of HIV-1 by these cells. These findings suggest that opioid immunomodulation directed at host T cells may be adjunctive to standard antiviral approaches to HIV-1 infection.

The delta1-opioid receptor antagonist, 7-(benzospiroindanyl)naltrexone [correction of 7-benzylspiroindanylnaltrexone], prolongs renal allograft survival in a rat model.

In this study we demonstrate allograft survival in a rat model of renal transplantation using the delta1-opioid receptor antagonist, 7-(benzospiroindanyl)naltrexone [corrected]. Treatment with 7-(benzospiroindanyl)naltrexone [corrected] caused 50% of the rats to survive longer than 100 days (untreated, 11 +/- 3 days). Naltrindole, a delta-opioid receptor antagonist without subtype selectivity, also promoted graft survival but was substantially less effective, suggesting that antagonism at delta1-opioid receptors is involved in allograft survival.