Gßγ subunits colocalize with RNA polymerase II and regulate transcription in cardiac fibroblasts.

Gßγ subunits mediate many different signaling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gßγ signaling, we investigated the functional role of Gßγ signaling in the regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. We identified a novel, negative, regulatory role for the Gß1γ dimer in the fibrotic response. Depletion of Gß1 led to derepression of the fibrotic response at the mRNA and protein levels under basal conditions and an enhanced fibrotic response after sustained stimulation of the angiotensin II type I receptor. Our genome-wide chromatin immunoprecipitation experiments revealed that Gß1 colocalized and interacted with RNA polymerase II on fibrotic genes in an angiotensin II-dependent manner. Additionally, blocking transcription with inhibitors of Cdk9 prevented association of Gßγ with transcription complexes. Together, our findings suggest that Gß1γ is a novel transcriptional regulator of the fibrotic response that may act to restrict fibrosis to conditions of sustained fibrotic signaling. Our work expands the role for Gßγ signaling in cardiac fibrosis and may have broad implications for the role of nuclear Gßγ signaling in other cell types.

In vivo detection of GPCR-dependent signaling using fiber photometry and FRET-based biosensors.

Genetically encoded fluorescent biosensors allow intracellular signaling dynamics to be tracked in live cells and tissues using optical detection. Many such biosensors are based on the principle of Förster resonance energy transfer (FRET), and we have recently developed a simple approach for in vivo detection of FRET-based biosensor signals using fiber photometry. By combining fiber photometry with FRET-based biosensors, we were able to track GPCR-dependent signaling pathways over time, and in response to drug treatments in freely-moving adult rats. Recording from specific neuronal populations, we can quantify intracellular signaling while simultaneously measuring behavioral responses. Our approach, described in detail here, uses adeno-associated viruses infused intracerebrally in order to express genetically-encoded FRET-based biosensors. After several weeks to allow biosensor expression, fiber photometry is used in order to record drug responses in real time from freely-moving adult rats. This methodology would be compatible with other mammalian species and with many biosensors. Hence, it has wide applicability across a spectrum of neuroscience research, ranging from the study of neural circuits and behavior, to preclinical drug development and screening.

High-Content Single-Cell Förster Resonance Energy Transfer Imaging of Cultured Striatal Neurons Reveals Novel Cross-Talk in the Regulation of Nuclear Signaling by Protein Kinase A and Extracellular Signal-Regulated Kinase 1/2.

Genetically encoded biosensors can be used to track signaling events in living cells by measuring changes in fluorescence emitted by one or more fluorescent proteins. Here, we describe the use of genetically encoded biosensors based on Förster resonance energy transfer (FRET), combined with high-content microscopy, to image dynamic signaling events simultaneously in thousands of neurons in response to drug treatments. We first applied this approach to examine intercellular variation in signaling responses among cultured striatal neurons stimulated with multiple drugs. Using high-content FRET imaging and immunofluorescence, we identified neuronal subpopulations with unique responses to pharmacological manipulation and used nuclear morphology to identify medium spiny neurons within these heterogeneous striatal cultures. Focusing on protein kinase A (PKA) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in the cytoplasm and nucleus, we noted pronounced intercellular differences among putative medium spiny neurons, in both the magnitude and kinetics of signaling responses to drug application. Importantly, a conventional "bulk" analysis that pooled all cells in culture yielded a different rank order of drug potency than that revealed by single-cell analysis. Using a single-cell analytical approach, we dissected the relative contributions of PKA and ERK1/2 signaling in striatal neurons and unexpectedly identified a novel role for ERK1/2 in promoting nuclear activation of PKA in striatal neurons. This finding adds a new dimension of signaling crosstalk between PKA and ERK1/2 with relevance to dopamine D1 receptor signaling in striatal neurons. In conclusion, high-content single-cell imaging can complement and extend traditional population-level analyses and provides a novel vantage point from which to study cellular signaling. SIGNIFICANCE STATEMENT: High-content imaging revealed substantial intercellular variation in the magnitude and pattern of intracellular signaling events driven by receptor stimulation. Since individual neurons within the same population can respond differently to a given agonist, interpreting measures of intracellular signaling derived from the averaged response of entire neuronal populations may not always reflect what happened at the single-cell level. This study uses this approach to identify a new form of cross-talk between PKA and ERK1/2 signaling in the nucleus of striatal neurons.

Stable long-term individual differences in 50-kHz vocalization rate and call subtype prevalence in adult male rats: Comparisons with sucrose preference.

Sucrose preference (SP) is a widely used measure of anhedonia in rat models of depression, yet depressed patients do not reliably show an analogous deficit. As an alternative affect-related measure, adult rat ultrasonic vocalizations (USVs) are attracting interest, but it is unclear whether SP and USVs provide independent measures. Here, we have assessed whether SP and USV emission are correlated in the absence of a depressogenic procedure. To this end, 24 male Long-Evans rats were tested daily for 24 days, with alternating SP tests and USV recordings; after a 3-month hiatus, USV emission was re-evaluated for 6 more days. SP was measured in simultaneous two-bottle choice tests, and USVs were recorded in an open field. The main measures were: SP, 50-kHz call rate, and relative prevalence of trill and flat call subtypes. These measures showed temporally-stable individual differences across the initial 24-day testing period, and at the 3-month USV follow-up tests. Correlational analysis revealed no significant relationships between SP and the three main USV measures. Rats differed consistently, not only in their 50-kHz call rates but also in their 50-kHz call profiles (i.e., the relative prevalence of 14 call subtypes); most rats preferentially emitted either trill or flat calls. Several inter-call subtype associations were detected, including a strong negative relationship between the relative prevalence of flat and trill calls. The 50-kHz call rate was correlated with the relative prevalence of only one call subtype (short calls, negative correlation), but was positively correlated with absolute emission rates for almost all subtypes. In conclusion, adult rats exhibited temporally-stable individual differences over weeks (SP) or months (USVs) of testing. This trait-like stability helped to reveal a lack of relationship between SP and the USV-related variables under study, suggesting that these measures may capture different constructs of possible relevance to animal models of depression.

A role for BET proteins in regulating basal, dopamine-induced and cAMP/PKA-dependent transcription in rat striatal neurons.

The activity of striatal medium-spiny projection neurons is regulated by D1 and D2 dopamine receptors. The D1 receptor (D1R) is a Gαs/olf-coupled GPCR which activates a cAMP/PKA/DARPP-32 signalling cascade that increases excitability and facilitates plasticity, partly through the regulation of transcription. Upon activation via D1R, PKA can translocate to the nucleus to regulate transcription through the phosphorylation of various targets. One candidate effector of PKA-dependent transcriptional regulation is the BET protein Brd4. It is known that when Brd4 is activated by phosphorylation, it binds more readily to acetylated histones at promoters and enhancers; moreover, in non-neuronal cells, PKA signalling has been shown to increase recruitment of Brd4 to chromatin. However, it is unknown whether BET proteins, or Brd4 specifically, are involved in transcriptional activation by cAMP/PKA in neurons. Here, we demonstrate that in adult rats, inhibition of BET proteins with the bromodomain inhibitor JQ1 suppressed the expression of ~25% of D1R-upregulated genes, while also increasing the expression of a subset of immediate-early genes. We further found that cAMP/PKA signalling promotes Brd4 recruitment to dopamine-induced genes in striatal neurons, and that knockdown of Brd4 attenuates D1R-induced gene expression. Finally, we report that JQ1 treatment downregulated expression of many GPCRs and also impaired ERK1/2 signalling in striatal neurons. Our findings identify the BET protein family, and Brd4 in particular, as novel regulators of basal and D1R-dependent transcription in rat striatal neurons, and delineate complex bi-directional effects of bromodomain inhibitors on neuronal transcription.

The Signaling and Pharmacology of the Dopamine D1 Receptor.

The dopamine D1 receptor (D1R) is a Gαs/olf-coupled GPCR that is expressed in the midbrain and forebrain, regulating motor behavior, reward, motivational states, and cognitive processes. Although the D1R was initially identified as a promising drug target almost 40 years ago, the development of clinically useful ligands has until recently been hampered by a lack of suitable candidate molecules. The emergence of new non-catechol D1R agonists, biased agonists, and allosteric modulators has renewed clinical interest in drugs targeting this receptor, specifically for the treatment of motor impairment in Parkinson's Disease, and cognitive impairment in neuropsychiatric disorders. To develop better therapeutics, advances in ligand chemistry must be matched by an expanded understanding of D1R signaling across cell populations in the brain, and in disease states. Depending on the brain region, the D1R couples primarily to either Gαs or Gαolf through which it activates a cAMP/PKA-dependent signaling cascade that can regulate neuronal excitability, stimulate gene expression, and facilitate synaptic plasticity. However, like many GPCRs, the D1R can signal through multiple downstream pathways, and specific signaling signatures may differ between cell types or be altered in disease. To guide development of improved D1R ligands, it is important to understand how signaling unfolds in specific target cells, and how this signaling affects circuit function and behavior. In this review, we provide a summary of D1R-directed signaling in various neuronal populations and describe how specific pathways have been linked to physiological and behavioral outcomes. In addition, we address the current state of D1R drug development, including the pharmacology of newly developed non-catecholamine ligands, and discuss the potential utility of D1R-agonists in Parkinson's Disease and cognitive impairment.

Nicotine-induced enhancement of a sensory reinforcer in adult rats: antagonist pretreatment effects.

RATIONALE AND OBJECTIVES: The reinforcement-enhancing effect (REE) of nicotine refers to the drug's ability to enhance the strength of other primary and conditioned reinforcers. The main aim was to investigate neuropharmacological mechanisms underlying nicotine's strengthening of a primary visual reinforcer (i.e., a light cue), using a subcutaneous (SC) dose previously shown to provide plasma nicotine levels associated with habitual smoking. METHODS: Adult male rats pressed an "active" lever to illuminate a brief cue light during daily 60-min sessions. Rats that showed a clear REE were tested with systemically administered pretreatment drugs followed by nicotine (0.1 mg/kg SC) or saline challenge, in within-subject counterbalanced designs. Pretreatments were mecamylamine (nicotinic, 0.1-1 mg/kg SC), SCH 39166 (D1-like dopaminergic, 0.003-0.2 mg/kg SC), naloxone (opioid, 1 and 5 mg/kg SC), prazosin (alpha1-adrenergic antagonist, 1 and 2 mg/kg IP), rimonabant (CB1 cannabinoid inverse agonist, 3 mg/kg IP), sulpiride (D2-like dopaminergic antagonist, 40 mg/kg SC), or propranolol (beta-adrenergic antagonist, 10 mg/kg IP). RESULTS: The nicotine REE was abolished by three antagonists at doses that did not impact motor output, i.e., mecamylamine (1 mg/kg), SCH 39166 (0.01 and 0.03 mg/kg), and naloxone (5 mg/kg). Prazosin and rimonabant both attenuated the nicotine REE, but rimonabant also suppressed responding more generally. The nicotine REE was not significantly altered by sulpiride or propranolol. CONCLUSIONS: In adult male rats, the reinforcement-enhancing effect of low-dose nicotine depends on nicotinic receptor stimulation and on neurotransmission via D1/D5 dopaminergic, opioid, alpha1-adrenergic, and CB1 cannabinoid receptors.

Effects of nicotine, nornicotine and cotinine, alone or in combination, on locomotor activity and ultrasonic vocalization emission in adult rats.

RATIONALE: The behavioral effects of the nicotine metabolites nornicotine and cotinine have not been investigated extensively. OBJECTIVES: To evaluate the effects of nicotine, cotinine, and nornicotine, given alone or in combination, on locomotor activity and emission of ultrasonic vocalizations in male adult rats. METHODS: Rats were first given home cage nicotine injections to make them tolerant to the drug's locomotor depressant effects. On subsequent days, locomotor activity (LMA) and ultrasonic vocalizations were recorded in an open field, for 60 min after challenge injection, using repeated measures designs. In single-drug experiments, subjects were tested with nicotine 0.05-0.4 mg/kg, cotinine 0.03-3 mg/kg, or nornicotine 0.1-10 mg/kg. In drug-combination experiments, saline or nicotine 0.2 mg/kg challenge was preceded by cotinine (0, 0.3, 3 mg/kg) or nornicotine (0, 0.1, 0.3, 1, 3 mg/kg) injection. RESULTS: High doses of nornicotine increased LMA and blunted the locomotor stimulant effect of nicotine. Less consistently, nicotine and high doses of nornicotine decreased the 50-kHz call rate, with no clear evidence of a nornicotine × nicotine interaction. Cotinine, given alone or before nicotine injection, altered neither LMA nor the call rate. No drug altered the relative prevalence of flat vs. trill 50-kHz call subtypes, except that the highest dose of nornicotine promoted flat calls over trills. No drug evoked 22-kHz calls. CONCLUSION: Nornicotine can exert an acute anti-nicotine effect in vivo, as previously reported in vitro. The finding that nicotine did not detectably alter the 50-kHz call profile appears consistent with this drug's mild subjective effects in human subjects.

Exploring functional consequences of GPCR oligomerization requires a different lens.

As the largest family of cell surface receptors, G protein-coupled receptors (GPCRs) represent an important strategic class of therapeutic targets. Attaining a clearer perspective of how such signaling complexes set molecular events in motion could have significant impact on our understanding and treatment of human diseases. As such, many experimental approaches have set out to better understand signaling networks associated with individual receptors to understand signaling architectures and their relationship to signaling outcomes. However, designing in vitro assays aimed at addressing signaling events downstream of single GPCRs must also take into account their propensity to form homo- and heterooligomeric complexes. In the context of GPCR oligomers, physical interactions with a partner protein can have a number of potential consequences, which we will explore in this review. We will also discuss methods used to identify putative dimer partners as well as the various techniques used to study the functional consequences of such complex formation. Since the full functional significance and physiological relevance of GPCR oligomers remains incompletely understood, owing in part to technical limitations, new tools to elucidate molecular mechanisms underlying allosteric co-regulation occurring between two GPCRs are required. Accordingly, using the example of the FP/AT1R heterodimer, we discuss the potential of the FlAsH-BRET approach as a simple tool to reveal how allosteric information is transmitted via conformational rearrangements within putative GPCR complexes and as a means to deorphanize receptors.

Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors.

As with many G protein-coupled receptors (GPCRs), the signalling pathways regulated by the dopamine D1 receptor (D1R) are dynamic, cell type-specific, and can change in the face of disease or drug exposures. In striatal neurons, the D1R activates cAMP/protein kinase A (PKA) signalling. However, in Parkinson's disease (PD), alterations in this pathway lead to functional upregulation of extracellular regulated kinases 1/2 (ERK1/2), contributing to L-DOPA-induced dyskinesia (LID). In order to detect D1R activation in vivo and to study the progressive dysregulation of D1R signalling in PD and LID, we developed ratiometric fiber-photometry with Förster resonance energy transfer (FRET) biosensors and optically detected PKA and ERK1/2 signalling in freely moving rats. We show that in Parkinsonian animals, D1R signalling through PKA and ERK1/2 is sensitized, but that following chronic treatment with L-DOPA, these pathways become partially desensitized while concurrently D1R activation leads to greater induction of dyskinesia.

Rats self-administer intravenous nicotine delivered in a novel smoking-relevant procedure: effects of dopamine antagonists.

Attempts to explain tobacco addiction have relied heavily on the assumption that each cigarette puff delivers a bolus of nicotine to the brain within seconds. However, nicotine transits from lungs to brain much more gradually than once thought. Nevertheless, animal self-administration studies continue to use rapid (e.g., <3-s) infusions, as well as high unit doses of nicotine (e.g., 15-30 microg/kg/infusion), each equivalent to one to two cigarettes. Here, we report that nicotine is self-administered across a range of infusion durations (3, 30, 60, and 120 s) in rats. Slow (30-s) infusions were preferred over fast (nominal 3-s) infusions and were self-administered across several reinforcement schedules, at doses as low as 3 microg/kg/infusion, equivalent to one to two puffs. A conventional "fast/high" self-administration procedure (3 s-30 microg/kg/infusion) was then compared with our new "slow/low" procedure (30 s-3 microg/kg/infusion) in rats trained on a progressive ratio schedule and acutely challenged with dopamine receptor antagonists. The D(1) antagonist R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH 23390) (6-25 microg/kg s.c.) reduced intake in both procedures and in rats self-administering cocaine (0.5 mg/kg/infusion). The D(2) antagonists spiperone (3-30 microg/kg s.c.) and sulpiride (5-20 mg/kg i.p.) increased intake of fast/high nicotine and cocaine, but markedly reduced intake of slow/low nicotine. In a final test, in which only infusion speed was varied, an acute spiperone challenge produced the same differential effect on nicotine self-administration. In conclusion, our new slow/low nicotine self-administration procedure, designed to better mimic smoking-associated nicotine intake, is pharmacologically distinct from the conventional fast delivery/high-dose procedure.

Rewarding and aversive effects of nicotine are segregated within the nucleus accumbens.

Forebrain dopamine plays a critical role in motivated behavior. According to the classic view, mesolimbic dopamine selectively guides behavior motivated by positive reinforcers. However, this has been challenged in favor of a wider role encompassing aversively motivated behavior. This controversy is particularly striking in the case of nicotine, with opposing claims that either the rewarding or the aversive effect of nicotine is critically dependent on mesolimbic dopamine transmission. In the present study, the effects of 6-hydroxydopamine lesions of nucleus accumbens core vs. medial shell on intravenous nicotine conditioned place preference and conditioned taste aversion were examined in male adult rats. Dopaminergic denervation in accumbens medial shell was associated with decreased nicotine conditioned place preference. Conversely, denervation in accumbens core was associated with an increase in conditioned place preference. In addition, dopaminergic denervation of accumbens core but not medial shell abolished conditioned taste aversion for nicotine. We conclude that nucleus accumbens core and medial shell dopaminergic innervation exert segregated effects on rewarding and aversive effects of nicotine. More generally, our findings indicate that dopaminergic transmission may mediate or enable opposing motivational processes within functionally distinct domains of the accumbens.

Reinforcement enhancement by nicotine in adult rats: behavioral selectivity and relation to mode of delivery and blood nicotine levels.

RATIONALE: Reinforcement-enhancing effects of nicotine occur in human subjects and laboratory rats. However, the doses used in animal studies typically exceed smoking-associated levels of exposure, and generalized behavioral activation by nicotine can potentially confound data interpretation. METHODS: During daily 60-min sessions, male adult rats pressed an "active" lever to illuminate a brief cue light. Pressing on either the active or inactive lever retracted both levers for 60 s. Nicotine (0.025-0.2 mg/kg) was given either by continuous intravenous (IV) infusion, or spaced IV pulses (3-s or 30-s/pulse), or pre-session subcutaneous (SC) injection. RESULTS: Almost all rats responded preferentially for the cue light for several weeks. After several home-cage nicotine injections, reinforcement enhancement occurred even within the first nicotine test session. Nicotine increased active lever responding without altering inactive lever responding, with effects reliably observed at doses as low as 0.1 mg/kg SC or 0.1 mg/kg/session IV. Within the session, the 0.1 mg/kg dose maximally increased active lever responding by 2-3-fold, coinciding with serum levels of 25 ng/ml. Intravenous nicotine (tested at 0.1 mg/kg/60-min session) was equally effective whether delivered by continuous infusion or in a series of equally spaced 0.003 mg/kg pulses each of 3-s or 30-s duration. CONCLUSIONS: Low doses of nicotine can potentiate responding for a primary sensory reinforcer without producing a generalized increase in lever pressing. Reinforcer enhancement by nicotine generalized to several modes of drug delivery, appeared to track circulating levels of drug, and occurred even at serum levels within the daytime range of moderate cigarette smokers.

Enhancement of a visual reinforcer by D-amphetamine and nicotine in adult rats: relation to habituation and food restriction.

RATIONALE AND OBJECTIVES: Nicotine and D-amphetamine can strengthen reinforcing effects of unconditioned visual stimuli. We investigated whether these reinforcement-enhancing effects reflect a slowing of stimulus habituation and depend on food restriction. METHODS: Adult male rats pressed an active lever to illuminate a cue light during daily 60-min sessions. Depending on the experiment, rats were challenged with fixed or varying doses of D-amphetamine (0.25-2 mg/kg IP) and nicotine (0.025-0.2 mg/kg SC) or with the tobacco constituent norharman (0.03-10 µg/kg IV). Experiment 1 tested for possible reinforcement-enhancing effects of D-amphetamine and norharman. Experiment 2 investigated whether nicotine and amphetamine inhibited the spontaneous within-session decline in lever pressing. Experiment 3 assessed the effects of food restriction. RESULTS: Amphetamine (0.25-1 mg/kg) and nicotine (0.1 mg/kg) increased active lever pressing specifically (two- to threefold increase). The highest doses of nicotine and amphetamine also affected inactive lever responding (increase and decrease, respectively). With the visual reinforcer omitted, responding was largely extinguished. Neither drug appeared to slow habituation, as assessed by the within-session decline in lever pressing, and reinforcement-enhancing effects still occurred if the drugs were given after this decline had occurred. Food restriction enhanced the reinforcement-enhancing effect of amphetamine but not that of nicotine. CONCLUSIONS: Responding remained goal-directed after several weeks of testing. Low doses of D-amphetamine and nicotine produced reinforcement enhancement even in free-feeding subjects, independent of the spontaneous within-session decline in responding. Reinforcement enhancement by amphetamine, but not nicotine, was enhanced by concurrent subchronic food restriction.

Effects of anxiogenic drugs on the emission of 22- and 50-kHz ultrasonic vocalizations in adult rats.

RATIONALE: Adult rat 22-kHz vocalizations are often associated with alarm or distress, whereas a subset of 50-kHz calls is preferentially emitted in response to amphetamine and other rewarding stimuli. Whether any 50-kHz calls reflect anxiety is unknown. OBJECTIVE: To determine the effects of anxiogenic drugs on 50-kHz call rate and call subtype profile, in comparison with D-amphetamine. METHODS: Adult male rats received systemic amphetamine (1 mg/kg) three times several days before testing. Ultrasonic vocalizations were then recorded after acute intraperitoneal injection of amphetamine or one of five anxiogenic drugs: yohimbine (2.5 mg/kg), N-methyl-ß-carboline-3-carboxamide (FG 7142, 5 mg/kg), pentylenetetrazol (PTZ, 20 mg/kg), m-chlorophenylpiperazine (mCPP, 1 mg/kg), caffeine (25 mg/kg), or vehicle. RESULTS: The duration of immobility was increased by FG 7142, PTZ, and mCPP; this measure was unchanged by yohimbine and reduced by the locomotor stimulant drugs amphetamine and caffeine. Conversely, the 50-kHz call rate was reduced by FG 7142, PTZ and mCPP, and increased by caffeine and amphetamine. Overall, the most common 50-kHz call subtypes were flat, trill, step-up, and complex. Consistent with previous reports, amphetamine increased the relative prevalence of trill calls while reducing the relative prevalence of flat calls. Yohimbine and caffeine reduced flat call prevalence, whereas mCPP reduced trill call prevalence. No other shifts in the call profile were observed, and no anxiogenic drug induced 22-kHz calls. CONCLUSION: Anxiogenic drugs, as a class, did not uniformly alter the 50-kHz call rate or subtype profile. Amphetamine-induced effects on 50-kHz call rate and profile do not reflect anxiety.

Effects of acute morphine withdrawal on ultrasonic vocalizations in adult rats: unchanged 50-kHz call rate and altered subtype profile.

RATIONALE: Adult rat 22- and 50-kHz ultrasonic vocalizations (USVs) are commonly considered as indices of negative and positive affect, respectively. More specifically, we have proposed that positive affective states are revealed by a predominance of trill over flat 50-kHz call subtypes. However, the 50-kHz call subtypes emitted during aversive drug states remain largely uninvestigated. OBJECTIVES: To determine whether acute morphine withdrawal affects 50-kHz call rates or alters the relative prevalence of trill and flat calls. METHODS: In experiment 1, adult male rats were given saline or morphine (6 mg/kg SC), then acutely challenged 4 h later with saline or naloxone (1 mg/kg SC), and recorded 10-30 min post-injection. In experiments 2 and 3, rats received saline or morphine (6 mg/kg), followed 4 h later by acute saline or naloxone (0.1 mg/kg) challenge; USVs were subsequently recorded during 30-min place conditioning sessions. RESULTS: Naloxone (0.1 mg/kg) produced a strong conditioned place aversion only after acute morphine pretreatment, consistent with antagonist-precipitated morphine withdrawal. The morphine-naloxone combination decreased the relative prevalence of trills and promoted flat calls. Naloxone given alone (0.1 and 1 mg/kg) inhibited trill calls but did not significantly alter the prevalence of flat calls, whereas morphine given alone (4 h pre-session) was largely without effect. Fifty-kHz call rates were inhibited by naloxone given alone, but otherwise unaffected. Twenty-two-kHz calls were sparse. CONCLUSIONS: The 50-kHz call subtype shift seen during antagonist-precipitated morphine withdrawal was opposite in direction to that previously associated with rewards, and hence may reveal negative affect.

Effects of repeated morphine on ultrasonic vocalizations in adult rats: increased 50-kHz call rate and altered subtype profile.

RATIONALE: Adult rat 50-kHz vocalizations have been proposed to indicate a positive affective state, putatively revealed by a predominance of trill calls over flat calls. However, short-term exposure to non-sedative doses of the euphorigen morphine suppresses calling, with no discernible shift in trill or flat call prevalence. OBJECTIVES: This study aimed to determine whether morphine acutely increases 50-kHz call rates or alters the relative prevalence of trill or flat calls, after long-term morphine exposure or acute pharmacological pretreatment. METHODS: Experiment 1 comprised 10 once-daily tests, alternating between saline and morphine, 1 mg/kg SC, followed by dose-response testing (0, 0.3, 1, and 3 mg/kg). Experiment 2 was similar but included additional testing with morphine in combination with the antinausea drug ondansetron or the peripheral opioid antagonist methylnaltrexone. In experiment 3, morphine was again combined with ondansetron or methylnaltrexone but in rats that were initially drug naïve. RESULTS: In animals that were initially drug naïve, morphine tended to suppress calling and did not alter the 50-kHz call subtype profile. In morphine-experienced rats, morphine acutely increased the 50-kHz call rate and promoted trills over flat calls; short calls were also inhibited. Neither ondansetron nor methylnaltrexone detectably altered any effect of morphine on calling, nor did these two drugs affect 50-kHz calling when given alone. CONCLUSIONS: With chronic exposure, morphine acutely enhances 50-kHz calling and differentially promotes trill calls, mainly at the expense of flat calls. These effects appear consistent with a positive affect interpretation of 50-kHz vocalizations.

Segregation of amphetamine reward and locomotor stimulation between nucleus accumbens medial shell and core.

Convergent evidence suggests that amphetamine (AMPH) exerts its rewarding and locomotor stimulating effects via release of dopamine in the nucleus accumbens. However, there is no consensus as to the relative contributions of core and medial shell subregions to these effects. Moreover, the literature is based primarily on intracranial administration, which cannot fully mimic the drug distribution achieved by systemic administration. In the present study, the effects of bilateral 6-hydroxydopamine lesions of the accumbens core or medial shell on rewarding and locomotor stimulating effects of systemically administered amphetamine (0.75 mg/kg, i.p.) were examined in a conditioned place preference (CPP) procedure relying solely on tactile cues (floor texture). Residual dopamine innervation was quantified by [125I]-RTI-55 binding to the dopamine transporter. When lesions were performed before the conditioning phase, AMPH-induced locomotor stimulation and CPP magnitude were positively correlated with residual dopamine transporter binding in core and medial shell, respectively. Medial shell lesions did not affect morphine CPP, arguing that a sensory or mnemonic deficit was not responsible for the lesion-induced reduction in AMPH CPP. Medial shell lesions performed between the conditioning phase and the test day reduced the expression of amphetamine CPP. These results suggest that after systemic amphetamine administration, rewarding and locomotor stimulating effects of the drug are anatomically dissociated within the nucleus accumbens: the medial shell contributes to rewarding effects, whereas the core contributes to behavioral activation.

Long-lasting rescue of age-associated deficits in cognition and the CNS cholinergic phenotype by a partial agonist peptidomimetic ligand of TrkA.

Previously, we developed a proteolytically stable small molecule peptidomimetic termed D3 as a selective ligand of the extracellular domain of the TrkA receptor for the NGF. Ex vivo D3 was defined as a selective, partial TrkA agonist. Here, the in vivo efficacy of D3 as a potential therapeutic for cholinergic neurons was tested in cognitively impaired aged rats, and we compared the consequence of partial TrkA activation (D3) versus full TrkA/p75 activation (NGF). We show that in vivo D3 binds to TrkA receptors and affords a significant and long-lived phenotypic rescue of the cholinergic phenotype both in the cortex and in the nucleus basalis. The cholinergic rescue was selective and correlates with a significant improvement of memory/learning in cognitively impaired aged rats. The effects of the synthetic ligand D3 and the natural ligand NGF were comparable. Small, proteolytically stable ligands with selective agonistic activity at a growth factor receptor may have therapeutic potential for neurodegenerative disorders.

Morphine-induced changes in delta opioid receptor trafficking are linked to somatosensory processing in the rat spinal cord.

An in vivo fluorescent deltorphin (Fluo-DLT) internalization assay was used to assess the distribution and regulation of pharmacologically available delta opioid receptors (deltaORs) in the rat lumbar (L4-5) spinal cord. Under basal conditions, intrathecal injection of Fluo-DLT resulted in the labeling of numerous deltaOR-internalizing neurons throughout dorsal and ventral horns. The distribution and number of Fluo-DLT-labeled perikaryal profiles were consistent with that of deltaOR-expressing neurons, as revealed by in situ hybridization and immunohistochemistry, suggesting that a large proportion of these cells was responsive to intrathecally administered deltaOR agonists. Pretreatment of rats with morphine for 48 hr resulted in a selective increase in Fluo-DLT-labeled perikaryal profiles within the dorsal horn. These changes were not accompanied by corresponding augmentations in either deltaOR mRNA or (125)I-deltorphin-II binding levels, suggesting that they were attributable to higher densities of cell surface deltaOR available for internalization rather than to enhanced production of the receptor. Unilateral dorsal rhizotomy also resulted in increased Fluo-DLT internalization in the ipsilateral dorsal horn when compared with the side contralateral to the deafferentation or to non-deafferented controls, suggesting that deltaOR trafficking in dorsal horn neurons may be regulated by afferent inputs. Furthermore, morphine treatment no longer increased Fluo-DLT internalization on either side of the spinal cord after unilateral dorsal rhizotomy, indicating that microOR-induced changes in the cell surface availability of deltaOR depend on the integrity of primary afferent inputs. Together, these results suggest that regulation of deltaOR responsiveness through microOR activation in this region is linked to somatosensory information processing.