Characterization of membrane-associated substrates of Ca2+-dependent kinases in astrocytes.

Membrane-associated kinase substrates are likely transducers of extracellular signals elicited by neuroimmunomodulators and other signaling molecules. Whereas specific signal transduction pathways in astrocytes are being defined, the global view is lacking. We, therefore, characterized membrane-associated substrates of Ca2+-dependent kinases in primary astrocytes using 2-dimensional gel electrophoresis. Ten proteins were phosphorylated in vitro and characterized with respect to their relative molecular mass (in the range 10 kDa - 100 kDa), isoelectric point (range 4.2 - 9.0) and four conditions of phosphorylation. They varied broadly in their requirements for phosphorylation displaying distinct kinase preferences. Eight phosphoproteins were substrates of protease kinase C. Judging by abundance and intensity of phosphorylation, the principal PKC substrates were three acidic proteins associated with the plasma membrane. These results suggest that a relatively small number of membrane-associated proteins serve as transducers of signals mediated by Ca2+-dependent kinases and most of them are PKC substrates in astrocytes.

Identification of myristoylated alanine-rich C kinase substrate (MARCKS) in astrocytes.

We have characterized membrane-associated substrates of Ca2+-dependent kinases in primary rat astrocytes by in vitro phosphorylation, 2-dimensional gel electrophoresis and autoradiography. The most prominent among these were three acidic, protein kinase C (PKC) substrates. These are important because they likely transduce cytokine and other neuro-immune modulatory signals mediated by PKC. We now show that one of these phosphoproteins is myristoylated alanine-rich PKC kinase substrate (MARCKS) or phosphomyristin C. The identity was corroborated by one- and 2- dimensional immunoblotting with an MARCKS-specific polyclonal antibody. Exposing primary astrocytes to phorbol 12-myristate 13-acetate stimulated phosphorylation of this protein. The level of MARCKS appeared inversely proportional to the proliferative potential of astrocytes because it was lower in spontaneously transformed as compared to passaged or confluent cells. These data are consistent with previous reports and indicate that one of three major acidic membrane-associated PKC substrates in astrocytes is MARCKS. Thus, MARCKS is likely near-proximal transducer of PKC-mediated signals in astrocytes.

Selective remodeling of rabbit frontal cortex: relationship between 5-HT2A receptor density and associative learning.

RATIONALE: Associative learning during classical trace eyeblink conditioning has been shown to be regulated by serotonin 5-HT(2A )receptors and to be critically dependent on the integrity of frontal cortex. Chronic administration of 5-HT(2A) ligands has been shown to produce a selective up- or down-regulation of 5-HT(2A) receptors in frontal cortex. OBJECTIVES: We examined whether alterations in 5-HT(2A) receptor density had a functional significance with respect to associative learning. METHODS: Animals received chronic injections of LSD, BOL or MDL11,939 and 1 day later began classical trace conditioning of the eyeblink response. RESULTS: The density of 5-HT(2A) receptors in frontal cortex was significantly increased at 1-4 days after the cessation of chronic injections of the selective 5-HT(2A) receptor ligand MDL11,939. Rabbits demonstrated an enhancement of associative learning when training began at 1 day after cessation of chronic MDL11,939 injections, but acquired at the same rate as controls when training began at 8 days after cessation of injections, a time when receptor density had returned to control levels. Animals that began training 1 day after chronic injections of BOL or LSD, drugs that produce decreases in 5-HT(2A) receptor density, demonstrated normal rates of acquisition. CONCLUSIONS: These results indicate that increases in the density of 5-HT(2A) receptors in frontal cortex are associated with increases in the rate of associative learning, and further support an important role for this receptor in cortical circuitry that mediates learning. More generally, these results suggest an approach for functional remodeling of brain regions in the adult animal.

Serotonin 5-HT2A receptors in the CA1 field of the hippocampus mediate head movements in the rabbit.

RATIONALE: Motor movements (head bobs) in the rabbit have been shown to be elicited by LSD-like hallucinogenic drugs through actions at central serotonin 5-HT(2A) receptors, though their central locus remains unknown. Serotonergic innervation of the hippocampus has been suggested to play an important role in motor programming including movements of the head. OBJECTIVES: We examined whether intrahippocampal injections of a 5-HT(2A) receptor agonist would elicit head bobs and whether elicitation of head bobs would be modified by increases in hippocampal 5-HT(2A) receptor density. METHODS: Animals received bilateral injections of DOI or its vehicle into the dorsal hippocampus either before or after chronic administration of MDL 11,939 or its vehicle. The number of head bobs was counted continuously for 60 min and reported in blocks of 10 min and this was compared with the density of 5-HT(2A) receptors in dorsal hippocampus. RESULTS: Infusion of DOI into the CA1 region of the dorsal hippocampus elicited head bobs that were blocked by prior intrahippocampal injection of the 5-HT(2A) receptor antagonist ketanserin. Receptor autoradiography revealed that chronic administration of MDL 11,939 produced a 2.5-fold up-regulation of 5-HT(2A) receptors in the CA1 field and dentate gyrus of the hippocampus. This 5-HT(2A) receptor up-regulation was associated with a nearly 2-fold increase in head bobs elicited by infusion of DOI into the CA1 field. CONCLUSIONS: These results indicate that 5-HT(2A) receptors located in the CA1 field of the hippocampus mediate a motor movement, head bobs, and that this mediation is functionally related to receptor density.

Cardiac norepinephrine release: modulation by ovariectomy and estrogen.

Previously, we have demonstrated that in contrast to male rats, female rats do not show an age-related reduction of depolarization-elicited norepinephrine (NE) release from cardiac synaptosomes (resealed nerve terminals). These results suggest that sex hormones such as estrogen may modulate NE release from cardiac synaptosomes prepared from female rats. The present study was designed to test the hypotheses that long-term estrogen depletion, resulting from ovariectomy, and estrogen replacement alters depolarization-elicited NE release from cardiac synaptosomes. Female F344 rats were divided into two groups, one of which underwent bilateral ovariectomy, whereas the other underwent a sham operation. Three ovariectomized subgroups received daily injections of conjugated equine estrogens, delta8,9-dehydroestrone or 17 alpha-dihydroequilenin. Another ovariectomized control subgroup and the sham-operated animals received daily injections of vehicle. After 90 or 270 days of treatment, the animals were sacrificed. Cardiac synaptosomes were prepared from each heart, incubated with [(3)H]-NE, and used to evaluate NE release capacity by exposure to 50 mM K(+). The effectiveness of the ovariectomy and the estrogenic actions of the test compounds was confirmed by evaluating vaginal smears, determining uterine weights, and measuring serum luteinizing hormone (LH) concentrations. Ovariectomy (after both 90 and 270 days) significantly increased depolarization-induced NE release compared with sham-operated rats. Treatment with all three estrogenic preparations reduced NE release in ovariectomized rats to values similar to those observed in sham-operated animals. Interestingly, NE release rates from rats treated with conjugated estrogens for 270 but not 90 days were significantly below that observed in age-matched sham animals. These results demonstrate that estrogen modulates depolarization-elicited NE release from cardiac nerve terminals. Such modulation may represent a protective action by estrogen at the cardiac synapse.

A 5-HT2C agonist elicits hyperactivity and oral dyskinesia with hypophagia in rabbits.

Serotonergic 5-HT2C and 5-HT1B receptors mediate inhibitory controls of eating. Questions have arisen about potential behavioral and neurological toxicity of drugs that stimulate the 2C site. We evaluated eating and other motor responses in male Dutch-belted rabbits after administration of m-chlorophenylpiperazine (mCPP). Studies conducted in vitro and in vivo assessed the pharmacological specificity of the ingestive actions of this agent. mCPP (0.15-10 micromol/kg sc) reduced consumption of chow and 20% sucrose solution with equal potencies (ED50 approximately equal 0.6 micromol/kg). In radioligand binding to rabbit cortex, mCPP displayed 15-fold higher affinity for 5-HT2C than for 5-HT1B receptors. The serotonin antagonist mesulergine (7000-fold selective for 5-HT2C) reversed the hypophagic action of mCPP, but the 5-HT1B/1D antagonist GR127,935 did not. GR127,935 (0.5 micromol/kg) did prevent hypophagia produced by the highly selective 5-HT1B/1D agonist GR46,611. Observational methods demonstrated that mCPP decreased the frequency of eating chow but increased other motor activities. When rabbits consumed sucrose, videoanalysis revealed that mCPP reduced total time licking and the duration of individual bouts, but not bout frequency or the actual rate of consumption. mCPP increased locomotor and other activities, and greatly increased vacuous oromotor stereotypies and tongue protrusions. Nonetheless, rabbits licked accurately at the spout for sucrose. When sucrose was infused intraorally through a cheek catheter, mCPP actually increased the peak amplitude and overall magnitude of jaw movements. We conclude that mCPP stimulates 5-HT2C receptors to reduce food intake in rabbits. This hypophagia involves disruption of appetitive components of eating and is accompanied by adverse motor actions. This profile raises questions about the use of the 5-HT2C receptor as a target for novel therapeutic agents for obesity.

A novel behavioral model that discriminates between 5-HT2A and 5-HT2C receptor activation.

2,5-Dimethoxy-4-iodoamphetamine (DOI), a serotonin (5-HT)2A/2C receptor agonist, elicits shaking behaviors in rodents, which have been reliably quantified as behavioral correlates of 5-HT2A receptor activation. Such studies are lacking in the rabbit. As part of our research examining the role of the 5-HT2 receptor in rabbits, we analyzed the behavioral effects of systemically administered DOI in rabbits. DOI (0.01-3 micromol/kg) or vehicle was injected, and two distinct behaviors, head bobs (vertical head movements) and body shakes (wet dog shakes), were counted for 90 min following the injection. DOI dose-dependently increased the number of head bobs and body shakes. The selective 5-HT2A receptor antagonist ketanserin (1-3 micromol/kg), 1 h before DOI (0.3 micromol/kg) challenge, significantly attenuated head bobs, but not body shakes. In contrast, the selective 5-HT2C receptor antagonists SDZ SER 082 (1-3 micromol/kg) and SB 206553 (1 micromol/kg) 30 min before challenge, significantly reduced body shakes but not head bobs produced by the same dose of DOI. This study establishes that, in rabbits, DOI mediates head bobs via 5-HT2A receptors and body shakes via 5-HT2C receptors. Thus, the rabbit provides a novel behavioral assay that discriminates between 5-HT2A and 5-HT2C receptor activation.

Role of central 5-HT2 receptors in mediating head bobs and body shakes in the rabbit.

Systemic administration of the 5-HT(2A/2C) agonist DOI [(1(2,5-dimethoxy-4-iodophenyl)-2-aminopropane)hydrochloride] in rabbits elicits head bobs and body shakes, which are mediated by 5-HT(2A) and 5-HT(2C) receptors, respectively. This study was designed to determine whether the receptors mediating these behaviors are primarily located in the brain or in the periphery. Systemic administration of the peripheral 5-HT(2A/2C) antagonist xylamidine 30 min before systemic DOI challenge attenuated DOI-elicited body shakes by 50% without an effect on head bobs, suggesting a central origin for head bobs and a partial peripheral and a partial central origin for body shakes. Central administration of DOI into the lateral ventricle (ICV) elicited head bobs but not body shakes, demonstrating that the receptors mediating head bobs are centrally located. Pretreatment with ICV xylamidine blocked head bobs elicited by ICV DOI, indicating that the lack of inhibition, when systemically administered, is due to xylamidine's failure to reach central receptors. ICV pretreatment with the 5-HT(2A) receptor antagonist ketanserin inhibited ICV DOI-elicited head bobs establishing that 5-HT(2A) receptors activation elicits head bobs. In conclusion, 5-HT(2A) receptors mediating head movements are located in the brain whereas 5-HT(2C) receptors mediating the body movements appear to be located at different central sites as well as in the periphery.

Phospholipase C mediates (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-, but not lysergic acid diethylamide (LSD)-elicited head bobs in rabbit medial prefrontal cortex.

The phenethylamine and indoleamine classes of hallucinogens demonstrate distinct pharmacological properties, although they share a serotonin(2A) (5-HT(2A)) receptor mechanism of action (MOA). The 5-HT(2A) receptor signals through phosphatidylinositol (PI) hydrolysis, which is initiated upon activation of phospholipase C (PLC). The role of PI hydrolysis in the effects of hallucinogens remains unclear. In order to better understand the role of PI hydrolysis in the MOA of hallucinogens, the PLC inhibitor, 1-[6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), was used to study the effects of two hallucinogens, the phenethylamine, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and the indoleamine, lysergic acid diethylamide (LSD). PI hydrolysis was quantified through release of [3H]inositol-4-phosphate from living rabbit frontocortical tissue prisms. Head bobs were counted after hallucinogens were infused into the medial prefrontal cortex (mPFC) of rabbits. Both DOI and LSD stimulated PI hydrolysis in frontocortical tissue through activation of PLC. DOI-stimulated PI hydrolysis was blocked by 5-HT(2A/2C) receptor antagonist, ketanserin, whereas the LSD signal was blocked by 5-HT(2B/2C) receptor antagonist, SB206553. When infused into the mPFC, both DOI- and LSD-elicited head bobs. Pretreatment with U73122 blocked DOI-, but not LSD-elicited head bobs. The two hallucinogens investigated were distinct in their activation of the PI hydrolysis signaling pathway. The serotonergic receptors involved with DOI and LSD signals in frontocortical tissue were different. Furthermore, PLC activation in mPFC was necessary for DOI-elicited head bobs, whereas LSD-elicited head bobs were independent of this pathway. These novel findings urge closer investigation into the intracellular mechanism of action of these unique compounds.

The stereoisomer (+)-naloxone potentiates G-protein coupling and feeding associated with stimulation of mu opioid receptors in the parabrachial nucleus.

Classically, opioids produce their effects by activating Gi-proteins that inhibit adenylate cyclase activity. Previous studies proposed that mu-opioid receptors can also stimulate adenylate cyclase due to an initial transient coupling to Gs-proteins. Treatment with ultra-low doses of the nonselective opioid antagonist (-)-naloxone or its inactive enantiomer (+)-naloxone blocks this excitatory effect and enhances Gi-coupling. Previously we reported that infusion of the mu-opioid receptor agonist [D-Ala2, N-Me-Phe4, Glycinol5]-Enkephalin (DAMGO) into the mu-opioid receptor expressing lateral parabrachial nucleus increases feeding. Pretreatment with (-)-naloxone blocks this effect. We used this parabrachial circuit as a model to assess cellular actions of ultra-low doses of (-)-naloxone and (+)-naloxone in modifying the effects of DAMGO. Our results showed that an ultra-low concentration of (-)-naloxone (0.001 nM) and several concentrations of (+)-naloxone (0.01-10 nM) enhanced DAMGO-stimulated guanosine-5'-0-(γ-thio)-triphosphate incorporation in parabrachial sections in vitro. Further, we analyzed the relevance of these effects in vivo. In the present study, we show that (+)-naloxone can potentiate DAMGO-induced feeding at doses at which (-)-naloxone was an antagonist. These results implicated (+)-naloxone as a novel tool for studying mu-opioid receptor functions and suggest that (+)-naloxone may have therapeutic value to enhance clinical actions of opiate drugs.

Serotonergic and dopaminergic distinctions in the behavioral pharmacology of (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and lysergic acid diethylamide (LSD).

RATIONALE: After decades of social stigma, hallucinogens have reappeared in the clinical literature demonstrating unique benefits in medicine. The precise behavioral pharmacology of these compounds remains unclear, however. OBJECTIVES: Two commonly studied hallucinogens, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and lysergic acid diethylamide (LSD), were investigated both in vivo and in vitro to determine the pharmacology of their behavioral effects in an animal model. METHOD: Rabbits were administered DOI or LSD and observed for head bob behavior after chronic drug treatment or after pretreatment with antagonist ligands. The receptor binding characteristics of DOI and LSD were studied in vitro in frontocortical homogenates from naïve rabbits or ex vivo in animals receiving an acute drug injection. RESULTS: Both DOI- and LSD-elicited head bobs required serotonin(2A) (5-HT(2A)) and dopamine(1) (D(1)) receptor activation. Serotonin(2B/2C) receptors were not implicated in these behaviors. In vitro studies demonstrated that LSD and the 5-HT(2A/2C) receptor antagonist, ritanserin, bound frontocortical 5-HT(2A) receptors in a pseudo-irreversible manner. In contrast, DOI and the 5-HT(2A/2C) receptor antagonist, ketanserin, bound reversibly. These binding properties were reflected in ex vivo binding studies. The two hallucinogens also differed in that LSD showed modest D(1) receptor binding affinity whereas DOI had negligible binding affinity at this receptor. CONCLUSION: Although DOI and LSD differed in their receptor binding properties, activation of 5-HT(2A) and D(1) receptors was a common mechanism for eliciting head bob behavior. These findings implicate these two receptors in the mechanism of action of hallucinogens.

Pharmacological and behavioral characterization of the 5-HT2A receptor in C57BL/6N mice.

RATIONALE: The serotonin (5-HT) 2A receptor is implicated in numerous psychiatric disorders, making it an important, clinically relevant target. Despite the availability of transgenic mouse lines, the native mouse 5-HT(2A) receptor is not well-characterized. OBJECTIVES: The goals of the current study were to determine 5-HT(2A) and 5-HT(2C) receptor densities in mouse cortex, establish a pharmacological profile of the mouse 5-HT(2A) receptor, and determine the effects of chronic drug treatment on 5-HT(2A) receptor density and 5-HT(2A) receptor-mediated behavior. METHODS: Receptor densities were determined in cortex and frontal cortex via saturation binding assays using [(3)H]ketanserin or [(3)H]mesulergine. A pharmacological profile was established by displacing [(3)H]ketanserin binding with several ligands. Chronic treatment with 5-HT(2A/2C) receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI), 5-HT(2A) receptor antagonist, MDL 11939, or vehicle was followed by 5-HT(2A) receptor density determination. Head twitch responses (HTRs) were counted on select days. RESULTS: Mice had high 5-HT(2A), but low 5-HT(2C) receptor densities. Ligand binding affinities for mouse 5-HT(2A) receptors correlated with rat, but not rabbit or human, affinities. Chronically DOI-treated mice displayed reduced HTRs and 5-HT(2A) receptor density compared to saline-treated mice. Receptor density was unchanged following chronic treatment with MDL 11939. CONCLUSIONS: The current study provides some basic information about mouse 5-HT(2A) and 5-HT(2C) receptors and provides comparisons to rats, rabbits, and humans. The current chronic agonist treatment study demonstrated an important similarity between the 5-HT(2A) receptor in mice, rats, and rabbits, while antagonist treatment revealed an interesting difference from previous studies in rabbits.

The role of serotonin-2 (5-HT2) and dopamine receptors in the behavioral actions of the 5-HT2A/2C agonist, DOI, and putative 5-HT2C inverse agonist, SR46349B.

RATIONALE: Atypical antipsychotic efficacy is often attributed to actions at serotonin-2 (5-HT(2)) and dopamine receptors, indicating a potential benefit of understanding the interplay between these systems. Currently, it is known that 5-HT(2) receptors modulate dopamine release, although the role of specific dopamine receptors in 5-HT(2)-mediated behavior is not well understood. OBJECTIVES: We examined the role of 5-HT(2A), 5-HT(2C), and dopamine (D1 and D2) receptors in the behavioral response to a 5-HT(2A/2C) agonist (DOI) and 5-HT(2A/2C) antagonist (SR46349B). MATERIALS AND METHODS: Effects were assessed by measuring rabbit head bobs (previously characterized as 5-HT(2A) receptor-mediated) and body shakes (5-HT(2C)-mediated). RESULTS: As expected, DOI produced head bobs and body shakes, and these DOI-elicited behaviors were attenuated by the SR46349B pretreatment. Unexpectedly, SR46349B also induced head bobs when administered alone. However, SR46349B-elicited head bobs are distinguishable from those produced by DOI since the 5-HT(2A) antagonist, ketanserin, only attenuated DOI-elicited head bobs. Conversely, 5-HT(2C) ligands (SB242084 and SB206553) inhibited SR46349B but not DOI-induced head bobs. Furthermore, when administered alone, SB206553 (a 5-HT(2C) inverse agonist) produced head bobs, indicating the behavior can be either 5-HT(2A) or 5-HT(2C) mediated. Next, it was revealed that D1 and D2 receptors play a role in DOI-elicited head bobs, but only D1 receptors are required for SR46349B-elicited head bobs. CONCLUSIONS: 5-HT(2A) receptor agonism and 5-HT(2C) inverse agonism produce the same behavior, likely due to similar downstream actions at D1 receptors. Consequently, 5-HT(2C) agonism or D1 agonism may be effective therapies for disorders, such as schizophrenia, currently being treated with 5-HT(2A) antagonists.

Intrahippocampal LSD accelerates learning and desensitizes the 5-HT(2A) receptor in the rabbit, Romano et al.

RATIONALE: Parenteral injections of d-lysergic acid diethylamide (LSD), a serotonin 5-HT(2A) receptor agonist, enhance eyeblink conditioning. Another hallucinogen, (±)-1(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI), was shown to elicit a 5-HT(2A)-mediated behavior (head bobs) after injection into the hippocampus, a structure known to mediate trace eyeblink conditioning. OBJECTIVE: This study aims to determine if parenteral injections of the hallucinogens LSD, d,l-2,5-dimethoxy-4-methylamphetamine, and 5-methoxy-dimethyltryptamine elicit the 5-HT(2A)-mediated behavior of head bobs and whether intrahippocampal injections of LSD would produce head bobs and enhance trace eyeblink conditioning. MATERIALS AND METHODS: LSD was infused into the dorsal hippocampus just prior to each of eight conditioning sessions. One day after the last infusion of LSD, DOI was infused into the hippocampus to determine whether there had been a desensitization of the 5-HT(2A) receptor as measured by a decrease in DOI-elicited head bobs. RESULTS: Acute parenteral or intrahippocampal LSD elicited a 5-HT(2A) but not a 5-HT(2C)-mediated behavior, and chronic administration enhanced conditioned responding relative to vehicle controls. Rabbits that had been chronically infused with 3 or 10 nmol per side of LSD during Pavlovian conditioning and then infused with DOI demonstrated a smaller increase in head bobs relative to controls. CONCLUSIONS: LSD produced its enhancement of Pavlovian conditioning through an effect on 5-HT(2A) receptors located in the dorsal hippocampus. The slight, short-lived enhancement of learning produced by LSD appears to be due to the development of desensitization of the 5-HT(2A) receptor within the hippocampus as a result of repeated administration of its agonist (LSD).

Inverse agonism at serotonin and cannabinoid receptors.

Contemporary receptor theory was developed to account for the existence of constitutive activity, as defined by the presence of receptor signaling in the absence of any ligand. In vitro studies with a variety of cell types have revealed the existence of constitutive activity and inverse agonism at a large number of receptors and also additional complexities of ligand-receptor interactions. Thus, ligands acting at a constitutively active receptor can act as agonists, antagonists, and/or inverse agonists, and these pharmacological characteristics can differ for an individual ligand depending upon the receptor response measured and the physiological state of the system under study. Studies with a variety of cell types have established that the serotonin 5-HT(2A) and 5-HT(2C) receptors and the cannabinoid CB1 receptor demonstrate constitutive activity and inverse agonism in vitro. Serotonin and cannabinoid receptors are involved in a large number of physiological and behavioral functions. The possible existence of constitutive activity and inverse agonism at these receptors in vivo would provide new avenues for drug development. Recent studies have provided compelling evidence that both the serotonin 5-HT(2A) and 5-HT(2C) receptors and cannabinoid CB1 receptor demonstrate inverse agonism and constitutive activity also in vivo. This chapter describes our current knowledge of constitutive activity in vitro and then examines the evidence for constitutive activity in vivo.

GTPgammaS incorporation in the rat brain: a study on mu-opioid receptors and CXCR4.

Chemokine and opioid receptors are G-protein-coupled receptors that play important roles in both the central nervous system and the immune system. The long-term goal of our research is to establish whether opioids regulate the activity of the chemokine receptor CXCR4 (one of the major HIV co-receptors) in the brain. In this research, we studied the anatomical distribution of functional receptors in young and adult animals by using the [(35)S]GTPgammaS "binding" assay as an indication of G-protein activation by CXCL12 (the natural CXCR4 ligand) or by mu-opioid agonists. Brain slices or homogenates from Holtzmann rats of different ages (from 2 to 21 days old and adult animals) were treated with CXCL12 (0.001-100 nM), D: -ala2,MePhe4,gly-ol5]enkephalin (DAMGO; 0.0003-10 microM) or morphine (0.0003-10 microM) and then processed for the assay. Our results show stimulation of both mu-OR and CXCR4 in several brain areas, including cortex and hippocampus (p < 0.001); this effect is dose and age dependent, and the magnitude of response varies among different brain regions. Furthermore, AMD3100 (100 ng/ml), a specific CXCR4 antagonist, abolished CXCL12 stimulation in all the brain regions analyzed (p < 0.001). Our findings suggest a similar pattern of expression for mu-OR and CXCR4 in the brain, supporting the possibility of an interaction between the two G-protein-coupled receptors in vivo. This might be relevant to the role of opiates in HIV neuropathogenesis.

A naloxonazine sensitive (mu1 receptor) mechanism in the parabrachial nucleus modulates eating.

The parabrachial nucleus (PBN) is an area of the brain stem that controls eating and contains endogenous opioids and their receptors. Previously, we demonstrated that acute activation of mu opioid receptors (MOPR) in the lateral PBN increased food consumption. MOPRs have been divided operationally into mu(1) and mu(2) receptor subtypes on the basis of the ability of naloxonazine (Nlxz) to block the former but not the latter. We used autoradiography to measure whether Nlxz blocks stimulation by the mu(1)/mu(2) agonist DAMGO (D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin) of the incorporation of [(35)S]-guanosine 5'(gamma-thio)triphosphate ([(35)S]-GTPgammaS) into sections of the PBN. In vitro, Nlxz dose dependently inhibited receptor coupling in all areas of the PBN. The 1 muM concentration of Nlxz reduced stimulation by 93.1+/-5% in the lateral inferior PBN (LPBNi) and by 90.5+/-4% in the medial parabrachial subregion (MPBN). Administration of Nlxz directly into the LPBNi decreased both food intake and agonist stimulated coupling, ex vivo, for the 24-h period after infusion. Infusion of Nlxz into the intended area reduced food intake by 42.3% below baseline values. Nlxz infusion prevented DAMGO stimulation of G-protein coupling in LPBNi and markedly reduced this stimulation in the MPBN. The incomplete inhibition of DAMGO-stimulated coupling in the MPBN is most likely due to the limited diffusion of Nlxz from the site of infusion (LPBNi) into this brain region. In conclusion, this study demonstrates that the mu(1) opioid receptor subtype is present in the parabrachial nucleus of the pons and that these receptors serve to modulate feeding in rats.

Behavioral response to emotional stress in rabbits: role of serotonin and serotonin2A receptors.

Exposure to a novel environment is a stressor which modulates behavior, increases stress hormones and enhances the release of several neurotransmitters including serotonin (5-HT). Exposing rabbits to a novel environment significantly increases head-bob behavior but fails to alter either grooming or wet dog shakes compared with those observed in the home-cage. The goal of this study was to determine the role of 5-HT and its receptors in mediating novelty-elicited head-bob behavior. Reduction of central 5-HT levels after treatment with the serotonergic neurotoxin 5,7-DHT significantly decreased novelty-elicited head bobs by 40% compared with those in sham-lesioned rabbits, indicating that 5-HT mediates, in part, this behavior. Additionally, pretreatment with the 5-HT1A partial agonist and clinically used anxiolytic buspirone also significantly attenuated novelty-elicited head bobs. Pretreatment with the selective 5-HT2A antagonist M 100,907 significantly reduced novel environment-elicited head bobs by 40%. Furthermore, agonist-induced reduction of cortical 5-HT2A receptor density resulted in a significant 40% reduction in the number of head bobs elicited by the novel environment. These data demonstrate that rabbit head-bob behavior, an index of the response to novelty stress, is mediated, in part, by 5-HT activation of 5-HT2A receptors.

The time-course for up- and down-regulation of the cortical 5-hydroxytryptamine (5-HT)2A receptor density predicts 5-HT2A receptor-mediated behavior in the rabbit.

5-Hydroxytryptamine (serotonin; 5-HT)2 receptor agonists such as (+/-)-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) injected systemically or directly into frontal cortex, elicit stereotyped head movements that are mediated by 5-HT2A receptors. Chronic administration of 5-HT2A receptor antagonists can produce either a down-regulation, e.g., d-2-bromolysergic acid diethylamide (BOL) or an up-regulation, e.g., alpha-phenyl-10(2-phenylethyl)-4-piperidinemethanol (MDL11,939) of cortical 5-HT2A receptors in the rabbit with no change in the density of the 5-HT2C receptor. We examined the degree to which the time course for the onset and offset of changes in cortical 5-HT2A receptor density was correlated with functional changes as measured by the magnitude of DOI elicited, 5-HT2A receptor-mediated head movements (head bobs). First, the magnitude of DOI-elicited head bobs was measured over 1 to 8 days after chronic BOL (5.8 micromol/kg), MDL11,939 (10 micromol/kg), or vehicle administration. Second, rabbits were injected with BOL, MDL11,939, or vehicle once daily for 8 days, and then, 1 to 8 days after the cessation of drug or vehicle, DOI-elicited head bobs were determined. Samples of frontal cortex were obtained for each animal immediately following behavioral testing, and 5-HT2A receptor density was measured using [3H]ketanserin. Thus, each animal provided a value for receptor density and number of head bobs, and these two measures showed a high degree of correlation between 0.94 for BOL and 0.95 for MDL11,939. This study establishes that the density of 5-HT2A receptors in cortex reflects their functional status.

Mu-opioid receptor cellular function in the nucleus accumbens is essential for hedonically driven eating.

Acute pharmacological studies have implicated mu-opioid receptors (MORs) in the shell of the nucleus accumbens (NAC) in mediating responses for palatable food and other natural and drug-induced rewards. However, the long-term behavioral effects of inactivating signal transduction via accumbal MORs, as quantified by an anatomically defined loss of cellular activity, have never been analysed. We combined microinfusion of the irreversible MOR antagonist, beta-funaltrexamine (beta-FNA; 8.0 nmol/0.8 microL, n=9; controls, n=6) with mapping by [35S]GTPgammaS autoradiography to demonstrate an anatomically specific loss of the coupling of MORs to their G-proteins in the dorsal caudomedial shell of the NAC in rabbits. beta-FNA did not alter the stimulated coupling of kappa-opioid receptors. This selective blockade of the cellular function of MORs persistently decreased consumption of a palatable sucrose solution by 40% during a daily 4-h test conducted 2, 3 and 4 days after infusion. beta-FNA did not alter body weight or 20-h consumption of standard chow or water. In 10 different rabbits, infusion of the selective, competitive MOR antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) into the same locus produced a reversible decrease in sucrose consumption, with normal intakes returning on the next day. Together, these data appear to establish that MORs in this accumbal subregion support responding for orosensory reward. Overall, these results visualize a discrete brain locus where cellular actions of endogenous opioids mediate behaviors involved in self-administration of foods and perhaps other hedonically valued substances, such as ethanol and drugs of abuse.