The medial preoptic area is necessary for motivated choice of pup- over cocaine-associated environments by early postpartum rats

Converging evidence suggests that the motivation to seek cocaine during the postpartum period is significantly impacted by the competing incentives of offspring, a stimulus unique to this life stage. In the present study, the functional role of the medial preoptic area (mPOA), a critical site involved in maternal responsiveness, on processing incentive value of pup-associated cues and influencing response allocation for pup- over cocaine-associated environments was investigated using a concurrent pup/cocaine choice conditioned place preference (CPP) paradigm. Early postpartum females with bilateral guide cannulae aimed into the mPOA or into anatomical control sites were conditioned, from postpartum days (PPD) 4 to 7, to associate different uniquely featured environments with pups or cocaine. CPP was tested on PPD8 following intra-mPOA infusions of either 2% bupivacaine or saline vehicle. In two additional experiments, the effects of intra-mPOA infusions of bupivacaine on expression of conditioned responding induced by environments associated with either pups or cocaine were examined separately. Transient inactivation of the mPOA selectively blocked the conditioned preferences for pup-associated environments, significantly contrasting the robust pup-CPP found in non-surgical and intra-mPOA vehicle-treated females. In contrast, mPOA inactivation failed to alter cocaine-CPP in postpartum females. When given a choice between environments associated with pups or cocaine, transient functional inactivation of the mPOA altered choice behavior, biasing the preference of females toward cocaine-associated environments, such that almost all preferred cocaine- and none the pup-associated option. The anatomical specificity was revealed when inactivation of adjacent regions to the mPOA did not affect CPP responses for pups. The findings support a critical role for the mPOA in mediating pup-seeking behavior, and further suggest that the competing properties of pups over alternative incentives, including drugs of abuse, rely on mPOA integrity to provide relevant pup-related information to the circuitry underlying the choice behavior between pups and alternative stimuli.     

Fos and glutamate AMPA receptor subunit coexpression associated with cue-elicited cocaine-seeking behavior in abstinent rats

Cocaine-associated cues acquire incentive motivational effects that manifest as craving in humans and cocaine-seeking behavior in rats. We have reported an increase in neuronal activation in rats, measured by Fos protein expression, in various limbic and cortical regions following exposure to cocaine-associated cues. This study examined whether the conditioned neuronal activation involves glutamate AMPA receptors by measuring coexpression of Fos and AMPA glutamate receptor subunits (GluR1, GluR2/3, or GluR4). Rats trained to self-administer cocaine subsequently underwent 22 days of abstinence, during which they were exposed daily to either the self-administration environment with presentations of the light/tone cues previously paired with cocaine infusions (Extinction group) or an alternate environment (No Extinction group). All rats were then tested for cocaine-seeking behavior (i.e. responses without cocaine reinforcement) and Fos and AMPA glutamate receptor subunits were measured postmortem using immunocytochemistry. The No Extinction group exhibited increases in cocaine-seeking behavior and Fos expression in limbic and cortical regions relative to the Extinction group. A large number of Fos immunoreactive cells coexpressed GluR1, GluR2/3, and GluR4, suggesting that an action of glutamate at AMPA receptors may in part drive cue-elicited Fos expression. Importantly, there was an increase in the percentage of cells colabeled with Fos and GluR1 in the anterior cingulate and nucleus accumbens shell and cells colabeled with Fos and GluR4 in the infralimbic cortex, suggesting that within these regions, a greater, and perhaps even different, population of AMPA receptor subunit-expressing neurons is activated in rats engaged in cocaine-seeking behavior.     

Neuronal and behavioral correlations in the medial prefrontal cortex and nucleus accumbens during cocaine self-administration by rats

Up to 31 neurons per animal were simultaneously recorded from the medial prefrontal cortex and nucleus accumbens in 15 rats during i.v. cocaine self-administration sessions, using a multi-channel, single-unit recording technique. Alterations of neuronal activity (both excitatory and inhibitory) were found a few seconds before each lever press for cocaine infusion; we have called these pre-lever press neuronal activations "anticipatory responses". A detailed video analysis revealed that these neuronal firing alterations were associated with specific portions of the behavioral sequence performed before each lever press in both recording areas. Some of the simultaneously recorded neurons displayed similar firing patterns in relation to a given behavioral episode within the behavioral sequence (turning, raising head, etc.), while others fired at different times relative to each behavioral event. Cross-correlational analyses revealed inter-regional and intra-regional correlated firing patterns between pairs of simultaneously recorded medial prefrontal cortex and nucleus accumbens neurons. This correlated firing occurred in the neurons with and without anticipatory responses, although the incidence of correlations between anticipatory neuron pairs was much higher than that between non-anticipatory neuron pairs (18.4% vs 3.6%). Many correlated neuron pairs displayed a time lag in the peak of correlational activity that indicated a temporal sequence in correlated activity. In contradiction to our hypothesis, the temporal pattern of correlation reveals that there are more cases in which nucleus accumbens neurons fired ahead of medial prefrontal cortex neurons.The results suggest that multiple mesocorticolimbic neuronal circuits may code sequential steps during the behavioral sequence performed to obtain an infusion of cocaine. The observed correlated firing between the medial prefrontal cortex and the nucleus accumbens indicates that dynamic, coherent activity occurs within the mesocorticolimbic circuit. Because this circuit is hypothesized to drive drug-seeking behavior, we suggest that this correlated firing between the nucleus accumbens and the medial prefrontal cortex may participate in the control of cocaine self-administration. In addition, the finding that correlated activity within the nucleus accumbens more often precedes that of the medial prefrontal cortex suggests that the nucleus accumbens may play a prime role in the initiation of cocaine self-administration.     

Development of cocaine sensitization before pregnancy affects subsequent maternal retrieval of pups and prefrontal cortical activity during nursing

Pups are a highly rewarding stimulus for early postpartum rats. Our previous work supports this notion by showing that suckling activates the mesocorticolimbic system in mothers. In the present study, we tested whether development of behavioral sensitization to cocaine before pregnancy affects the neural response to pups during the early postpartum days (PD). Virgin rats were repeatedly administered cocaine for 14 days (15 mg kg(-1)) and withdrawn from treatment during breeding and pregnancy. The neural response to suckling was measured at PD 4-8 using blood-oxygen-level-dependent (BOLD) MRI or microdialysis. Our results show that BOLD activation in the medial prefrontal cortex (PFC), septum and auditory cortex was curtailed in cocaine-sensitized dams. No differences between cocaine sensitized and saline control dams were observed in the nucleus accumbens, olfactory structures, or in 48 additional major brain regions that were analyzed. Baseline, but not pup-stimulated, dopamine (DA) levels in the medial PFC were lower in cocaine-sensitized dams than in controls. When tested for maternal behaviors, cocaine-sensitized dams showed significantly faster retrieval of pups without changes in other maternal behaviors such as grouping, crouching and defending the nest. Taken together, the present findings suggest that maternal motivation to retrieve pups was enhanced by repeated cocaine exposure and withdrawal, a result reminiscent of 'cross-sensitization' between the drug and a natural reward. Changes in retrieval behavior in cocaine-sensitized mothers might be associated with a hypo-responsive medial PFC.     

Fos expression induced by cocaine-conditioned cues in male and female rats

Previous studies have shown that female rats exhibit different patterns of drug seeking during multiple phases of cocaine addiction when compared with males. However, the underlying mechanisms for these sex differences remain largely unknown. Here, we used a cocaine self-administration/reinstatement model to examine neuronal activation, as determined by Fos expression, following cue-induced reinstatement of cocaine seeking in male and female rats. Fos expression revealed both similarities between sexes in some brain regions, as well as selective sexually dimorphic patterns. As compared to no cue control subjects, conditioned cues induced higher Fos expression in the Cg1 region of the anterior cingulate cortex, but lower expression in the nucleus accumbens in both males and females. Females exhibited higher Fos expression than males in multiple brain regions, including the agranular insular cortex, dorsal medial caudate-putamen, nucleus accumbens shell, ventral tegmental area, dorsal subiculum, and ventral CA1 and CA3 regions of the hippocampus. Notably, only Fos expression in the prelimbic cortex, nucleus accumbens shell, basolateral amygdala, and ventral subiculum correlated positively with lever responding in response to conditioned cues across males and females. These findings indicate that while sexually dimorphic Fos activation does occur, the relationship between cue-induced cocaine seeking and neuronal activation may be similar for males and females in key brain regions of the relapse circuit.     

Relationship of cocaine-induced c-Fos expression to behaviors and the role of serotonin 5-HT2A receptors in cocaine-induced c-Fos expression

Serotonin 5-HT2A receptor antagonists have been shown to attenuate the locomotor stimulant effects of cocaine in rats. The present study used the expression of c-Fos protein as a marker to identify brain areas through which 5-HT2A receptors may modulate cocaine-induced behaviors. Significant correlations were observed between cocaine-induced hyperactivity and c-Fos expression in the nucleus accumbens core (NAcC), caudate-putamen (CPu), and subthalamic nucleus. In a separate experiment, a low, behaviorally relevant dose of cocaine was found to increase c-Fos immunoreactivity in the medial CPu, NAcC, and nucleus accumbens shell (NAcSh). The selective 5-HT2A receptor antagonist M100907 significantly attenuated cocaine-induced c-Fos expression in the medial CPu and in the NAcSh. These data suggest that 5-HT2A receptors in the NAcSh and CPu or in afferents to these regions may contribute to genomic responses to cocaine in the brain as well as to cocaine-induced locomotor activity.     

Experience-dependent effects of cocaine self-administration/conditioning on prefrontal and accumbens dopamine responses

Experiments were performed to examine the effects of cocaine self-administration and conditioning experience on operant behavior, locomotor activity, and nucleus accumbens (NAcc) and prefrontal cortex (PFC) dopamine (DA) responses. Sensory cues were paired with alternating cocaine and nonreinforcement during 12 (limited training) or 40 (long-term training) daily operant sessions. After limited training, NAcc DA responses to cocaine were significantly enhanced in the presence of cocaine-associated cues compared with nonreward cues and significantly depressed after cocaine-paired cues accompanied a nonreinforced lever response. PFC DA levels were generally nonresponsive to cues after the same training duration. However, after long-term training, cocaine-associated cues increased the magnitude of cocaine-stimulated PFC DA levels significantly over levels observed with nonreinforcement cues. Conversely, conditioned cues no longer influenced NAcc DA levels after long-term training. In addition, cocaine-stimulated locomotor activity was enhanced by cocaine-paired cues after long-term, but not after limited, training. Findings demonstrate that cue-induced cocaine expectation exerts a significant impact on dopaminergic and behavioral systems, progressing from mesolimbic to mesocortical regions and from latent to patent behaviors as cocaine and associative experiences escalate.     

Neuroadaptive changes in mesocorticolimbic dopamine and acetylcholine neurons following cocaine or saline self-administration are dependent on pre-existing individual differences

Previously, we demonstrated that stress-induced self-grooming behaviour in rats predicted an enhanced motivation to self-administer cocaine as determined under a progressive ratio schedule of reinforcement. The enhanced motivation of high grooming (HG) rats was associated with a reduced reactivity of dopaminergic neurons in the medial prefrontal cortex and amygdala, but not nucleus accumbens. In the present study, we studied the effect of cocaine and saline self-administration on these pre-existing differences in neurochemical profile by determining the electrically evoked release of [3H]dopamine and [14C]acetylcholine from superfused slices of the nucleus accumbens shell and core, medial prefrontal cortex and amygdala of HG and low grooming (LG) rats. Although HG and LG rats did not differ in acquisition of cocaine and saline self-administration, both conditions induced substantially different neuroadaptations in these rats. Differences in depolarisation-induced dopamine and acetylcholine release were maintained in the medial prefrontal cortex, emerged in the nucleus accumbens and dissipated in the amygdala. These results indicate that altered reactivity of mesocorticolimbic dopaminergic and cholinergic neurons due to exposure to cocaine and environmental stimuli (saline) is dependent on pre-existing neurochemical differences and displays region-specificity. These pre-existing differences and the cocaine- and environmental-induced neuroadaptations seem to act in concert to produce an enhanced motivational state to self-administer cocaine.     

Cocaine reward and hyperactivity in the rat: sites of mu opioid receptor modulation

Opioid receptor agonists and antagonists have profound effects on cocaine-induced hyperactivity and conditioned reward. Recently, the role specifically of the mu opioid receptor has been demonstrated based on the finding that i.c.v. administration of the selective mu opioid receptor antagonist, d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), can attenuate cocaine-induced behaviors. The purpose of the present study was to determine the location of mu opioid receptors that are critical for cocaine-induced reward and hyperactivity. Adult male Sprague-Dawley rats received injections of CTAP into the caudate putamen, the rostral or caudal ventral tegmental area (VTA) or the medial shell or core of the nucleus accumbens prior to cocaine to determine the role of mu opioid receptors in cocaine-induced reward and hyperactivity. Cocaine-induced reward was assessed using an unbiased conditioned place preference procedure. Results demonstrate that animals pre-treated with CTAP into the nucleus accumbens core or rostral VTA, but not the caudal VTA, caudate putamen or medial nucleus accumbens shell, during conditioning with cocaine showed an attenuation of the development of cocaine-induced place preference. In contrast, CTAP injected into the nucleus accumbens shell but not the core attenuated the expression of cocaine place preference. Intra-nucleus accumbens core, caudate putamen or caudal VTA CTAP significantly attenuated cocaine-induced hyperactivity. In addition, the number of cFos positive cells was increased in the motor cortex, medial and ventromedial aspects of the nucleus accumbens shell, basolateral amygdala and caudal VTA during the expression of cocaine place preference, and this increase was attenuated in the animals that received intra-accumbens core CTAP during daily cocaine conditioning. These results demonstrate the importance of mu opioid receptors in the nucleus accumbens and VTA in cocaine-induced reward and hyperactivity and suggest that some aspects of the behavioral effects of cocaine are mediated by endogenous activation of mu opioid receptors in these brain regions.     

Comparison of two positive reinforcing stimuli: pups and cocaine throughout the postpartum period.

This set of experiments investigated the appetitive or motivational processes underlying the performance of maternal behavior. The place preference paradigm was adapted to simultaneously investigate the reinforcing properties of cocaine and pups for maternal, lactating dams. These modifications allowed the authors to assess which stimulus, either a 10 mg/kg s.c. injection of cocaine or 3 pups, had the strongest reinforcing value. At Postpartum Days 10 and 16, the dams preferred the cocaine cue-associated chamber, whereas the dams tested at Postpartum Day 8 preferred the pup cue-associated chamber. Overall, the data revealed an interaction between the postpartum period at testing and the exhibited preference for cocaine or pups. Further testing will investigate the neural circuitry underlying the appetitive processes of each stimulus.     

Conditioned increases in behavioral activity and accumbens dopamine levels produced by intravenous cocaine

In vivo microdialysis, behavioral activity assessments, and a conditioned place preference (CPP) test were used to investigate dopaminergic correlates of cocaine-conditioned behaviors. Over 12 days, rats were given either intravenous cocaine (4.2 mg/kg) or saline (6 cocaine and 6 saline infusions) daily in distinctively different environments. The following day, rats were tested in the cocaine- and saline-paired environments; 48 hr later, CPP was determined. The cocaine-associated environment elicited greater nucleus accumbens dopamine (NAcc DA) levels, hyperactivity, and place preference, though the emergence of DA increases was not in synchrony with peak behavioral activation. Although conditioned behavioral effects after repeated cocaine are well documented, direct evidence of increased NAcc DA in response to a cocaine-paired environment has not been previously reported. Discrepancies with previous work are attributed to a number of methodological differences.     

A similar pattern of neuronal Fos activation in 10 brain regions following exposure to reward- or aversion-associated contextual cues in mice

Relapse triggered by drug-paired cues is a major obstacle for successful treatment of drug abuse. Patterns of brain activation induced by drug-paired cues have been identified in human and animal models, but lack of specificity poses a serious problem for craving or relapse interpretations. The goal of this study was to compare brain responses to contextual cues paired with a rewarding versus an aversive stimulus in a mouse model to test the hypothesis that different patterns of brain activation can be detected. Mice were trained to associate a common environmental context with an intraperitoneal injection of saline, lithium chloride or cocaine. After measuring each animal for conditioned place preference or aversion, mice were re-exposed to the context (CS+ or CS−) in absence of the reinforcer to analyze patterns of Fos expression in 10 brain regions chosen from previous literature. Levels of Fos in the cingulate cortex, paraventricular thalamic nucleus, paraventricular hypothalamic nucleus, and dentate gyrus differed in CS+ versus CS− groups, but the direction of the differences was the same for both lithium chloride (LiCl) and cocaine reinforcers. In the cingulate cortex, Fos was positively correlated with degree of place preference for cocaine or aversion to LiCl whereas in the periaqueductal gray the relationship was positive for LiCl and negative for cocaine. Results confirm Fos responses to reward- or aversion-paired cues are similar but specificity is detectable. Future studies are needed to comprehensively establish neuroanatomical specificity in conditioned responses to drugs as compared to other reinforcers.     

Cholinergic interneurons of the nucleus accumbens and dorsal striatum are activated by the self-administration of cocaine

The nucleus accumbens, a major component of the ventral striatum, and the dorsal striatum are primary targets of the mesolimbic dopamine pathway, which is a pathway that plays a critical role in reward and addiction. The shell compartment of the nucleus accumbens and the ventromedial striatum, in particular, receive extensive afferent projections from the ventral tegmental area, which is the major afferent source of the mesolimbic pathway [Prog Brain Res 99 (1993) 209; J Neurosci 7 (1987) 3915]. The present study focused on striatal cholinergic interneurons as potential key neurons involved in the neural basis of drug reinforcement. The main finding of this study is that cholinergic interneurons located in the shell compartment of the nucleus accumbens and the ventromedial striatum were activated, as measured by Fos labeling, following a 1 h session of the self-administration of cocaine in rats. A direct correlation existed between the percent of cholinergic interneurons that were activated and the amount of cocaine that was self-administered. The greatest amount of administered cocaine (approximately 10 mg/kg) resulted in the activation of approximately 80% of the cholinergic neurons. No such correlation existed in the group of animals that self-administered saline. In addition, activation was not found in the core compartment of the nucleus accumbens or the dorsolateral striatum, which receive extensive innervation from the substantia nigra and thus are more closely tied to the motor effects of the drug.

In conclusion, cocaine-driven neuronal activation was specific to the shell compartment of the nucleus accumbens (R²=0.9365) and the ventromedial striatum (R²=0.9059). These findings demonstrate that cholinergic interneurons are involved in the initial stage of cocaine intake and that these neurons are located in areas of the nucleus accumbens and dorsal striatum that are more closely tied to the rewarding and hedonic effects rather than the motor effects of cocaine intake.     

c-Fos expression in supramammillary and medial mammillary nuclei following spatial reference and working memory tasks

To investigate brain substrates of spatial memory, neuronal expression of c-Fos protein was studied. Two groups of rats were trained in two spatial memory tasks in the Morris water maze, where the rats have to apply a reference memory rule or a working memory rule. In addition to the experimental groups, two control groups were used to study c-fos activation not specific to the memory processes studied. After immunohistochemical procedures, the number of c-Fos positive neuronal nuclei was quantified in the mammillary body (MB) region (medial mammillary nucleus [MMn] and supramammillary nucleus [SuM]). The results have shown that some MMn neurons expressed c-Fos nuclear immunoreactivity related to spatial working memory but not to spatial reference memory. The increased number of c-Fos immunoreactive neuronal nuclei in the SuM was related to spatial training but not to either working or reference memory demands of the tasks.     

Juvenile rats show reduced c-fos activity in neural sites associated with aversion to pups and inhibition of maternal behavior

Juvenile rats (18-23 days old) interact avidly with pups as novel stimuli and show maternal behavior after only 1-3 days of pup exposure; adults initially avoid pups and require 3-9 days of pup exposure. Upon exposure to pups as novel stimuli, adults had more c-Fos-immunoreactive neurons in the hypothalamus and amygdala--regions associated with aversion to pups--than adults exposed to familiar pup stimuli (maternal) or not exposed to pups (p < .05). In juvenile rats exposed to pups as novel stimuli, only the medial amygdala had a small significant increase of c-Fos neurons. In juveniles, this blunted engagement of c-Fos neurons may reflect the diminished activation of inhibitory neurons, facilitating the interaction of juveniles with pups as novel stimuli and onset of maternal behavior.     

Gender differences in prodynorphin but not proenkephalin mRNA expression in the striatum of adolescent rats exposed to prenatal cocaine

The objective of this study was to determine if prenatal cocaine affects the levels of prodynorphin and proenkephalin mRNA in male and female adolescent rats. Pregnant dams received cocaine or vehicle from gestational days 8–22 and upon delivery, the pups were fostered. At postnatal days 42–44, pups were killed and brains removed and frozen. Sections of striatum and nucleus accumbens were processed for prodynorphin and proenkephalin mRNA expression. Prenatal cocaine did not affect the expression of proenkephalin mRNA, but males showed higher expression than females. However, prodynorphin mRNA was lower in female rats exposed to cocaine compared to controls. Prenatal cocaine appears to have unique effects on neuropeptides during adolescence.     

Neuroanatomical specificity of conditioned responses to cocaine versus food in mice

Neural circuits implicated in drug conditioning, craving and relapse overlap extensively with those involved in natural reward and reinforcement. To determine whether specificity could be detected in conditioned brain responses to drugs versus food, male outbred HSD:ICR mice were conditioned to a common environment using either 20 mg/kg cocaine (ip) or a familiar food (under food restriction). The mice were then re-exposed to the same environment without the reinforcer and patterns of brain activation were compared using immunohistochemical detection of Fos. Conditioned place preference tests were conducted first to establish relative potency of each reward and facilitate analysis of correlations between Fos and motivation. Place preference was stronger for cocaine than food. Food- but not cocaine-paired cues increased Fos in the paraventricular hypothalamic nucleus whereas the opposite occurred for prefrontal, cingulate and piriform cortices. Individual differences in cocaine place preference were negatively correlated with Fos in the prefrontal cortex. One difference between drugs and natural reinforcers may be lack of feedback from the periphery for drugs which may circumvent control from the hypothalamus in the development of reinforcement circuits.     

Experience-dependent plasticity: differential changes in activation associated with repeated reinforcement

Experience-dependent change in blood-oxygen-level-dependent (BOLD) signal is increasingly being employed in neuroimaging research to examine questions about function and plasticity. In this investigation, plasticity was examined during consecutive visual cue presentations that preceded correct button presses and subsequent reinforcer deliveries. Using functional neuroimaging and a modified repeated acquisition methodology, 10 adult subjects learned, through trial and error, a series of novel cue-response-reinforcer relations. Separate BOLD responses were obtained to consecutive cues and reinforcers. Repeated measures analysis of variance highlighted differential BOLD response changes. Consecutive visual cue presentations elicited rapid bilateral increases in activation in the anterior cingulate and medial frontal gyrus and moderate increases in medial temporal lobe structures and the striatum. Consecutive reinforcer presentations elicited rapid increases in activation in the left precuneus, lingual and fusiform gyri and moderate increases in medial temporal lobe structures and striatum. Within the medial temporal lobe, cues elicited a gradual increase then an abrupt decrease in activation and rewards elicited abrupt and then sustained activation. Consideration of experience-dependent BOLD response change and variability provides basic research a new perspective from which to examine regional plasticity and further explore dynamic experience-dependent shifts among cognitive processes. Furthermore, BOLD change and variability offer many clinical research areas novel supplemental indices of neuropathology.