Acute postnatal exposure to di(2-ethylhexyl) phthalate adversely impacts hippocampal development in the male rat.

The distribution of CA3 hippocampal axonal terminal fields undergo a period of widespread connectivity-based changes in the early postnatal stages of life. The purpose of the current study was to examine the effect of acute phthalate exposure during this period of hippocampal development (postnatal days 16-22 (p16-p22)) on morphological outcomes in male and female Long Evans rats. The reproductive toxicity of exposure to phthalates early in life has been well-documented; however, much less is known about the effects of phthalates on brain development. The present research demonstrated that exposure to di(2-ethylhexyl) phthalate (DEHP; 10 mg/kg, i.p.) from p16 to p22 reduced axonal markers in the CA3 distal stratum oriens (SO) and reduced cell density of both immature and mature neurons in the dentate gyrus (DG) and CA3, respectively, in male rats. The same markers in the hippocampus of female rats were similar in saline and DEHP-treated animals. These data suggest that DEHP has a negative impact on the development of the hippocampus in males but not females and recommend more extensive animal studies on phthalate exposure during the vulnerable post-natal developmental period when rapid structural and functional changes are taking place.

Differential patterns of extracellular signal-regulated kinase-1 and -2 phosphorylation in rat limbic brain regions after short-term and long-term inhibitory avoidance learning.

Activation of the extracellular signal-regulated kinase-1 and -2 has been shown to be required for neural plasticity and memory. Previous pharmacological studies have demonstrated that inhibition of extracellular signal-regulated kinase-1 and -2 blocks inhibitory avoidance retention. The aim of the present study was to investigate the different neural substrates underlying short- and long-term inhibitory avoidance learning and memory in rats using phosphorylated extracellular signal-regulated kinase-1 and -2 labeling as an index of plasticity. Short- and long-term retention tests were given 10 min or 24 h after inhibitory avoidance training. A significant elevation in the number of phosphorylated extracellular signal-regulated kinase-1 and -2-immunoreactive neurons was observed in area 1 of anterior cingulate cortex, the secondary motor cortex, lateral orbital cortex, claustrum, and the medial amygdala nucleus after the short-term inhibitory avoidance test. After the long-term retention test, phosphorylated extracellular signal-regulated kinase-1 and -2-immunoreactive neurons were localized in area 1 of anterior cingulate cortex, prelimbic cortex, and the central nucleus of amygdala. This suggests that phosphorylated extracellular signal-regulated kinase-1 and -2-immunoreactivity may reveal different brain regions involved in the storage of short- and long-term aversive memories.

A pharmacological analysis of the substrates underlying conditioned feeding induced by repeated opioid stimulation of the nucleus accumbens.

It has previously been demonstrated that stimulation of opiate receptors within the nucleus accumbens results in marked hyperphagia, perhaps reflecting enhancement of taste palatability. Rats that have received multiple morphine treatments also increase feeding in response to environmental stimuli that have been associated with the morphine injections. The present investigation further examined this phenomenon. In Experiment 1, it was shown that induction of conditioned feeding was dose-dependent; significant conditioned feeding was obtained with repeated (n = 5) intra-accumbens injections of 5 or 10 microg/microl morphine but not with saline or 1 microg. The conditioned feeding response was blocked by systemic naltrexone (5 mg/kg). In the second experiment, co-treatment with either a D-1 (SCH 23390, 0.1 mg/kg) or D-2 (haloperidol, 0.25 mg/kg) antagonist did not block the development of conditioned feeding, nor did these drugs block morphine-induced feeding. In Experiment 3, it was found that systemic naltrexone blocked the expression of conditioned feeding (confirming Experiment 1), as did SCH-23390, whereas haloperidol did not affect expression of conditioned feeding. In the fourth experiment, we observed that significant conditioned feeding was induced with repeated treatment with the selective mu agonist D-Ala2, NMe-phe4, Glyol5-enkephalin (DAMGO, 2.5 microg), but not with the delta agonist D-Pen2,5-enkephalin (DPEN, 3.1 microg). The final experiment tested the diurnal variability of the expression of conditioned feeding, and it was found that the magnitude of the effect depended on time of day. In summary, the development of opioid-induced conditioned feeding depends on mu opiate receptor stimulation, but not dopamine receptor stimulation. Its expression, however, involves both opiate and D-1 receptor activation. These findings are considered in terms of putative neural mechanisms governing conditioned meal initiation, and implications for compulsive eating and bulimia are also discussed.

Morphine-associated environmental cues elicit conditioned gene expression.

Drug-associated contextual cues can exert a powerful influence on behavior through associative pairing between the drug and the environment. However, the anatomical and molecular substrates for these effects are not well characterized. Using a drug-conditioning paradigm, we examined the expression of the immediate early gene product, Fos, within specific brain circuits using immunocytochemical detection. Rats were given either morphine (5 mg/ml/kg) or saline once a day for 10 days. The drug administration was always paired with a specific environment (activity monitors) different from the home cage. Following this treatment, the rats were returned to the cages at various times thereafter, with only a mock injection. Conditioned behavioral activation was observed in rats at 3, 5, and 7 days following treatment with morphine. In rats showing the conditioned motor response, several cortical and limbic areas showed substantial increases in the number of Fos positive cells, indicating that these regions were more active during exposure to the drug-paired environment. Areas that were most activated included prefrontal cortex, cingulate cortex, nucleus accumbens, and preoptic area. Further analysis showed that this increase in Fos expression was not directly related to the increase in motor activity, and that the drug-associated conditioning and Fos expression was lessened at 7 days and absent by 14 days post-treatment. These results are discussed in terms of their relevance to the problem of relapse in drug addiction.

N-methyl-D-aspartate receptor-dependent plasticity within a distributed corticostriatal network mediates appetitive instrumental learning.

The effect of microinfusion of the N-methyl-D-aspartate (NMDA) antagonist 2-amino-5-phosphonopentanoic acid (AP-5) into the amygdala, medial prefrontal cortex, and dorsal and ventral subiculum on acquisition of a lever-pressing task for food in rats was examined. Serial transmission between the basolateral amygdala and nucleus accumbens core was also examined in an asymmetric infusion design. AP-5 administered bilaterally into either the amygdala or medial prefrontal cortex markedly impaired learning, whereas administration into the dorsal or ventral subiculum had no effect. Unilateral infusion of AP-5 into either the nucleus accumbens core or amygdala was also sufficient to impair learning. These data provide novel evidence for NMDA receptor-dependent plasticity within corticostriatal networks in the acquisition of appetitive instrumental learning.

Response-reinforcement learning is dependent on N-methyl-D-aspartate receptor activation in the nucleus accumbens core.

The nucleus accumbens, a site within the ventral striatum, is best known for its prominent role in mediating the reinforcing effects of drugs of abuse such as cocaine, alcohol, and nicotine. Indeed, it is generally believed that this structure subserves motivated behaviors, such as feeding, drinking, sexual behavior, and exploratory locomotion, which are elicited by natural rewards or incentive stimuli. A basic rule of positive reinforcement is that motor responses will increase in magnitude and vigor if followed by a rewarding event. It is likely, therefore, that the nucleus accumbens may serve as a substrate for reinforcement learning. However, there is surprisingly little information concerning the neural mechanisms by which appetitive responses are learned. In the present study, we report that treatment of the nucleus accumbens core with the selective competitive N-methyl-D-aspartate (NMDA) antagonist 2-amino-5-phosphonopentanoic acid (AP-5; 5 nmol/0.5 microl bilaterally) impairs response-reinforcement learning in the acquisition of a simple lever-press task to obtain food. Once the rats learned the task, AP-5 had no effect, demonstrating the requirement of NMDA receptor-dependent plasticity in the early stages of learning. Infusion of AP-5 into the accumbens shell produced a much smaller impairment of learning. Additional experiments showed that AP-5 core-treated rats had normal feeding and locomotor responses and were capable of acquiring stimulus-reward associations. We hypothesize that stimulation of NMDA receptors within the accumbens core is a key process through which motor responses become established in response to reinforcing stimuli. Further, this mechanism, may also play a critical role in the motivational and addictive properties of drugs of abuse.

Enhanced reward-related responding following cholera toxin infusion into the nucleus accumbens.

In recent years, considerable focus has been directed to understanding how drugs of abuse affect neuronal function at the molecular level. For example, repeated administration of stimulants or opiates can induce long-lasting alterations in gene expression, transcription factors, and signal transduction pathways. Our laboratory previously showed that intraaccumbens infusion of cholera toxin (CTX), which alters the Gs protein such that production of cyclic Adenosine Monophosphate (AMP) is upregulated, causes pronounced, long-lasting motor activation and sensitization to stimulants. In the present experiments, the effect of intraaccumbens infusion of cholera toxin on reward-related responding was investigated. The conditioned reinforcement (CR) paradigm was employed, which measures an animal's instrumental response to obtain presentation of a stimulus previously paired with a primary reward. When this stimulus supports acquisition of a new operant response (lever-pressing), it is termed a conditioned reinforcer (CR). In the first experiment, the effects of bilateral intraaccumbens infusion of CTX (100 ng/1 microliter) were examined on previously-established responding. CTX treatment resulted in enhanced responding for the CR. This enhancement developed over several days and reached its peak 3 days following infusion. In the second experiment, the influence of CTX was examined on acquisition of responding for the CR. The group treated with CTX (100 ng) discriminated between the CR and control (NCR) lever earlier than the vehicle-infused group, and showed greater levels of responding on the CR lever. In the third experiment, it was determined that infusion of CTX (300 ng bilaterally) into the anterior dorsal striatum did not affect levels of responding, although a later test with cocaine in these animals (25 mg/kg, intraperitoneally) (i.p.) indicated that they were capable of potentiated responding. These data are interpreted as evidence that the G(S) protein-cyclic AMP second messenger system within the nucleus accumbens is directly involved in reward-related behavior.

Microinfusion of corticotropin-releasing factor into the nucleus accumbens shell results in increased behavioral arousal and oral motor activity.

Corticotropin-releasing factor (CRF) is a 41 amino acid peptide postulated to be involved in integrating the physiological and behavioral responses to stress. The purpose of this experiment was to determine the effects of CRF microinfused into the nucleus accumbens core (AcbC) and shell (AcbSh) subregions. Rats were tested for general motor activity, cage crossings, and rearing following CRF (0, 125, 250, or 500 ng). Behavioral observations were also made to determine the profile of activity caused by CRF infusion into the Acb. CRF in the AcbSh but not the AcbC regions elicited an increase in general motor activity that lasted approximately 2 h. When compared with ventricular injections, CRF in the AcbSh had greater activating effects. The CRF-induced behavioral profile consisted of increases in grooming, sniffing, and oral behavior. Results are discussed as they pertain to the involvement of the AcbSh in stress, motivated behavior, and drug sensitization.

Injections of nociceptin into nucleus accumbens shell or ventromedial hypothalamic nucleus increase food intake.

The novel opioid receptor ORL1 is widely distributed throughout the CNS of the rat, and is present in high densities in several brain regions known to participate in the control of food intake. We injected a recently identified endogenous agonist of this receptor, nociceptin, into two of these feeding-related areas. Microinjections of nociceptin (2.5-25 nmol) into either the ventromedial hypothalamic nucleus or the nucleus accumbens shell significantly increased food intake in rats. We believe this to be the first report of a specific effect of nociceptin on a motivated behavior.