Ultrastructural evidence that androgen receptors are located at extranuclear sites in the rat hippocampal formation.

Like estrogens in female rats, androgens can affect dendritic spine density in the CA1 subfield of the male rat hippocampus [J Neurosci 23:1588 (2003)]. Previous light microscopic studies have shown that androgen receptors (ARs) are present in the nuclei of CA1 pyramidal cells. However, androgens may also exert their effects through rapid non-genomic mechanisms, possibly by binding to membranes. Thus, to investigate whether ARs are at potential extranuclear sites of ARs, antibodies to ARs were localized by light and electron microscopy in the male rat hippocampal formation. By light microscopy, AR immunoreactivity (-ir) was found in CA1 pyramidal cell nuclei and in disperse, punctate processes that were most dense in the pyramidal cell layer. Additionally, diffuse AR-ir was found in the mossy fiber pathway. Ultrastructural analysis revealed AR-ir at several extranuclear sites in all hippocampal subregions. AR-ir was found in dendritic spines, many arising from pyramidal and granule cell dendrites. AR-ir was associated with clusters of small, synaptic vesicles within preterminal axons and axon terminals. Labeled preterminal axons were most prominent in stratum lucidum of the CA3 region. AR-containing terminals formed asymmetric synapses or did not form synaptic junctions in the plane of section analyzed. AR-ir also was detected in astrocytic profiles, many of which apposed terminals synapsing on unlabeled dendritic spines or formed gap junctions with other AR-labeled or unlabeled astrocytes. Collectively, these results suggest that ARs may serve as both a genomic and non-genomic transducer of androgen action in the hippocampal formation.

Dentate hilar mossy cells and somatostatin-containing neurons are immunoreactive for the alpha8 integrin subunit: characterization in normal and kainic acid-treated rats.

Integrins are heterodimeric cell surface receptors composed of different alpha and beta subunits that mediate cell-cell and cell-extracellular matrix interactions. They have been implicated in the regulation of neuronal migration, differentiation, process outgrowth, and plasticity. The alpha8 integrin subunit associates exclusively with the beta1 subunit to form a receptor (alpha8beta1) for fibronectin, vitronectin, tenascin, and osteopontin. In a previous study, we demonstrated that hippocampal dentate hilar neurons are immunoreactive for alpha8. The present study identifies the major types of alpha8-immunoreactive hilar neurons and characterizes the effects of kainic acid-induced seizures on alpha8-immunoreactivity in these cells. Examination of the hilus in normal rats revealed alpha8-immunoreactivity in the somatodendritic compartments of large hilar neurons identified as mossy cells, including a subset of dendritic thorny excrescences that were contacted by large mossy fiber terminals. alpha8-immunoreactivity also was found in approximately 71% of somatostatin-containing hilar cells. Kainic acid-induced seizures dramatically and rapidly altered the levels and distribution of alpha8-immunoreactivity in hilar neurons. After 1.5 h of seizures, alpha8-immunoreactivity in their dendrites was reduced greatly. One day after kainic acid treatment, labeling was diminished throughout the somatodendritic compartments of most hilar cells. This decrease appeared to be transient, since alpha8 labeling returned to normal levels in surviving hilar neurons within 2 weeks of treatment. In addition, many alpha8-immunoreactive hilar neurons, particularly in caudal dentate regions, were lost 3-5 weeks after kainic acid treatment. Our findings suggest that alpha8beta1 may mediate adhesive interactions of the dendritic processes of mossy cells and somatostatin-containing hilar neurons with other cellular elements or with extracellular matrix components. They also suggest that alpha8 may be susceptible to activity-dependent proteolysis that could modulate its function in the somatodendritic compartment of these cells.

Antinociceptive and behavioral activation responses elicited by d-Pro(2)-endomorphin-2 in the ventrolateral periaqueductal gray are sensitive to sex and gonadectomy differences in rats.

Sex differences have been observed in antinociception after morphine administered into either the lateral ventricles, rostral ventromedial medulla, or ventrolateral periaqueductal gray such that male rats exhibit significantly greater antinociception than female rats. Adult gonadectomy produced small, but significant changes in morphine antinociception relative to same-sex sham-operated controls. The present study examined whether sex and adult gonadectomy differences were observed in antinociceptive responses after D-Pro(2)-Endomorphin-2 (1-50 microg) elicited from the ventrolateral periaqueductal gray (vlPAG) on the tail-flick and jump tests in rats, and compared these effects with morphine antinociception. D-Pro(2)-Endomorphin-2 antinociception in the vlPAG was significantly greater in estrous-phase, sham-operated and ovariectomized female rats relative to sham-operated and castrated male rats on the tail-flick, but not jump test that differed markedly from the greater magnitude of morphine antinociception noted for male rats on both tests. In testing whether D-Pro(2)-Endomorphin-2's antinociceptive sex differences were secondary to alterations in activity, similar decreases in the pattern of total activity were observed after D-Pro(2)-Endomorphin-2 in the vlPAG in male and female rats. In evaluating whether male and female rats differed in their behavioral activation responses after D-Pro(2)-Endomorphin-2 in the vlPAG, significantly more excessive grooming, seizures, barrel rolls and explosive running behaviors were observed after D-Pro(2)-Endomorphin-2 in male, but not female rats during the precise periods of time when they were failing to display robust antinociceptive responses on the tail-flick test. Thus, the different patterns of sex differences after D-Pro(2)-Endomorphin-2 in the vlPAG appear to be attributable to sex-dependent alterations in behavioral activation rather than nociceptive processing per se.

Actions of NMDA and cholinergic receptor antagonists in the rostral ventromedial medulla upon beta-endorphin analgesia elicited from the ventrolateral periaqueductal gray.

Analgesia elicited by morphine in the ventrolateral periaqueductal gray is mediated in part by NMDA and cholinergic receptors in the rostral ventromedial medulla because selective receptor antagonists applied to the latter structure reduced morphine analgesia elicited from the former structure. Previous studies have demonstrated that morphine and beta-endorphin employ different anatomical and neurochemical pathways in exerting their supraspinal analgesic effects. The present study evaluated whether pretreatment with either competitive (AP7, 3-10 microg) or non-competitive (MK-801, 3-10 microg) NMDA antagonists, or muscarinic (scopolamine, 5 microg) or nicotinic (mecamylamine, 1 microg) cholinergic antagonists administered into the rostral ventromedial medulla altered beta-endorphin (15 microg) analgesia elicited from the ventrolateral periaqueductal gray as measured by the tail-flick and jump tests in rats. Whereas AP7 produced minimal (11%) and transient (30 min) reductions in beta-endorphin analgesia on the jump test, MK-801 produced minimal (9%) and transient (30 min) reductions in beta-endorphin analgesia on the tail-flick test. Whereas mecamylamine failed to reduce beta-endorphin analgesia on either measure, scopolamine produced small (23%) and transient (30 min) reductions in beta-endorphin analgesia on the tail-flick test. Each of these antagonists administered into the rostral ventromedial medulla at comparable or lower doses virtually eliminated morphine analgesia elicited from the ventrolateral periaqueductal gray. The opioid mediation of beta-endorphin analgesia in the ventrolateral periaqueductal gray was confirmed by its sensitivity to naltrexone (1-20 microg) pretreatment into the same structure. These data provide further evidence for dissociations between the descending neuroanatomical and neurochemical circuitry mediating the supraspinal analgesic responses induced by morphine and beta-endorphin, and indicate that the latter response is mediated by either non-cholinergic and non-NMDA synapses within the rostral ventromedial medulla, and/or by brainstem sites outside of the rostral ventromedial medulla.

Analysis of dopamine receptor antagonism upon feeding elicited by mu and delta opioid agonists in the shell region of the nucleus accumbens.

The nucleus accumbens (NAcc) has been implicated as an important reward site for the mediation of unconditioned reinforcers such as food. Although both mu-selective and delta-selective opioid agonists in the NAcc induce spontaneous and palatable feeding, these effects are mediated by multiple opioid receptor subtypes within the nucleus. A role for dopaminergic mediation of feeding in the NAcc is based upon selective antagonist-induced suppression of feeding induced by systemic amphetamine. The present study investigated whether feeding elicited by infusion of either mu ([D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin) or delta(2) ([D-Ala(2), Glu(4)]-deltorphin) opioid receptor subtype agonists in the shell region of the NAcc would be modified by intra-accumbens pretreatment with equimolar (12-100 nmol) doses of either D(1)-selective (SCH23390) or D(2)-selective (raclopride) antagonists. Both opioid agonists displayed comparable magnitudes and durations of feeding responses in the NAcc. SCH23390 significantly and dose-dependently reduced mu agonist-induced feeding in the NAcc with significant reductions noted following the two higher, but not two lower doses. In contrast, raclopride pretreatment produced inconsistent effects upon mu agonist-induced feeding with limited actions across doses and test times. Further, neither SCH23390 nor raclopride pretreatment in the NAcc affected feeding elicited by the delta(2) opioid agonist. These data indicate that the role of dopamine receptors in mediating opioid-induced feeding within the shell region of the NAcc is both dependent upon the dopamine receptor subtype that was blocked (D(1) vs. D(2)) as well as the opioid receptor subtype which was being stimulated mu vs. delta(2)).

Mercaptoacetate induces feeding through central opioid-mediated mechanisms in rats.

The endogenous opioid system has been implicated in the mediation of food intake elicited by such regulatory challenges as glucoprivation induced by 2-deoxy-D-glucose (2DG) or food deprivation in rodents. Administration of the free fatty acid oxidation inhibitor, mercaptoacetate (MA), produces a potent short-term increase in feeding in rats, the mechanisms of which have been dissociated from that elicited by 2DG. The present study evaluated whether MA-induced feeding in rats was mediated by the endogenous opioid system through systemic administration of the general opioid antagonist, naltrexone, through central administration of either general, mu, mu(1), kappa(1) or delta opioid antagonists, and through central administration of antisense oligodeoxynucleotide (AS ODN) probes directed against specific exons of either the mu (MOR-1), kappa (KOR-1), kappa(3) (KOR-3/ORL-1) or delta (DOR-1) opioid receptor clones. MA-induced feeding was significantly and dose-dependently reduced by systemic naltrexone (0.005-5 mg/kg); these ingestive effects were quite selective since neither total, ambulatory nor stereotypic activity was affected by either MA itself or MA paired with naltrexone. MA-induced feeding was significantly reduced by central pretreatment with either naltrexone (0.1-20 microgram) or mu-selective (beta-funaltrexamine, 0.1-20 microgram), mu(1)-selective (naloxonazine, 1-20 microgram), kappa(1)-selective (nor-binaltorphamine, 0.1-20 microgram), or delta-selective (naltrindole, 1-20 microgram) opioid receptor antagonists. MA-induced feeding was significantly reduced by AS ODN probes directed against either exons 1, 2 or 3, but not exon 4 of the MOR-1 clone, exon 3, but not exons 1 or 2 of the KOR-1 clone, exons 1 or 2, but not exon 3 of the KOR-3/ORL-1 clone, and exon 1, but not exons 2 or 3 of the DOR-1 clone. These data are discussed in terms of opioid mediation of ingestive responses related to fat, and in terms of potential central sites of action at which lipoprivic ingestive responses might act.

gamma-Aminobutyric acid receptor subtype antagonists differentially alter opioid-induced feeding in the shell region of the nucleus accumbens in rats.

Food intake is significantly increased by administration of mu-selective opioid agonists into the nucleus accumbens, particularly its shell region. Pretreatment with either opioid (mu, delta(1), delta(2) or kappa(1)) or dopaminergic (D(1)) receptor antagonists in the nucleus accumbens shell reduce mu opioid agonist-induced feeding. Selective GABA(A) (muscimol) and GABA(B) (baclofen) agonists administered into the nucleus accumbens shell each stimulate feeding which is respectively and selectively blocked by GABA(A) (bicuculline) and GABA(B) (saclofen) antagonists. The present study investigated whether feeding elicited by the mu-selective opioid agonist, [D-Ala(2),NMe(4),Gly-ol(5)]-enkephalin in the nucleus accumbens shell was decreased by intra-accumbens pretreatment with an equimolar dose range of either GABA(A) or GABA(B) antagonists, and further, whether general opioid or selective GABA antagonists decreased feeding elicited by GABA(A) or GABA(B) agonists in the nucleus accumbens shell. Feeding elicited by the mu-selective opioid agonist was dose-dependently increased following intra-accumbens pretreatment with GABA(A) (bicuculline) antagonism; this enhancement was significantly blocked by pretreatment with general or mu-selective opioid antagonists. In contrast, mu opioid agonist-induced feeding elicited from the nucleus accumbens shell was dose-dependently decreased by GABA(B) (saclofen) antagonism. Neither bicuculline nor saclofen in the nucleus accumbens shell altered baseline food intake. Whereas muscimol-induced feeding elicited from the nucleus accumbens shell was reduced by bicuculline and naltrexone, but not saclofen pretreatment, baclofen-induced feeding elicited from the nucleus accumbens shell was reduced by saclofen, but not by bicuculline or naltrexone. These data indicate that GABA(A) and GABA(B) receptor subtype antagonists differentially affect feeding elicited by mu opioid receptor agonists within the nucleus accumbens shell in rats.

Excitatory amino acid receptor subtype agonists induce feeding in the nucleus accumbens shell in rats: opioid antagonist actions and interactions with mu-opioid agonists.

Administration of mu-opioid receptor subtype agonists into the nucleus accumbens shell elicits feeding which is dependent upon the normal function of mu-, delta- and kappa-opioid receptors, D(1) dopamine receptors and GABA(B) receptors in the nucleus accumbens shell for its full expression. Whereas the AMPA antagonist, DNQX administered into the nucleus accumbens shell elicits a transient, though intense feeding response, feeding is elicited by excitatory amino acid agonists administered into the lateral hypothalamus. The present study examined whether excitatory amino acid agonists elicited feeding following administration into the nucleus accumbens shell of rats, whether such feeding responses were altered by opioid antagonist pretreatment, and whether such feeding responses interacted with feeding elicited by mu-opioid agonists. Both AMPA (0.25-0.5 microg) and NMDA (1 microg) in the nucleus accumbens shell significantly and dose-dependently increased food intake over 4 h. Both feeding responses were blocked by naltrexone pretreatment in the nucleus accumbens shell. The mu-opioid agonist, [D-Ala(2),NMe-Phe(4),Gly-ol(5)]-enkephalin in the nucleus accumbens shell significantly increased food intake which was significantly enhanced by AMPA cotreatment. This enhanced feeding response was in turn blocked by pretreatment with either general or mu-selective opioid antagonists. In contrast, cotreatment of NMDA and the mu-opioid agonist in the nucleus accumbens shell elicited feeding which was significantly less than that elicited by either treatment alone. These data indicate the presence of important interactions between excitatory amino acid receptors and mu-opioid receptors in the nucleus accumbens shell in mediating feeding responses in nondeprived, ad libitum-fed rats.