over the past several years , we have developed several animal models for acute , subacute , and chronic exposure to specific drugs of abuse , with emphasis on cocaine , morphlne and heroin , and alcohol . one of these models , which we have developed , validated , and used extensively in our studies , is  binge -pattern cocaine administration mimicking the most common pattern of human abuse . in studies from our laboratory in which animals were allowed to self - administer cocaine with presentation of high , as well as moderate , and the usual low doses of cocaine , and with extended access ( 10 hours ) provided , we found that animals will escalate their use of cocaine . in fact , by 5 days of extended access to high doses of cocaine rats will self - administer more than twice the dose which we had usually used in our chronic binge - pattern  cocaine administration ( 15 mg / kg x3 , that is 45 mg / kg / day ) . we have extended this  binge - pattern ,  using both the steady - dose and escalating - dose  binge - pattern  administration of cocaine . we have been able to study various behavioral factors , as well as impact on gene expression , comparing these two models . one of the most important early findings from our laboratory ( and others ) on gene expression has been the finding of significant increased preprodynorphin gene expression in the striatum of rodents after acute , subacute , and chronic cocaine administration ( eg , refs 20,21 ) . this is especially important since we and others have shown that dynorphin peptides , which are the natural endogenous opioid ligands of the kappa - opioid receptors , serve to modulate dopaminergic tone and countermodulate cocaine - induced dopaminergic surges . in a recent study , we examined the effects of steady - dose versus escalating - dose binge - pattern cocaine administration upon striatal preprodynorphin messenger ribonucleic acid ( mrna ) levels , and also on behavioral stereotypy . we found that both steady - dose and escalating - dose binge cocaine administration resulted in increased preprodynorphin mrna levels in the caudate - putamen , but not in the nucleus accumbens . these are similar to all our earlier studies of the impact of acute , subacute , and chronic cocaine administrations . in this study , there were no significant differences in preprodynorphin mrna levels when escalating doses ( up to 30 mg / kg x 3 , or a total of 90 mg / day ) were administered during the last five days of 14-day chronic dosing , compared with a total of 45 mg / day , the steady dose these data showed that the enhancement of gene expression of dynorphin response to cocaine has probably reached its maximum level at a dose of 45 mg / kg / day of cocaine , and may or may not be dose - dependent at lower doses . further , in this study it was found that cocaine significantly affected body weight in both paradigms , and that both resulted in expression of behavioral stereotypy . however , of note , one component of stereotypy , that is , intense rapid head movements , was found to be both doseand time - dependent , with more profound effects in the escalating - dose model . extending our much earlier studies in the rat , the effects of the natural kappa - opioid receptor agonist , dynorphin a ( l-17 ) , on both basal striatal dopamine levels and on cocaine - induced increases in striatal dopamine levels , as well as on cocaine - induced conditioned place preference , was studied in c57bl/6j mice . in earlier studies conducted in the rat , we had shown that dynorphin applied directly into the striatum causes a dose - dependent reduction in dopaminergic levels . in this recent study , dynorphin , at four different doses , was infused into the caudate - putamen , and dopamine levels were quantitatively measured , using high - performance liquid chromatography , in the extracellular fluid obtained during in vivo microdialysis in that brain region . also , the effect of a relatively high dose of dynorphin a on increases in dopamine levels caused by 15 mg / kg of cocaine was measured using in vivo microdialysis . in related studies , the effect of this dose of dynorphin a on cocaine - induced conditioned place preference was studied . we found that dynorphin significantly decreased basal dopamine levels in a dose - dependent manner and by more than 60% at the highest dose . further , this effect was blocked by preinjection with a selective kappa - opioid receptor antagonist , nor - binaltorphimine ( nor - bni ) . further , it was found that the highest dose of dynorphin studied ( 4.4 nanomolar ) resulted in a complete block of the cocaineinduced increases in dopamine levels , and also attenuated locomotor activity induced by 15 mg / kg of cocaine , and blocked the formation of cocaine - induced conditioned place preference . these findings suggest that a dynorphin agonist might be helpful in managing cocaine and other stimulant dependency by preventing cocaine or other stimulant - induced dopamine surges . however , on the other hand , any significant lowering of basal dopaminergic tone could lead to dysphoria , and thus more craving for a drug of abuse such as cocaine . therefore , it has made our laboratory suggest that a potentially effective kappa - opioid receptor - directed compound for management of cocaine addiction would probably be a kappa partial agonist , that is , with modest agonist activity , but also antagonist activity , which should render stable basal dopaminergic tones , yet significantly attenuate cocaineor other stimulant - induced dopamine surges , as well as  liking of  cocaine . in related studies , zhang et al studied a related potent synthetic kappa - agonist , r-84760 , on cocaine - induced increases in striatal dopamine levels in cocaine - induced conditioned place preference in c57bl/6j mice . r-84760 is a novel nonpeptidic potent synthetic selective kappa - opioid receptor agonist that has been studied to a limited extent in humans for other indications . it was found that , similarly to dynorphin itself , this compound would effect a dose - dependent reduction in dopaminergic tone , as measured during in vivo microdialysis in the striatum . also , it was shown that , like dynorphin , a low dose ( 0.1 mg / kg ) of r-84760 would block cocaineinduced increases in the dopamine levels . also , it was found that similarly low doses of r-84760 would completely prevent the development of cocaine - induced conditioned place preference and would attenuate locomotor activity in the conditioning chamber . further , it was documented that these effects of r-84760 on lowering dopaminergic tone and cocaine - induced surges were completely blocked by a selective kappa - antagonist , norbni . thus , these effects were documented to be mediated exclusively by the kappa - opioid receptor . in different studies , we further explored the impact of extended - access ( 10 hours ) versus short - access ( 3 hours ) and also highversus low - dose cocaine impact on self - administration , cocaine - induced reinstatement , and on brain mrna levels . it was again found that the escalation of cocaine self - administration under long - access conditions was greater than under short - access , and was dose - dependent . further , we showed that such longaccess , with animals who were allowed self - administration for 10 hours at high doses , resulted in an increased susceptibility to drug - induced relapse . there were also differences in neurobiological indices , specifically levels of gene expression in those animals who were allowed to have long access and high doses , compared with short access . there were significant increases in proenkephalin gene expression in the caudate - putamen following longaccess and high - dose self - administration . further , it was found that dopamine d2 receptor mrna levels in the caudate - putamen and nucleus accumbens were significantly correlated with cocaine reinstatement . however , there was no significant correlation between neuropeptide mrna levels and cocaine - induced reinstatement . body weight progressively declined in the long - access self - administering rats . in parallel to these findings , food consumption was also significantly reduced in each group during self - administration , but the reduction in food intake was much greater in the long - access rats . during the 10-day extinction period , food consumption was significantly greater in the long - access , high - dose rats compared with both the short - access and the low - dose rats , and , in fact , food consumption during extinction in the high - dose group was significantly greater than pre - self - administration baseline levels . these findings are similar to observations made by our group in human cocaine addicts in a controlled research setting . they have negative implications for some groups of people , where the desire for thinness , or the desire for attaining the self - image of thinness , may contribute to continued cocaine ( or other stimulant ) self - administration . the many findings from these long - access , high - dose cocaine self - administration rodent studies , both our more recent ones , as well as our earlier ones , along with the studies from other groups , particularly those of koob and of miczek , suggest that the findings may not only be relevant potentially for the human situation , but provide new insights for further study both in laboratory - based and human research paradigms . we have conducted studies in collaboration with the laboratory of dr paul greengard in which we have studled the impact of change of a single amino acid in an important signal transduction protein on ( i ) both dopaminergic responses to binge - pattern cocaine ; as well as ( ii ) acquisition ; and then ( iii ) persistence and amounts of self - administration of cocaine . these studies were conducted in four separate lines of mutant mice , each with a mutation code for alanine introduced into the gene for the protein darpp-32 at four sites of phosphorylation . the four sites of phosphorylation chosen were : ( i ) the protein kinase a site , threonine 34a ; ( ii ) the cyclin - dependent kinase-3 site , threonine 75a ; ( iii ) the kinase ck2 site , serine 97a ; and finally , ( iv ) the kinase ck1 site , serine 130a . in each case , animals were bred so that both the mutant strain , as compared with the wildtype strain , could be studied , with the single amino acid change introduced into one of these four sites of critical phosphorylation involved in different pathways of the dopamine d1 receptor signal transduction through the darpp-32 cascade pathway acquisition of self - administration required significantly more time in the threonine 34a-/- mice . however , once self - administration was established , both threonine 34a and the serine 130a darpp-32 mutant mice administered significantly more cocaine than did their wild - type controls . this became especially apparent after training each of these strains on a high dose of cocaine ( 1 mg / kg ) and then starting the self - administration studies for each strain using an even higher dose of cocaine per injection ( 2 mg / kg ) , but then progressing downward in concentration to 1.0 , .05 , and .01 mg / kg per injection . as the dose was reduced below 1.0 mg / kg per injection , both the threonine 34a and the serine 130a mice significantly increased lever pressing to obtain more cocaine than did their matched wild - type controls . such an increase during reduction of cocaine concentration was not seen in either the threonine 75a or the serine 97a mice . this suggests that although somewhat slower to acquire self - administration , both the threonine 34 site and the serine 130 site of darpp-32 phosphorylation are important for the persistence of , and though not studied , possibly also to relapse to , cocaine self - administration . further , and in support of these findings , studies using microdialysis in the freely - moving mice could be carried out in three of the four strains ( the fourth strain was not available in adequate numbers for study . ) when this was performed , it was found that the same two strains that administered more cocaine , that is , the threonine 34a and the serine 130a , experienced a much lower rise in extracellular fluid dopamine after each of three binge cocaine injections than did the control mixed wild - type animals . further , this did not happen in the threonine 75a ; these animals had a much higher level of dopamine achieved after each dose of binge cocaine , and these were animals that showed no difference between the single amino acid change mutant strain and the wild - type strain . these findings suggest that a single amino acid change of a critical phosphorylation site may alter the behaviors of self - administration ; they also give further support to the concepts of many groups , that a lower dopaminergic tone either at rest , or achieved after any normal ( for instance , a liked or desired food ) or abnormal ( for instance , cocaine ) self - administration , may result in a lesser increase in dopamine tone . thus , such animals ( or possibly people ) could be expected to seek more activation of this pleasure - related dopaminergic system , and thus have a greater vulnerability to developing an addiction . we have conducted studies in which morphine was self - administered by animals and was available 18 hours / session / day . in these studies , animals were allowed to select a more concentrated or less concentrated morphine solution and once stable choice was established , the concentrations were increased . the animals allowed such a choice both escalated their morphine use to a much greater extent than did steady - dose animals . after 14 days the animals were self - administering extremely large amounts of morphine in the extended - access and escalating high - dose model . these studies showed that the average daily morphine self - administration increased from 22.5 mg on day 1 up to 66.4 mg by day 14 . in addition to our neurobiological studies of drug addiction by more traditional methods , such as gene expression , we have been collaborating with dr virginia pickel 's laboratory in the use of immunogold electron microscopy ( em ) to study drug - induced receptor trafficking . in these studies we have been exploring the effects of chronic intermittent self - administration of escalating doses of morphine on ionotropic glutamate receptor subunit trafficking in postsynaptic ( ie , dendritic ) sites in neurons , a process that is emerging as a critical cellular substrate of neural plasticity . because immunogold em can be used to localize receptors near intracellular organelles , as well as presumably functional areas of the plasma membrane , this approach provides a more functional view than many of the more conventional methods of measuring receptor levels . we have been using immunogold em to study glutamate receptor localization in neurons in portions of limbic - autonomic brain areas , namely the reciprocally connected nucleus tractus solitarius ( nts ) and central ( cea ) and basolateral ( bla ) nuclei of the amygdala , a brain circuit that may play a critical role in homeostatic adaptations associated with repetitive drug use . we have reported that the n - methyl - d - aspartate ( nmda)-nrl receptor subunit is decreased on the dendritic plasma membrane of nts neurons in animals self - administering morphine , compared with control animals not exposed to morphine . further , morphine self - administering rats showed regiondependent changes in the subcellular location of the ampa - glur1 receptor subunit in the amygdala . specifically , there was an increase in ampa - glur1 labeling on the dendritic plasma membrane of bla neurons and a concomitant decrease in dendritic ampaglur1 in cea neurons from animals self - administering morphine compared with control animals . these findings suggest that chronic opiate self - administration is associated with a redistribution of postsynaptic plasma membrane glutamate receptor subunits that play an important role in neural plasticity in brain circuitry regulating homeostatic processes . these adaptations may be an important neural substrate for alterations in drug reward , autonomic function , and behavioral processes , each of which may be associated with the acquisition and persistence of an addiction . 
  in four separate earlier studies from our laboratory we have shown that chronic ( 14 days ) binge - pattern cocaine administration increases mu - opioid receptor mrna levels and also increases density of mu - opioid receptors in specific brain regions where there are abundant dopaminergic terminals from neurons located in the ventral tegmental area . in recent studies , bailey and our group have shown that early withdrawal from chronic binge cocaine administration results in a recurrence of an increase in mu - opioid receptor mrna levels in the rat frontal cortex , but only in this region . in further studies , bailey found that there is a persistent upregulation of mu - opioid receptors following long - term withdrawal from escalating - dose binge - pattern cocaine . in these studies , animals were treated with our new modified paradigm of escalating - dose binge cocaine over 14 days , which also results in an increase of mu - opioid receptor density , but with no increase in endogenous endorphin levels . following 14 days of withdrawal , there was still a highly significant increase in mu - opioid receptor density , and primarily in specific brain regions , again where there are dopaminergic terminals from the ventral tegmental area neurons and in fields in close proximity to both muopioid receptor mrna levels in the neurons producing mu - opioid receptors and presenting them on the cell surface . 
  in a further set of studies , bailey explored changes in the kappa - opioid receptors following 14-day withdrawal from escalating - dose binge - pattern cocaine . here , very different findings were made . whereas in multiple studies from our laboratory we have found both increases in gene expression of dynorphin , and increases in kappaopioid receptor densities , and a correlated increase in kappa - opioid receptor mrna levels , with kappa , unlike mu - opioid receptors , which are found to be persistently increased in density following 14 days of withdrawal from binge - pattern escalating - dose cocaine , in this study there was lowering of kappa - opioid receptors in two specific brain regions in animals in long - term withdrawal from cocaine . such a decrease in density was not found in other regions , but also with no persistence of increase in density . these selective brain regions of decrease in kappa - opioid receptor might contribute , in part , to the biological substrate for the development of dysphoria , which is usually observed in drug - free former cocainedependent individuals . in our recent studies , we have also further explored the relative role of dopamine d1 and dopamine d2 receptors in various specific neurobiological changes , or neural plasticity , resulting from chronic exposure to cocaine . since it has been well established that dopamine plays a major role in the rewarding properties of cocaine , and since it has been established for a long time that one of cocaine 's prlmary sites of action is the presynaptic reuptake transporter for dopamine , where cocaine , by blocking reuptake , effects a flooding of perlsynaptlc space with dopamine , we have tried to dissect out the relative role of dopamine d1-like versus dopamine d2-like receptors in some of the resultant changes , both in behaviors , but also in gene expression and neuropeptide levels . during the last 5 years , we have completed further studies of the effects of selective dopamine d1-like and also dopamine d2-like receptor antagonists during acute binge - pattern cocaine administration on corticotropin - releasing factor ( crf ) mrna levels and pro - opiomelanocortin ( pomc ) mrna levels in the hypothalamus . in earlier studies , we have found that both dopamine d1-like and also dopamine d2-like antagonists attenuate the chronic binge - pattern cocaine - induced increase in adrenocorticotropin hormone ( acth ) and corticosterone levels . further , we have shown that an attenuation of cocalne - induced changes in stress hormone levels similarly occurs in animals with complete deletion of the darpp-32 protein , which is involved directly in dopamine d1 receptor signal transduction . in our recent studies , we again found that both dopamine d1-like and dopamine d2-like antagonists attenuated the elevation of corticosterone levels by acute , as well as in our earlier studies of chronic , binge - pattern cocaine . the previously identified acute binge cocaine - induced increases in hypothalamic crf mrna levels were not found in rats pretreated either with a dopamine d1-like or d2-llke antagonist . further , we found that neither the dopamine d1-like or dopamine d2-like receptor antagonists alone , in the absence of cocaine , altered mrna levels of crf in the hypothalamus . thus , these results further support our earlier concept , that both dopamine d1 receptors and dopamine d2 receptors mediate acute as well as chronic cocaine 's stimulatory effects on the hypothalamic - pituitary - adrenal ( hpa ) axis . 
  since neurobiological evidence has suggested that there are functional interactions between the dopaminergic and opioid systems regulating preproenkephalin and preprodynorphin expression in the striatum , and since there is increasing evidence there may be direct connections between the dopaminergic system in the striatum and the stress - responsive components of the hypothalamus , we also raised the question of whether dopamine d1-like or dopamine d2-like antagonists could play a role in regulation of pomc mrna levels in the hypothalamus . in this part of these studies , it was found that dopamine d2-like receptor blockade increased the pomc mrna levels in the hypothalamus , a site with a different function than pomc mrna levels in the anterior pituitary . these findings suggest that activation of the dopamine d2 receptor may play a tonic inhibitory tone on hypothalamic pomc gene expression . however , neither dopamine d2 blockade nor acute binge cocaine altered pomc mrna levels in the amygdala , the anterior pituitary , or the neurolntermedlate level of the pituitary . also , dopamine d1 receptor blockade had no impact on hypothalamic pomc expression . thus , these results both suggest a possible specific role for dopamine d2 in at least acute cocaine effects on hypothalamic pomc gene expression . 
  to further our studies on the relative role of the d1-like and d2-like ( and also d3-like , which are d2-like ) dopamine receptors in the setting of drug abuse , and , specifically the impact of binge - pattern cocaine administration , we have conducted studies using d1-/- or d3-/- selective dopamine receptor gene deletion mice . in these studies , we examined mu - opioid receptor gene expression in response to binge - pattern cocaine . we found that , at basal state , there was a significant increase in mu - opioid receptor mrna levels in the frontal cortex of both the d1-/- and d3-/- dopamine receptor gene deletion mice , as compared with each of their wild - type controls . however , there were no differences in basal levels of mu - oploid gene expression in the nucleus accumbens or in the caudate - putamen in these gene deletion mice . strikingly , and in an opposite direction from some of our earlier findings in wild - type rat models , acute binge cocaine 15 mg / kg x 3 doses resulted in the restoration of frontal cortex mu - opioid receptor mrna levels in the gene deletion mice to the levels of those in wildtype mice . further , in the nucleus accumbens core , after acute binge cocaine , there was an actual decrease in mu - opioid receptor levels in the d1-/- mutant mice , whereas in that brain region there was an increase in mu - opioid receptor gene expression in d3-/- mice . the opposite pertained in the caudate - putamen , with an increase in mu - opioid receptor levels after binge cocaine in the caudate - putamen of the d1-/- mice and a decrease in the dopamine d3-/- mice . in addition , a decrease in basal orexin mrna levels was found in the lateral hypothalamus of the d3-/- mice , which did not change with cocaine . these findings suggested that both d1 and d3 receptors are involved in mu - opioid receptor gene regulation in the frontal cortex , and also that d1 and d3 receptors may play opposite roles in the effects of cocaine on mu - opioid receptor gene expression in two striatal areas , the caudate - putamen and the nucleus accumbens core . in the control wild - type mice for the d1 receptor gene deletion , binge - pattern cocaine , as expected , increased mu - opioid receptor gene expression . however , in the wild - type controls for the dopamine d3 receptor knockout mice , there was a very modest , but not significant , increase in mu - opioid gene expression after binge cocaine , which was unexpected . these findings were made both in the caudate - putamen and in the nucleus accumbens core , suggesting that in the actual breeding of the wild - type animals , the controls for the d3 knockout groups may have been substantially different from the wild - type mice which were the controls to the d1 knockout mice . of particular note was the finding of increased basal levels of mu - oploid gene expression in both the d1 and d3 knockout mice , though only in the frontal cortex . these curious findings need to be studied further in d1 and d3 gene deletion mice , and also in dlfferent strains of wild - type mice . most of the other studies of the impact of drug - induced stress on many different parameters , with emphasis on documentations of specific changes or evidence of neuroplasticlty , have been conducted in rat models . in one sequence of studies , we examined the effects of acute morphine administration ; chronic intermittent escalating - dose morphine ( from 7.5 mg / kg / day on day 1 up to 120 mg / kg / day on day 10 ) ; and spontaneous 12-hour withdrawal from chronic morphine administration , using the escalating dose 10-day paradigm . there were no changes in mu - opioid receptor mrna levels in the lateral hypothalamus , the nucleus accumbens core , the caudate - putamen , or the amygdala following acute single injection of morphine , nor after chronic 10-day intermittent escalatlng - dose morphine . however , after 12 hours of withdrawal from 10-day chronic morphine administration , several indices documenting stress response in the hpa axis were found , including increased pomc mrna levels in the anterior pituitary , coupled with increases in acth levels , and also increased mu - opioid receptor mrna levels in the lateral hypothalamus , the nucleus accumbens core , and the caudate - putamen . the changes in mu - opioid receptor gene expression suggest both a rebound from the abrupt withdrawal from large doses of the exogenous opioid morphine , as well as changes integral to the hpa stress - responsive axis , as well as in the hypothalamus . several studies from other laboratories have demonstrated a role of lateral hypothalamic orexin ( hypocretin ) activation in drug - related positive reward , as well as in withdrawal effects ; therefore gene expression of this peptide was also studied . it has been established by others that around half of the lateral hypothalamic orexin neurons concomitantly express mu - opioid receptors . in parallel to the increase in mu - opioid receptor gene expression found in the lateral hypothalamus in acute morphine withdrawal , similarly the levels of orexin mrna in the lateral hypothalamus were also found to be increased . no changes were found in the lateral hypothalamic levels of preprodynorphin mrna , a gene which is known to be usually coexpressed with orexin in that hypothalamic region . these findings suggest that many different responses to the stress of morphine withdrawal occur , or , alternatively , changes which occur in the setting of withdrawal may drive the hpa axis activation and stress of withdrawal , just as we have found to be the case in our clinical studies . further , they suggest that in the lateral hypothalamus activation of orexin gene expression occurs in parallel to mu - oploid receptor gene expression . in a subsequent series of studies , a similar but somewhat different opioid administration paradigm was used . in these studies , heroin , the most common human opiate of abuse , was used , coupled with a chronic , intermittent escalating - dose administration paradigm and conducted with doses of heroin ranging on day 1 from 7.5 mg / kg up to 60 mg / kg by day 10 ( it should be noted that in this intermittent morphine escalating - dose paradigm , the starting dose was the same for heroin and morphine ( 7.5 mg / kg ) , but after 10 days , the escalation was up to 120 mg / kg when morphine was used , and 60 mg / kg when heroin was used . one group of animals was then studled at the end of chronic escalating heroin administration ; other animals were studied during early f 2-hour withdrawal from such chronic heroin exposure ; and a third group was studied after late 10 days of withdrawal from chronic heroin exposure . in this study , it was found that arglnine vasopressin mrna levels were significantly increased during early spontaneous withdrawal , and , of several brain regions examined , only in the amygdala . further , separate studies showed that arglnine vasopressin mrna levels were increased not only in early spontaneous withdrawal from heroin in the amygdala , but also following foot - shock in rats withdrawn from heroin self - administration . such findings were not made in the self - admlnistration control , heroin - nave rats . this increase in arglnine vasopressin mrna levels was no longer observed following 10 days of withdrawal from chronic heroin . as in earlier studies , pomc mrna levels in the anterior pituitary were found to be increased , both 30 mm after chronic heroin administration , which probably is a sign of very early withdrawal , as well as at 12 hours of withdrawal from heroin . similarly , acth levels were increased in early withdrawal , coupled with a significant increase in plasma corticosterone , after 12 hours of withdrawal . although the levels of both acth and corticosterone at the end of the chronic heroin administration , and thus 30 min , after the last dose , were somewhat greater than those in the salinetreated controls , these changes were not significant . 
  in much earlier basic clinical research studies , performed in a stress - minimized research unit , documented that plasma levels of acth and cortisol became elevated before any signs and symptoms of opioid abstinence were observed or reported following very - low - dose opioid antagonist administration in opioid - dependent persons , suggesting that hpa axis activation drives , in part , the stress of opioid withdrawal , rather than reflecting a response to that stress . 
  in separate , but related , studies , a model of heroin self - administration was used . the dose of heroin administration was 0.05 mg / kg per infusion , and 7 dally short - access ( 3-hour ) sessions were used . since vasopressin mrna elevations had been observed in animal models of heroin withdrawal , these studies were designed to look at the effects of a vasopressin receptor ( v1b receptor ) antagonist , ssr149415 , in that setting . administration of this compound was before the first extinction , or drug withdrawal , session . the vasopressin receptor antagonist dose - dependently attenuated foot - shock - induced reinstatement and blocked heroin - induced reinstatement . all these data suggest that arginine vasopressin activation may occur during withdrawal from opiates , and suggest that this peptide also may contribute to relapse or reinstatement . further , it is shown that , in the stress of withdrawal , when foot - shock is added , there is a significant increase in gene expression , and thus probably in the arginine vasopressin peptide . most important , the data suggest that a vasopressin antagonist might attenuate either stress ( in these experiments , withdrawal - induced and foot - shock - induced ) , and also drug - induced reinstatement and relapse to opiate self - administration or use . in other separate studies , possible alterations of arginine vasopressin mrna levels in the amygdala were studied in animals undergoing acute withdrawal from cocaine . in these studies , our model of steady - dose binge - pattern ( 15 mg / kg every hour x 3 hours with no cocaine for 22 hours ) administration for 14 days was used , followed by acute withdrawal ( 3 hours ) , subacute withdrawal ( 24 hours ) , and long - term withdrawal ( 10 days ) . it was found that , although there were no changes in arginine vasopressin mrna levels in the amygdala immediately following 14 days of cocaine administration , there were increases in arginine vasopressin mrna levels in acute withdrawal ( 3 hours ) from cocaine . further , it was found that the selective opioid receptor antagonist naloxone blocked this increase . as found in previously reported studies from our laboratory , chronic cocaine did not result in increased mu - opioid mrna levels in the amygdala , nor did acute withdrawal from cocaine in these studies . at 24 hours of withdrawal , significant increases in arginine vasopressin mrna levels in the amygdala were still observed . however , these levels had returned to normal after 10 days of withdrawal . as found in our previous studies , adaptation or tolerance to the cocaine effects on the hpa axis activation as expected , naloxone produced modest elevations in acth levels in cocane - nave rats ; naloxone did not have such an effect in the acute or subacute cocaine - withdrawn animals . there were no changes in arginine vasopressin , or pomc , or mu - opioid receptor mrna levels in the hypothalamus following chronic cocaine administration , and acute withdrawal from cocaine . these findings suggested that opioid receptors may mediate the increase in arginine vasopressin in the amygdala during acute cocaine withdrawal , and suggest a potential role for arginine vasopressin in the amygdala in some of the adversive effects of withdrawal from cocaine as well as in withdrawal from opiates . a recent set of laboratory - based studies in rats affirm , and further suggest a mechanism , for observations which we have made in two separate clinical studies , around 7 years apart , and in two parts of the world . we have determined that steady - state methadone may attenuate or eliminate the liking of cocaine , and may do so by a mu - opioid receptor - mediated mechanism in several earlier studies , as discussed above , we have shown that chronic binge - pattern cocaine administration results in an increase in mu - opioid receptor density in multiple , but not all , brain regions , and specifically in regions where there are abundant dopaminergic terminals from dopamine neurons in the ventral tegmental area and substantia nigra compecta . further , we have shown that acute and subacute , but not chronic , cocaine administration results in an increase in mu - opioid receptor mrna levels . in these recent studies , rats were implanted with either salineor methadone - filled osmotic minipumps and then conditioned with 1 , 5 , or 20 mg / kg cocaine intraperltoneally . animals with the 20 mg / kg / day or 55 mg / kg / day methadone - filled osmotic pumps did not express cocaine - induced place preference . however , methadone pumps at two doses ( 30 and 55 mg / kg / day ) did not alter intravenous self - administration of cocaine using a continuous schedule of reinforcement with different doses of cocaine ( 0.1 , 0.5 , and 2.0 mg / kg / infusion ) studied . mu - opioid receptor mrna levels were measured in animals treated with cocaine as part of conditioning for place preference . as in earlier studies , it was shown that this subacute cocaine administration resulted in increased mu - opioid receptor mrna levels in the nucleus accumbens core and in the frontal cortex 10 days after cocaine conditioning . however , this increase in mu - opioid receptor mrna levels was attenuated or eliminated by the steady - dose infusion of methadone . earlier studies have shown that the dose of 55 mg / kg / day subcutaneously by pump in the rat results in a plasma level similar to that in patients seen in methadone maintenance . these studies showed that , although high doses of methadone delivered by pump did not alter the direct reinforcing effects of cocaine as seen in self - administration , those doses of methadone did block both spontaneous and cocaine - induced  seeking  or  liking  10 days after cocaine conditioning . further , we have suggested that this may be through the mechanism of methadone attenuating or preventing the relative endorphin deficiency resulting from the increased mu - opioid receptor density preceded by increased mu - opioid receptor gene expression , but with no concomitant increase in the endogenous opioids that bind to the mu receptor , that is , no increase in beta - endorphin or in the enkephalin peptides . these studies also build upon the early and also much more recent findings that , despite the fact that up to 70% of all persons in the middle atlantic states , as well as currently in tel aviv , israel , have concomitant dependence upon cocaine , when presenting for treatment for longstanding dependence on heroin , after 1 year or more of methadone treatment , as expected , the numbers using heroin dropped precipitously , to less than 20% of patients using heroin at any time ( as contrasted to heroin use by all patients 3 to 6 times a day prior to entry ) . this was accompanied by the more surprising findings that during steady - dose methadone maintenance treatment , the percentage of persons dependent on cocaine drops down to less than 20% , and those using any cocaine to less than 30 % . although these beneficial results of methadone maintenance on managing cocaine addiction were always attributed to the counseling and other psychosocial benefits derived from a good methadone maintenance program , we have , over the last decade , hypothesized that a pharmacological mechanism also is in place , a hypothesis based on our findings that binge cocaine increases acutely mu - opioid receptor gene expression and on a chronic basis , mu - opioid receptor density , and further , that a relative endorphin deficiency thus develops in humans , since there is no concomitant increase of beta - endorphin or enkephalins , as may be directly documented by stress - responsive metyrapone testing . these findings suggest that possibly an opioid agonist such as methadone , or possibly a partial agonist , such as buprenorphine , might be able to be effectively used to treat very severe , long - term , cocaine - dependent persons who have not responded to any other available current treatment . since there are no effective targeted pharma - cotherapies for cocaine addiction , the potential target of the mu - opioid receptor , with now a neurobiological basis for such treatment , might be warranted . in other studies , conducted by a collaboration with our colleagues at the karolinska institute in stockholm , sweden , yet another potential target for future therapeutic use , a noclceptin / orphanin fq receptor agonist ( ro64 - 6198 ) was found to reduce alcohol self - administration , and , further , and importantly , to prevent relapse to alcohol drinking in a rat model . other orphanin - nociceptin ( orl-1 ) receptor agonists may be found to have effectiveness in treatment of alcoholism and possibly other specific addictive diseases , which involve interactions between the dopaminergic system and different components of the opioid and opioid - like system . corticotropin - releasing factor ( crf ) , synthesized and released in the hypothalamus , passes through the portal blood system to the anterior pituitary , where it effects processing and release of the single gene product of the pomc gene ( reviewed in ref 7 ) . this large peptide is then further processed to yield many biologically active and important neuropeptides , including the major stress - responsive and glucocorticoid - regulated peptide , acth , as well as the longest ( 31 amino acids ) of the endogenous opioids , and a primary ligand of the endogenous mu - opioid receptor , beta - endorphin . acth and beta - endorphin are released in equimolar amounts from the anterior pituitary sites in humans ( who , unlike rodents , do not possess an intermediate lobe in the pituitary except transiently during pregnancy . ) acth impacts directly upon the adrenal cortex to bring about the processing and release of the major glucocorticoid in humans , cortisol , in addition to altering and enhancing the biotransformation and release of several other steroid hormones . there is some evidence that there may be retrograde passage of these two neuropeptides back into the hypothalamic region , which in human and nonhuman primates , but not in rodents , lies partially outside the brain barrier . glucocorticoids have been documented for a very long time to negatively regulate the hpa axis in a negative - feedback mode , with cortisol being the primary glucocorticoid in humans , non - human primates and guinea pigs , and corticosterone , the primary glucocorticoid having this effect in rats and mice . thus , cortisol acts at both the hypothalamic sites of crf production and at the anterior pituitary sites of pomc processing and release , to transiently attenuate or inhibit the release of these hormones . a 24-hour circadian rhythm is thus achieved , with the lowest levels of crf , acth , beta - endorphin and thus cortisol in the late afternoon and early evening in humans , and with levels rising again in the early morning hours , the opposite times pertain in rodents , with highest hormone levels at night , at the beginning of the activity period . based on early findings of volavka , our group and a few others years ago began to study the possible role of the endogenous opioid system , in particular , the mu - opioid receptor system , in also modulating the hpa axis . in several studies we have shown that the hpa axis is inhibited by the mu - opioid receptor system ( reviewed in refs 5,7,8 ) . in one study from our group , we looked at high and very high doses of two different selective mu - opioid receptor antagonists , both of which can be administered intravenously in humans , naloxone and nalmefene . studies using nonhuman primate membranes and , more recently , studies using cloned human genes in proper molecular - cellular constructs , have shown that , in contrast to rodents , naloxone binds almost exclusively to the mu - opioid receptor and acts as an antagonist . nalmefene , on the other hand , binds to both mu - and kappa - opioid receptors . very recently , in collaboration with the group of bidlack , we have shown that the kappa opioid receptor effect of nalmefene is that of a partial agonist ( that is , with some agonist and some antagonist properties ) , whereas the mu component is pure mu - opioid receptor antagonist . since we have studied both of these compounds in several earlier clinical research studies , we elected to use high and very high doses of each , to be sure that the ceiling of the effective doses in humans was exceeded . we found , as we and others had shown before , that naloxone activates the hpa axis by disinhibition and causes significant increases in both acth and cortisol . of great interest in this study , however , was the finding that nalmefene causes a significantly greater activation of the hpa axis , with higher resultant peripheral levels of acth and cortisol . our more recent studies , in which we found that the kappa component of nalmefene is a partial agonist , suggest that whereas the mu antagonists act at mu - opioid receptors of the hypothalamic and anterior pituitary sites , and through the mechanism of disinhibition bring about the increased release of crf and acth and beta - endorphin , the kappa partial agonist component of nalmefene may act directly to enhance release of crf and/or of the pomc peptides , acth and beta - endorphin , thus directly activating the hpa stress - responsive axis , which has been suggested by several workers in preclinical studies . this possibility has not , however , been well studied with any of the very few selective kappa agonists which have ever been introduced to human use , and only a few additional studies of these kappa agonists or partial agonists have been conducted in nonhuman primates . in earlier studies , it has been shown that activation of the hpa axis , with increased levels of plasma acth and cortisol , occurs after administration of alcohol or cocaine , and many groups have made similar findings in animal models . further , we have shown that tolerance develops to this hpa activation effect of both cocaine and alcohol . in other studies , we have suggested that activation of the hpa axis is sought by the rat or mouse , and by the human . in human studies conducted , in collaboration , by o'malley at yale in a clinical research setting , naltrexone , a selective mu - opioid antagonist with some kappa antagonist activity , was administered for 1 week to alcoholics and compared with placebo administered for one week to a similar group . then a laboratory session was conducted in which limited alcohol self - administration was permitted for up to 2 hours . we found , just as in the numerous field trials , that alcoholics receiving naltrexone drank significantly fewer drinks . because of the naltrexone disinhibition of the hypothalamic - pituitary sites of the hpa axis , there was a significant increase in levels of acth and cortisol in alcoholics treated with naltrexone after consumption of fewer than two drinks , whereas the much larger amounts of alcohol consumed by the alcoholics receiving placebo resulted in no significant activation of this axis . further , on responding to specific questionnaires , the alcoholics receiving naltrexone , and who had consumed only a small amount of alcohol , but had experienced modest activation of the hpa axis , felt no further  craving ,  or desire to drink alcohol , and this decrease in craving was correlated to the increase of serum cortisol levels . the opposite pertained in those alcoholics receiving a placebo , who had consumed more alcohol , but had no activation of the hpa axis , and no increase in cortisol , a significant urge to drink alcohol persisted . many of our earlier studies have shown that short - acting opiates , opposite from the effects of cocaine and alcohol in the hpa axis , profoundly attenuate or suppress the hpa axis , resulting in lowered levels of acth and cortisol after opiate administration . however , after tolerance and physical dependence have developed , in the setting of withdrawal from opiates , profound activation of the hpa axis occurs with increases in levels of acth and cortisol . the neuroendocrine changes of opiate withdrawal look very similar to the normal response to a specific mu opioid receptor antagonist , such as naltrexone , when given to a healthy volunteer . therefore , it is not surprising , as we had predicted , that most opiate addicts will not willingly accept chronic daily naltrexone or other opioid antagonist treatment once experienced , whereas alcoholics would accept such treatment , and might be directly benefited . giving an opioid antagonist to any opiate - dependent person is contraindicated , because profound activation of the stress - responsive axis will occur and creates a very adversive and noxious experience . in many of our earlier studies , we have shown that during chronic methadone maintenance treatment , which provides steady perfusion with a synthetic ligand of the mu - opioid receptor , complete normalization of the hpa axis occurs , including normalization of basal levels of hormones , as well as responsivity in various functional tests . to dissect further the relative contribution of the glucocorticoid system contrasted to the mu - opioid receptor endogenous ligands , that is , beta - endorphins and enkephalins , we have conducted further studies using metyrapone . in humans , metyrapone blocks the final step of cortisol synthesis , that is , 11--hydroxylation . in the single oral dose test using metyrapone , the synthesis of cortisol is blocked for about 8 hours , and then returns to normal . therefore , one can measure the levels of acth ( which also reflect the equimolar release and levels of beta - endorphin ) following metyrapone administration which are elevated because with cortisol synthesis blocked , and the normal negative feedback is transiently cut off . in healthy human beings , with normal endogenous opioid systems , the mu - opioid receptor system responds to bring a check , or brake , to the increased release and levels of acth ( and beta - endorphin ) . however , we had shown in several earlier studies that in medication - free , drug - free former heroin addicts , there is no such mu - opio!d receptor - mediated brake , and thus hyper - responsivity to metyrapone testing is observed ( reviewed in refs 5,7 ) . further , we had reported that in abstinent cocaine addicts a similar hyper - responsivity to metyrapone testing exists . this hyper - responsivity , therefore , suggests a relative endorphin deficiency , which our laboratory -based studies also support . as discussed above , we have found that chronic binge cocaine administration causes an increase in gene expression in the mu - opioid receptor , as well as an increase in density in mu - opioid receptors , in specific brain regions with abundant dopaminergic terminals , and , further , in recent studies , we have found that this increase in mu - opioid receptor density persists for a protracted period of time after last cocaine exposure . however , we have also shown that there is no increase of the endogenous opioids that bind at the mu receptor . thus a relative endorphin deficiency develops ( or possibly was present a priori on a genetic or environmentally - induced basis ) . subsequently , frost and colleagues , using positron emission tomography ( pet ) showed similarly the mu - opioid receptor density being increased in recently - abstinent cocaine addicts , and further more recently have shown that this increase persists for protracted periods of time into successful cocaine abstinence . thus , a relative endorphin deficiency has been documented both in humans as well as in rodent models , in humans directly shown by testing of the stress - responsive system . in several studies , we have found that metyrapone responsivity is abnormal in opiate addicts , but becomes normalized in methadone maintenance patients ( reviewed in refs 5,7 ) . we also have shown that abnormal hyper - responsivity occurs in cocaine addicts . in a more recent study we also conducted studies in a subgroup of methadone maintenance patients who continued during 6-month treatment or more to meet the criteria of cocaine dependence . this group was maintained on moderate doses of methadone ( 60 to 90 mg / day ) . as discussed above , an early clinical study from our laboratory , a very recent clinical study from our laboratory , and a recent laboratory - based study have all suggested that increasing the dose of methadone may decrease cocaine addiction in maintenance patients with dual - dependency , and further , in the rodent model , that the addition of steady - state methadone may prevent alterations in mu - opioid receptor gene expression and attenuate or prevent conditioned place preference to cocaine . 
  in another set of studies reported in the last decade we have re - explored the glucocorticoid negative feedback both in methadone - maintained former heroin addicts , as well as those with ongoing cocaine dependence . in all our earlier studies , we found , surprisingly , that all of the methadone - maintained patients had normal suppression to dexamethasone and , in this study , we also used two lower doses than the usual suppression dose , that is , 0.5 and .125 mg and found that all subjects suppressed completely ( as reviewed in refs 5,7,57 ) . although not significant , the glucocorticoid feedback effects in the cocaine - dependent , methadone - maintained patients , and also in the otherwise well - stabilized methadone - maintained patients appeared to be greater than the normal volunteers in the late afternoon , suggesting that there may be a modestly altered , or enhanced , negative feedback by glucocorticolds , in at least some subjects . this , in turn , may contribute to the observed attenuation of both basal and cocaine - induced responsivity of the hpa axis in humans and in rodents in other studies from our laboratory and others . in another study , we examined the effect of corticotropin - releasing factor in methadone - maintained versus control subjects . in this study , we found differences between long - term well - stabilized methadone - maintained subjects as compared with normal control subjects . in this study , two doses of crf were used ; one lower than the usual dose ( 0.5 jug / kg ) and one dose higher ( 2.0 mg / kg ) than usually used in the neuroendocrine diagnostic procedure ( 100 jug , irrespective of weight ) . there was no difference in hormonal measurements between the two groups following placebo administration , nor during low - dose hcrf administration . however , following high - dose crf administration , the methadone - maintained patients displayed a significantly greater increase in plasma acth levels than did the normal volunteers . this suggested that in long - term methadone - maintained patients some abnormalities in hpa axis responsivity may pertain , in this case , a greater sensitivity of the anterior pituitary to crf stimulation . in turn , these findings suggest that the basal and peak levels of crf may be slightly reduced in stable methadone maintenance patients , possibly related to the increased sensitivity to negative feedback by glucococorticoids , as discussed above , or due to the steady but high and exogenous opioid tone in patients in treatment with the long - acting mu agonists . further studies to explore this altered sensitivity in other persons with specific addictive diseases , not in treatment , as well as in treatment , are in progress . in another series of studies , we have been able to pursue in humans findings which we and others had made in rodents , that is , that dynorphin , the natural endogenous opioid ligand of the kappa - opioid receptor , may directly act to alter ( lower ) dopaminergic tone . we have been able to access dynorphin a(1 - 13 ) , a natural - sequenced dynorphin four residues shorter than the natural dynorphin a(1 - 17 ) for research use under an investigator - initiated investigational new drug application ( ind ) approved by the us food and drug administration . building upon the established biological fact that , in humans , prolactin release is almost exclusively under dopaminergic tone , and thus , that a lowering of dopamine in the tuberoinfundibular dopaminergic region results in a rise in prolactin levels , we conducted studies first in healthy volunteers using two different doses of intravenously - administered dynorphin a(1 - 13 ) ( 120 g / kg and 500 g / kg ) . since in humans some of the hypothalamus lies outside the blood - brain barrier , we assumed that the peptide dynorphin would be able to act on this tuberoinfundibular dopaminergic system . when we conducted these studies in a stress - minimized environment of our rockefeller hospital clinical research center , we found that peripheral administration of dynorphin a(l13 ) gave a prompt dose - dependent increase in serum prolactin levels , which then returned to normal within 120 minutes . this duration of action was much longer than we predicted , based on our in vitro biotransformation studies in which we established the probable half -life of dynorphin a(1 - 13 ) in human blood . of interest , with respect to the possible effect of dynorphin on the hpa axis , we found no increment in acth or crf following peripheral dynorphin administration . to document whether the dynorphin effect was modulated by the endogenous opioid system , we conducted studies using the lower dose of dynorphin a(1 - 13 ) following pretreatment with either naloxone or nalmefene , both selective mu - opioid receptor antagonists , and one ( nalmefene ) with partial kappa - opioid agonist activity we found pretreatment with either of these compounds attenuated the rise in serum prolactin . in these initial studies , a further very provocative ( but not completely unexpected given the physiological differences ) observation was made , an unusual finding in our human studies of addictive diseases , specifically that female subjects had modestly higher basal levels of prolactin than males , but when given dynorphin , gave a significantly exaggerated response , with higher levels of prolactin achieved after dynorphin administration and thus the reduction of dopamine in the tuberoinfundibular region . our subsequent studies of dynorphin effects now must be done always considering males and females separately . in a second set of studies , we have addressed the question of whether or not the dynorphin responsivity , with respect to lowering dopaminergic tone , will occur similarly in healthy long - term well - stabilized methadonemaintained subjects . two doses of dynorphin again were used for study in both a new group of healthy volunteer subjects and in a group of long - term stable methadonemaintained patients . again , in the healthy volunteer subjects , a dose - dependent rise in serum prolactin was observed after dynorphin administration . similarly , in the methadone - maintained patients ( receiving 80 to 120 mg / day of methadone ) , a dose - dependent rise in prolactin occurs . because years ago ( published in 1978 by our group ) , we had shown that methadone itself , acting as a mu opioid receptor agonist , acts to lower dopaminergic tone , causes increase in serum prolactin , which occurs at time of peak plasma levels of methadone ( that is , around 2 to 4 hours after oral methadone dose ) , in the dynorphin studies , we withheld the methadone dose until 60 minutes after the dynorphin was given . in these subjects , as in our much earlier studies , we showed a second and separate brisk rise in prolactin levels , beginning at 2 hours after methadone administration and remaining elevated at 5 hours after methadone administration . again , in the methadone - maintained patients , as in both groups of healthy volunteer subjects , there was a dose - dependent dynorphin - induced rise in prolactin levels which returned to basal levels by 90 to 120 minutes . thus , in this study , we were able to observe both the dynorphin- and methadone - induced lowering of tuberoinfundibular dopaminergic tone , resulting in both rises in serum prolactin levels . in yet another series of studies , we had observed that when given to healthy volunteers nalmefene caused a small but modest rise in serum prolactin levels . therefore , we entered into a collaboration with bidlack , and in that collaboration addressed directly the issue of whether the kappa opioid receptor activity of nalmefene is antagonist , or possibly , as we hypothesized , partial agonist . it was found clearly that nalmefene possesses kappa - opioid receptor partial agonist activity in in vitro studies using appropriate molecular cellular constructs . it was reconfirmed that the mu opioid receptor action of nalmefene is only that of antagonism ; the kappa opioid receptor action is both agonism ( partial agonist ) and antagonism . further , we were able to show that nalmefene effects a modest elevation of prolactin levels , suggesting a modest lowering of dopaminergic tone . this suggests , however , that nalmefene or other mu - opioid receptor antagonists , which have kappa - partial agonism ( probably also true for naltrexone ) may have augmented benefit for management of alcoholism , and possibly even for treatment for stimulant , such as cocaine , dependency , since a modest lowering of dopaminergic tone could be helpful in decreasing or attenuating the  reward  effect , whereas the inhibition of the mu - opioid receptor regulation of the stress - responsive hpa axis could provide modest activation of this axis , which we have directly documented to be sought by alcoholics , and in our animal modeling suggests is also sought by the cocaine self - administering animals . in these basic clinical research studies , we have again found an extremely important role of the mu - opioid receptor system , as well as identifying a previously not - appreciated role of the kappa - opioid receptor system in modulation of the human stress - responsive hpa axis . our genetics work , including our work in physiogenetics , has not been discussed herein , but has been reviewed elsewhere , as discussed above . all these findings have taught us that physiogenetics may occur , that is , difference in our response to our own proteins , peptides , neurotransmitters , or steroids , based on a polymorphism of a receptor or some polymorphism of the ligand or the pathway producing the ligand . further , such studies , in the future , may give us increasing insights into targets for therapeutics , as well as providing a basis for effective primary prevention of specific addictive diseases .
the articulated goals of dialogues in clinical neuroscience are to serve as  an interface between clinical neuropsychiatry and the neurosciences by providing state - of - the - art information and original insights into relevant clinical , biological , and therapeutic aspects .  my laboratory 
 , the laboratory of the biology of addictive diseases at the rockefeller university , has for years been focused on  bi - directional translational research ,  that is , learning by careful observations and study in patient populations with the disorders under study , in this case primarily specific addictive diseases , and then using that knowledge to create improved animal models or other laboratory - based research paradigms , while , at the same time , taking research findings made at the bench into the clinic as promptly as that is appropriate and feasible . in this invited review , 
 therefore , the focus will be on perspectives of our laboratory of the biology of addictive diseases and related national institutes of health / national institute on drug abuse research center , including laboratory - based molecular neurobiological research , research using several animal models designed to mimic human patterns of drug abuse and addiction , as well as basic clinical research , intertwined with treatment - related research .