Imaging dopamine's role in drug abuse and addiction.

Dopamine is involved in drug reinforcement but its role in addiction is less clear. Here we describe PET imaging studies that investigate dopamine's involvement in drug abuse in the human brain. In humans the reinforcing effects of drugs are associated with large and fast increases in extracellular dopamine, which mimic those induced by physiological dopamine cell firing but are more intense and protracted. Since dopamine cells fire in response to salient stimuli, supraphysiological activation by drugs is experienced as highly salient (driving attention, arousal, conditioned learning and motivation) and with repeated drug use may raise the thresholds required for dopamine cell activation and signaling. Indeed, imaging studies show that drug abusers have marked decreases in dopamine D2 receptors and in dopamine release. This decrease in dopamine function is associated with reduced regional activity in orbitofrontal cortex (involved in salience attribution; its disruption results in compulsive behaviors), cingulate gyrus (involved in inhibitory control; its disruption results in impulsivity) and dorsolateral prefrontal cortex (involved in executive function; its disruption results in impaired regulation of intentional actions). In parallel, conditioning triggered by drugs leads to enhanced dopamine signaling when exposed to conditioned cues, which then drives the motivation to procure the drug in part by activation of prefrontal and striatal regions. These findings implicate deficits in dopamine activity-inked with prefrontal and striatal deregulation-in the loss of control and compulsive drug intake that results when the addicted person takes the drugs or is exposed to conditioned cues. The decreased dopamine function in addicted individuals also reduces their sensitivity to natural reinforcers. Therapeutic interventions aimed at restoring brain dopaminergic tone and activity of cortical projection regions could improve prefrontal function, enhance inhibitory control and interfere with impulsivity and compulsive drug administration while helping to motivate the addicted person to engage in non-drug related behaviors.

Heat shock gene regulation by nascent polypeptides and denatured proteins: hsp70 as a potential autoregulatory factor.

Heat shock genes encode proteins (hsp's) that play important structural roles under normal circumstances and are essential to the cells' ability to survive environmental insults. Evidence is presented herein that transcriptional regulation of hsp gene expression is linked with the regulation of overall protein synthesis as well as with the accumulation of proteins denatured by stressful events. The factor that connects the three processes appears to be one of the hsp's, presumably a member(s) of the hsp70 family. Biochemical experiments demonstrate that complexes containing hsp70 and heat shock transcription factor, the specific regulator of hsp gene activity, are formed in the cells.

Pineal serotonin N-acetyltransferase: expression cloning and molecular analysis.

Pineal serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, or AA-NAT) generates the large circadian rhythm in melatonin, the hormone that coordinates daily and seasonal physiology in some mammals. Complementary DNA encoding ovine AA-NAT was cloned. The abundance of AA-NAT messenger RNA (mRNA) during the day was high in the ovine pineal gland and somewhat lower in retina. AA-NAT mRNA was found unexpectedly in the pituitary gland and in some brain regions. The night-to-day ratio of ovine pineal AA-NAT mRNA is less than 2. In contrast, the ratio exceeds 150 in rats. AA-NAT represents a family within a large superfamily of acetyltransferases.

Activation of human heat shock genes is accompanied by oligomerization, modification, and rapid translocation of heat shock transcription factor HSF1.

Transcriptional activity of heat shock (hsp) genes is controlled by a heat-activated, group-specific transcription factor(s) recognizing arrays of inverted repeats of the element NGAAN. To date genes for two human factors, HSF1 and HSF2, have been isolated. To define their properties as well as the changes they undergo during heat stress activation, we prepared polyclonal antibodies to these factors. Using these tools, we have shown that human HeLa cells constitutively synthesize HSF1, but we were unable to detect HSF2. In unstressed cells HSF1 is present mainly in complexes with an apparent molecular mass of about 200 kDa, unable to bind to DNA. Heat treatment induces a shift in the apparent molecular mass of HSF1 to about 700 kDa, concomitant with the acquisition of DNA-binding ability. Cross-linking experiments suggest that this change in complex size may reflect the trimerization of monomeric HSF1. Human HSF1 expressed in Xenopus oocytes does not bind DNA, but derepression of DNA-binding activity, as well as oligomerization of HSF1, occurs during heat treatment at the same temperature at which hsp gene expression is induced in this organism, suggesting that a conserved Xenopus protein(s) plays a role in this regulation. Inactive HSF1 resides in the cytoplasm of human cells; on activation it rapidly translocates to a soluble nuclear fraction, and shortly thereafter it becomes associated with the nuclear pellet. On heat shock, activatable HSF1, which might already have been posttranslationally modified in the unstressed cell, undergoes further modification. These different process provide multiple points of regulation of hsp gene expression.

Activation of the DNA-binding ability of human heat shock transcription factor 1 may involve the transition from an intramolecular to an intermolecular triple-stranded coiled-coil structure.

Heat stress regulation of human heat shock genes is mediated by human heat shock transcription factor hHSF1, which contains three 4-3 hydrophobic repeats (LZ1 to LZ3). In unstressed human cells (37 degrees C), hHSF1 appears to be in an inactive, monomeric state that may be maintained through intramolecular interactions stabilized by transient interaction with hsp70. Heat stress (39 to 42 degrees C) disrupts these interactions, and hHSF1 homotrimerizes and acquires heat shock element DNA-binding ability. hHSF1 expressed in Xenopus oocytes also assumes a monomeric, non-DNA-binding state and is converted to a trimeric, DNA-binding form upon exposure of the oocytes to heat shock (35 to 37 degrees C in this organism). Because endogenous HSF DNA-binding activity is low and anti-hHSF1 antibody does not recognize Xenopus HSF, we employed this system for mapping regions in hHSF1 that are required for the maintenance of the monomeric state. The results of mutagenesis analyses strongly suggest that the inactive hHSF1 monomer is stabilized by hydrophobic interactions involving all three leucine zippers which may form a triple-stranded coiled coil. Trimerization may enable the DNA-binding function of hHSF1 by facilitating cooperative binding of monomeric DNA-binding domains to the heat shock element motif. This view is supported by observations that several different LexA DNA-binding domain-hHSF1 chimeras bind to a LexA-binding site in a heat-regulated fashion, that single amino acid replacements disrupting the integrity of hydrophobic repeats render these chimeras constitutively trimeric and DNA binding, and that LexA itself binds stably to DNA only as a dimer but not as a monomer in our assays.

Synergistic activation of transcription is mediated by the N-terminal domain of Drosophila fushi tarazu homeoprotein and can occur without DNA binding by the protein.

Synergistic activation of transcription by Drosophila segmentation genes in tissue culture cells provides a model with which to study combinatorial regulation. We examined the synergistic activation of an engrailed-derived promoter by the pair-rule proteins paired (PRD) and fushi tarazu (FTZ). Synergistic activation by PRD requires regions of the homeodomain or adjacent sequences, and that by FTZ requires the first 171 residues. Surprisingly, deletion of the FTZ homeodomain does not reduce the capacity of the protein for synergistic activation, although this mutation abolishes any detectable DNA-binding activity. This finding suggests that FTZ can function through protein-protein interactions with PRD or other components of the homeoprotein transcription complex, adding a new layer of mechanisms that could underlie the functional specificities and combinatorial regulation of homeoproteins.

Tissue-specific transgenic knockdown of Fos-related antigen 2 (Fra-2) expression mediated by dominant negative Fra-2.

Fos-related antigen 2 (Fra-2) is a member of the Fos family of immediate-early genes, most of which are rapidly induced by second messengers. All members of this family act by binding to AP-1 sites as heterodimeric complexes with other proteins. However, each appears to have a distinct role. The role and biology of Fra-2 are less well understood than those of its relatives c-Fos, Fra-1, and FosB; moreover, Fra-2 target genes remain largely unknown, as does the basis of its selective effects on transcriptional activity. To pursue these issues, we created a transgenic rat line (NATDNF2) in which a dominant negative fra-2 (DNF2) gene is strongly expressed in the pineal gland; tissue selectivity was achieved by putting the DNF2 gene under the control of the rat arylalkylamine N-acetyltransferase (AANAT) regulatory region, which targets gene expression to a very restricted set of tissues (pineal gland >> retina). Expression of AANAT is normally turned on after the onset of darkness in the rat; as a result, pineal DNF2 expression occurs only at night. This was associated with marked suppression of the nocturnal increase in fra-2 mRNA and protein levels, indicating that DNF2 expression inhibits downstream effects of Fra-2, including the maintenance of high levels of fra-2 gene expression. Analysis of 1,190 genes in the NATDNF2 pineal gland, including the AANAT gene, identified two whose expression is strongly linked to fra-2 expression: the genes encoding type II iodothyronine deiodinase and nectadrin (CD24).

Clockless yeast and the gears of the clock: how do they mesh?

In spite of its apparent weakness as a clock model, the budding yeast has spawned a technique that has revolutionized our ability to study specific protein-protein interactions like those at the core of the molecular timekeeping mechanisms. Here, the author will summarize the evolution, power, and limitations of this technique and highlight its potential and actual contributions to the field of chronobiology.

Melatonin synthesis: analysis of the more than 150-fold nocturnal increase in serotonin N-acetyltransferase messenger ribonucleic acid in the rat pineal gland.

In vertebrates, the circadian rhythm in the activity of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87] drives the daily rhythm in circulating melatonin. We have discovered that expression of the AA-NAT gene in the rat pineal gland is essentially turned off during the day and turned on at night, resulting in a more than 150-fold rhythm. Expression is regulated by a photoneural system that acts through an adrenergic-cAMP mechanism in pinealocytes, probably involving cAMP response element-binding protein phosphorylation. Turning off AA-NAT expression appears to involve de novo synthesis of a protein that attenuates transcription. A approximately 10-fold night/day rhythm in AA-NAT messenger RNA occurs in the retina, and AA-NAT messenger RNA is also detected at low levels in the brain.

Differential regulation of fos family genes in the ventrolateral and dorsomedial subdivisions of the rat suprachiasmatic nucleus.

Extensive studies have established that light regulates c-fos gene expression in the suprachiasmatic nucleus, the site of an endogenous circadian clock, but relatively little is known about the expression of genes structurally related to c-fos, including fra-1, fra-2 and fosB. We analysed the photic and temporal regulation of these genes at the messenger RNA and immunoreactive protein levels in rat suprachiasmatic nucleus, and we found different expression patterns after photic stimulation and depending on location in the ventrolateral or dorsomedial subdivisions. In the ventrolateral suprachiasmatic nucleus, c-fos, fra-2 and fosB expression was stimulated after a subjective-night (but not subjective-day) light pulse. Expression of the fra-2 gene was prolonged following photic stimulation, with elevated messenger RNA and protein levels that appeared unchanged for at least a few hours beyond the c-fos peak. Unlike c-fos and fra-2, the fosB gene appeared to be expressed constitutively in the ventrolateral suprachiasmatic nucleus throughout the circadian cycle; immunohistochemical analysis suggested that delta FosB was the protein product accounting for this constitutive expression, while FosB was induced by the subjective-night light pulse. In the dorsomedial suprachiasmatic nucleus, c-fos and fra-2 expression exhibited an endogenous circadian rhythm, with higher levels during the early subjective day, although the relative abundance was much lower than that measured after light pulses in the ventrolateral suprachiasmatic nucleus. Double-label immunohistochemistry suggested that some of the dorsomedial cells responsible for the circadian expression of c-Fos also synthesized arginine vasopressin. No evidence of suprachiasmatic nucleus fra-1 expression was found. In summary, fos family genes exhibit differences in their specific expression patterns in the suprachiasmatic nucleus, including their photic and circadian regulation in separate cell populations in the ventrolateral and dorsomedial subdivisions. The data, in combination with our previous results [Takeuchi J. et al. (1993) Neuron 11, 825-836], suggest that activator protein-1 binding sites on ventrolateral suprachiasmatic nucleus target genes are constitutively occupied by DeltaFosB/JunD complexes, and that c-Fos, Fra-2, FosB and JunB compete for binding after photic stimulation. The differential regulation of fos family genes in the ventrolateral and dorsomedial suprachiasmatic nucleus suggests that their circadian function(s) and downstream target(s) are likely to be cell specific.

Evidence for a role of Hsp70 in the regulation of the heat shock response in mammalian cells.

Heat and other environmental insults (stress) cause unfolding of proteins, triggering the activation of heat shock transcription factor HSF (HSF1 in vertebrates) that, in higher eukaryotes, involves trimerization of the factor and acquisition of heat shock element (HSE) DNA-binding ability. Interaction of activated HSF1 with HSEs in promoters of genes encoding heat shock proteins (Hsps) enhances their expression. It was suggested that Hsp70 may function as the negative regulator of HSF1. In the simplest model, stress-unfolded proteins would compete with monomeric HSF1 for Hsp70 binding. This competition would result in dissociation of an HSF1-Hsp70 complex, allowing trimerization of released HSF1 monomers. In support of this model, we present evidence herein that 1) non-activated HSF1 forms a 1:1 complex with Hsp70, 2) both rates of heat-induced appearance of HSF1 oligomers and rates of disappearance of HSF1 heterodimers and monomers decrease when concentrations of unengaged Hsps are increased, and 3) transient overexpression of Hsp70 inhibits heat activation of HSF1.

The rat arylalkylamine N-acetyltransferase E-box: differential use in a master vs. a slave oscillator.

The rat arylalkylamine N-acetyltransferase (AA-NAT) gene encodes the key enzyme whose rhythmic expression drives the nocturnal production of melatonin. It is of interest that this enzyme is expressed virtually exclusively in two phylogenetically related tissues: retinal photoreceptors, which harbor an endogenous clock, and pinealocytes which, in higher vertebrates, function strictly in response to the master oscillator in the suprachiasmatic nucleus (SCN). While much is known about AA-NAT transcriptional regulation in the rat pineal gland (a slave oscillator), a full understanding of the mechanisms controlling AA-NAT gene expression in the retina by the clock is lacking. In the present study we have identified a functional E box in the first intron of the rat AA-NAT gene which is capable of mediating transcriptional upregulation via the action of a bMAL/CLOCK heterodimer. This is the first report to characterize the AA-NAT gene as a possible direct transcriptional target of the biological clock loop in a master oscillator.

cDNA array analysis of pineal gene expression reveals circadian rhythmicity of the dominant negative helix-loop-helix protein-encoding gene, Id-1.

The pineal gland is a major output of the endogenous vertebrate circadian clock, with melatonin serving as the output signal. In many species, elevated nocturnal melatonin production is associated with changes in pineal gene expression. In the current study, cDNA array analysis was used in an attempt to identify additional genes that exhibit day/night differential expression in the rat pineal gland. This revealed 38 candidate genes, including Id-1 (inhibitor of DNA binding and differentiation). Id-1 encodes a helix-loop-helix (HLH) protein that lacks a basic DNA binding domain and could affect pineal physiology via a dominant negative trans-acting regulatory activity. For this reason Id-1 was selected for further analysis. Id-1 was expressed in a major population of pineal cells and the Id-1 protein was associated with a nuclear complex. The levels of Id-1 mRNA and protein exhibit approximately six-fold day/night rhythms. In contrast, the related genes Id-2 and Id-3 do not exhibit marked day/night differences in pineal expression. Rhythmic Id-1 expression is primarily limited to a C-terminally extended splice variant of Id-1, which would restrict the functional output of the rhythm to protein binding partners of this isoform of Id-1. Our findings add to the body of evidence indicating that transcriptional regulators play a role in neuroendocrine rhythms, and extend this by introducing the concept of a dominant negative HLH involvement. The rhythm in Id-1 in the pineal gland provides an experimental opportunity to identify Id-1-binding partners which may also be involved in Id-1 activity in other functional contexts.

Addiction science: Uncovering neurobiological complexity.

Until very recently addiction-research was limited by existing tools and strategies that were inadequate for studying the inherent complexity at each of the different phenomenological levels. However, powerful new tools (e.g., optogenetics and designer drug receptors) and high throughput protocols are starting to give researchers the potential to systematically interrogate "all" genes, epigenetic marks, and neuronal circuits. These advances, combined with imaging technologies (both for preclinical and clinical studies) and a paradigm shift toward open access have spurred an unlimited growth of datasets transforming the way we investigate the neurobiology of substance use disorders (SUD) and the factors that modulate risk and resilience. This article is part of a Special Issue entitled 'NIDA 40th Anniversary Issue'.

Obesity and addiction: neurobiological overlaps.

Drug addiction and obesity appear to share several properties. Both can be defined as disorders in which the saliency of a specific type of reward (food or drug) becomes exaggerated relative to, and at the expense of others rewards. Both drugs and food have powerful reinforcing effects, which are in part mediated by abrupt dopamine increases in the brain reward centres. The abrupt dopamine increases, in vulnerable individuals, can override the brain's homeostatic control mechanisms. These parallels have generated interest in understanding the shared vulnerabilities between addiction and obesity. Predictably, they also engendered a heated debate. Specifically, brain imaging studies are beginning to uncover common features between these two conditions and delineate some of the overlapping brain circuits whose dysfunctions may underlie the observed deficits. The combined results suggest that both obese and drug-addicted individuals suffer from impairments in dopaminergic pathways that regulate neuronal systems associated not only with reward sensitivity and incentive motivation, but also with conditioning, self-control, stress reactivity and interoceptive awareness. In parallel, studies are also delineating differences between them that centre on the key role that peripheral signals involved with homeostatic control exert on food intake. Here, we focus on the shared neurobiological substrates of obesity and addiction.

Food and drug reward: overlapping circuits in human obesity and addiction.

Both drug addiction and obesity can be defined as disorders in which the saliency value of one type of reward (drugs and food, respectively) becomes abnormally enhanced relative to, and at the expense of others. This model is consistent with the fact that both drugs and food have powerful reinforcing effects-partly mediated by dopamine increases in the limbic system-that, under certain circumstances or in vulnerable individuals, could overwhelm the brain's homeostatic control mechanisms. Such parallels have generated significant interest in understanding the shared vulnerabilities and trajectories between addiction and obesity. Now, brain imaging discoveries have started to uncover common features between these two conditions and to delineate some of the overlapping brain circuits whose dysfunctions may explain stereotypic and related behavioral deficits in human subjects. These results suggest that both obese and drug-addicted individuals suffer from impairments in dopaminergic pathways that regulate neuronal systems associated not only with reward sensitivity and incentive motivation, but also with conditioning (memory/learning), impulse control (behavioural inhibition), stress reactivity, and interoceptive awareness. Here, we integrate findings predominantly derived from positron emission tomography that shed light on the role of dopamine in drug addiction and in obesity, and propose an updated working model to help identify treatment strategies that may benefit both of these conditions.

Auto-delayed-type hypersensitivity induced in immunodeficient mice with modified self-antigens. III. Suppressive T-cell factor controls the autoreactivity against self-antigens.

X-irradiated (250 rad), cyclophosphamide-treated or ATx A mice injected with syngeneic trinitrophenylated spleen cells (TNP-SC) and footpad challenged with syngeneic lymphoblasts generated delayed-type hypersensitivity (DTH) responses 24, 48 and 72 h after challenge. The syngeneic-DTH (syn-DTH) response was mediated by Lyt-1+ cells and suppressed with Lyt-1+2+3+, I-Jk+ cells. The suppressor cells were obtained from spleens or thymuses of normal syngeneic mice. Suppressor factor (SF) was extracted or released from Lyt-1+2+3+, I-Jk+ cells obtained from normal A mice (but not from X-irradiated A mice). The factor blocked the DTH responses of X-irradiated mice injected with syngeneic TNP-SC and challenged with syngeneic lymphoblasts when injected into the mice both at the induction phase and the elicitation phase of the DTH. The factor failed to abrogate allogeneic and xenogeneic DTH. However, allogeneic factor (derived from C57BL/6 mice) abolished the syn-DTH response of mice injected with syngeneic TNP-SC and challenged with syngeneic lymphoblasts. The SF was produced by Lyt-1+2+3+, I-Jk+ T cells or by thymocytes. The combined extracted product of Lyt-1+ and Lyt-2+ cells did not abrogate the syn-DTH response. Normal spleen cells depleted of phagocytes by a magnetic procedure also produced the SF. These findings indicate, therefore, that suppressive factor (or factors; see Discussion in the accompanying paper, Ref. 17) controls the immunological autoreactivity against syngeneic TNP-SC.

Circadian expression of transcription factor Fra-2 in the rat pineal gland.

Physiological changes in Fos-like immunoreactivity in the rat pineal gland are shown here to be due primarily to changes in a 42/46-kDa Fos-related antigen (Fra). Studies are presented that indicate this 42/46-kDa Fra is Fra-2, a poorly understood member of the Fos family of transcription factors. Both Fra-2 mRNA and protein are absent during the day and increase robustly at night on a circadian basis; organ culture studies indicate that regulation is mediated by an adrenergic-->cyclic AMP mechanism. AP-1 binding activity changes in parallel to changes in the level of Fra-2 protein.

The rat arylalkylamine N-acetyltransferase gene promoter. cAMP activation via a cAMP-responsive element-CCAAT complex.

A 10-100-fold rhythm in the activity of arylalkylamine N-acetyltransferase (AA-NAT; EC 2.3.1.87) controls the rhythm in melatonin synthesis in the pineal gland. In some mammals, including the rat, the high nocturnal level of AA-NAT activity is preceded by an approximately 100-fold increase in AA-NAT mRNA. The increase in AA-NAT mRNA is generated by norepinephrine acting through a cAMP mechanism. Indirect evidence has suggested that cAMP enhances AA-NAT gene expression by stimulating phosphorylation of a DNA-binding protein (cAMP-responsive element (CRE)-binding protein) bound to a CRE. The nature of the sites involved in cAMP activation was investigated in this report by analyzing the AA-NAT promoter. An approximately 3700-base pair fragment of the 5'-flanking region of the rat AA-NAT gene was isolated, and the major transcription start points were mapped. The results of deletion analysis and site-directed mutagenesis indicate that cAMP activation requires a CRE.CCAAT complex consisting of a near-perfect CRE and an inverted CCAAT box located within two helical turns.