Corticotropin-releasing factor projections from limbic forebrain and paraventricular nucleus of the hypothalamus to the region of the ventral tegmental area.

Corticotropin-releasing factor (CRF) is a peptide neurotransmitter with high numbers of cell bodies found in limbic regions of the rat brain including the oval nucleus of the bed nucleus of the stria terminalis (BNSTov) and central nucleus of the amygdala (CeA) as well as in the paraventricular nucleus of the hypothalamus (PVN). CRF systems are activated in response to acute stressors and mediate a wide variety of physiological and behavioral responses to acute stress including aversive responses and responses that support appetitive behaviors. CRF is released in the ventral tegmental area (VTA), the cell body region of the mesocorticolimbic dopaminergic neurons, in response to acute stress and plays a role in stress-activation of appetitive behavior [Wang B, Shaham Y, Zitzman D, Azari S, Wise RA, You ZB (2005) Cocaine experience establishes control of midbrain glutamate and dopamine by corticotropin-releasing factor: a role in stress-induced relapse to drug seeking. J Neurosci 25:5389-5396]. However, although it is known that the VTA region contains significant levels of CRF-immunoreactive fibers [Swanson LW, Sawchenko PE, Rivier J, Vale WW (1983) Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinology 36:165-186], the source of CRF input to the region has not been identified. We used infusions of a fluorescent retrograde tracer, fluorogold, into the VTA region, combined with fluorescent immunocytochemistry for CRF to identify sources of this input. Double-labeled cells were found in BNSTov, CeA and PVN. The percent of fluorogold-labeled cells in each region that were CRF-positive was 30.8, 28.0 and 16.7% respectively. These data point to diffusely distributed sources of CRF-containing fibers in the VTA.

Netrin receptor deficient mice exhibit functional reorganization of dopaminergic systems and do not sensitize to amphetamine.

Netrins are guidance cues that play a fundamental role in organizing the developing brain. The netrin receptor, DCC (deleted in colorectal cancer), is highly expressed by dopaminergic (DA) neurons. DCC may therefore participate in the organization of DA circuitry during development and also influence DA function in the adult. Here we show that adult dcc heterozygous mice exhibit a blunted behavioral response to the indirect DA agonist amphetamine and do not develop sensitization to its effects when treated repeatedly. These behavioral alterations are associated with profound changes in DA function. In the medial prefrontal cortex, dcc heterozygotes exhibit increased tyrosine hydroxylase (TH) protein levels and dramatic increases in basal concentrations of DA and DA metabolites. In contrast, in the nucleus accumbens, dcc heterozygotes show no changes in either TH or DA levels, but exhibit decreased concentrations of DA metabolites, suggesting reduced DA activity. In addition, dcc heterozygous mice exhibit a small, but significant reduction in total number of TH-positive neurons in midbrain DA cell body regions. These results demonstrate for the first time that alterations in dcc expression lead to selective changes in DA function and, in turn, to differences in DA-related behaviors in adulthood. These findings raise the possibility that changes in dcc function early in life are implicated in the development of DA dysregulation observed in certain psychiatric disorders, such as schizophrenia, or following chronic use of drugs of abuse.

Effects of sex and hormonal status on astrocytic basic fibroblast growth factor-2 and tyrosine hydroxylase immunoreactivity after medial forebrain bundle 6-hydroxydopamine lesions of the midbrain dopamine neurons.

We examined astrocytic basic fibroblast growth factor immunoreactivity (FGF-2-IR) and tyrosine hydroxylase immunoreactivity (TH-IR) in the cell-body region of midbrain dopaminergic neurons after unilateral infusions of the neurotoxin 6-hydroxydopamine into the medial forebrain bundle in male and female rats. In addition, to determine whether neonatal exposure to gonadal hormones has consequences on the expression of astrocytic FGF-2 and cell loss in response to injury in adulthood, we studied the effects of these lesions in adult male and female rats that had been exposed or not to testosterone in the neonatal period. In both males and females there was a progressive loss of TH-expressing cells that peaked 5 weeks after the lesions. Females showed less loss of TH-expressing cells than males, but this effect was not estrogen dependent. Lesions led to an increase in expression of astrocytic FGF-2 that was greater in males than in females. Finally, it was found that, regardless of genetic sex, rats exposed to testosterone neonatally showed greater astrocytic FGF-2 expression after lesions than those not exposed, and that among those not exposed to testosterone, estrogen treatment had a modest protective effect. Analysis of behavior and striatal dopamine content showed that the percent of striatal dopamine depletion 14 days after the lesion correlated with the amount of behavioral asymmetry displayed by animals on all tests conducted after lesioning. In groups killed 2 and 5 weeks after the lesion, the amount of behavioral asymmetry correlated with the percent loss of TH-IR cells and with the percent increase in FGF-2-IR cells in the midbrain. These relationships were not evident in groups killed 3 and 7 days after the lesion, possibly because the changes in the number of FGF-2- and TH-IR cells were not fully manifested. The present findings show that hormonal events early in life can alter the response of midbrain dopamine neurons to insult and injury in adult life and suggest that the slow degeneration of these neurons may release signals triggering a sustained activation of adjacent astrocytes which, in turn, may lead to induction of astrocytic FGF-2.

A role for the CRF-containing pathway from central nucleus of the amygdala to bed nucleus of the stria terminalis in the stress-induced reinstatement of cocaine seeking in rats.

RATIONALE: We reported previously that bilateral injection of a corticotropin-releasing factor (CRF)-receptor antagonist, D-Phe CRF(12-41), into the bed nucleus of the stria terminalis (BNST) blocks the reinstatement of cocaine seeking induced by footshock, whereas the injection of CRF into the same region induces reinstatement. One source of CRF in the BNST arises from a CRF-containing projection originating in the central nucleus of the amygdala (CeA). OBJECTIVE: To determine whether the CRF-containing projection from the amygdala to the BNST is involved in the mediation of stress-induced reinstatement of cocaine seeking by functionally interrupting the pathway. METHODS: Rats trained to self-administer cocaine (1 mg/kg, IV, 9 days) were given extinction sessions after a 10- to 11-day drug-free period, followed by tests for stress-induced reinstatement (footshock: 15 min intermittent 0.8-mA footshocks given immediately before presentation of the previously active lever). Before the tests, animals were pretreated with either: (1) TTX (2.5 ng) in amygdala (including the CeA) in one hemisphere and D-Phe CRF(12-41) (50 ng) in BNST in the other, (2) unilateral TTX, or (3) unilateral D-Phe. RESULTS: Footshock reinstated cocaine seeking following unilateral injections of either TTX in amygdala or D-Phe in BNST, but following the injection of both TTX in amygdala and D-Phe in BNST the effects of footshock were greatly attenuated. CONCLUSION: These results suggest that the CRF-containing pathway from CeA to BNST is involved in mediating the effects of CRF and its receptor antagonist in the BNST on the reinstatement of cocaine seeking.

Ovariectomy of adult rats leads to increased expression of astrocytic basic fibroblast growth factor in the ventral tegmental area and in dopaminergic projection regions of the entorhinal and prefrontal cortex.

Changes in astrocytic function may underlie the neurochemical and morphological alterations in limbic and cortical areas after estrogen loss in adult females. We assessed whether increased expression of basic fibroblast growth factor (bFGF), an astrocytic response involved in injury-induced neuronal plasticity, occurs after ovariectomy. We examined bFGF immunoreactivity (IR) in ovariectomized rats with oil or estradiol benzoate (5 microgram every 4 d; Experiment 1) and in ovariectomized and intact animals (Experiment 2). In the ventral tegmental area (VTA), bFGF-IR and glial fibrillary acidic protein (GFAP)-IR were greater in ovariectomized animals than in animals with estrogen replacement. bFGF-IR in the VTA was greater in ovariectomized than in intact females. In the dorsal raphe, no differences between groups were found in GFAP-IR or bFGF-IR. In mesolimbic dopaminergic target areas within entorhinal cortex (Ent), prefrontal cortex, and nucleus accumbens, bFGF-IR was higher in Ent of ovariectomized animals 4 weeks after surgery in both experiments, but no differences were seen in nucleus accumbens or in an occipital cortical, control, area in either study. In Experiment 2, small increases in bFGF-IR were seen in the prefrontal cortex after ovariectomy. In the VTA and Ent, changes in bFGF-IR developed gradually, peaking at 4 weeks and waning at 40 weeks. Furthermore, increased dendritic arbor of Ent layer II/III pyramidal cells was found in ovariectomized females with the use of a modified Golgi-Cox staining procedure. These findings suggest that, within specific regions, ovariectomy induces astrocytic responses similar to those observed after injury that may affect neuronal chemistry and morphology.

The effects of gonadal hormones on the development and expression of the stimulant effects of morphine in male and female rats.

Previous studies have shown that the behavioral activating effects of the stimulant drug amphetamine are augmented in female rats by estradiol. Here we studied the effects of gonadectomy and gonadal hormone replacement on the stimulant effects of morphine in both females and males. Groups of intact, ovariectomized (Ovex), and Ovex females given estradiol benzoate (EB) (5 micrograms), and groups of intact, castrated, and castrated males given testosterone propionate (30 micrograms) were administered five injections of morphine sulphate (10 mg/kg, i.p.) or saline at 3-day intervals. Activity was monitored on each occasion for 2 h. Among females treated with morphine, intact and Ovex-EB animals showed progressive enhancement of activity over sessions, whereas Ovex animals showed no change. Three days after the last pre-exposure session, all animals received 5 mg/kg morphine in a test for sensitization. In spite of the lower levels of activity in Ovex animals, animals from all groups previously exposed to morphine showed a sensitized response to morphine compared with those receiving morphine for the first time. These findings are virtually identical to our previous findings in female rats treated with amphetamine. Among males, only intact animals showed a progressive increase in morphine-induced activity and only in the second hour of testing, but, overall, there was no significant effect of either group or drug during the pre-exposure phase. On the test for sensitization, as seen in females, those males that had been exposed to morphine previously showed a sensitized responses to morphine. There were, however, no differences in activity levels between the groups of males. We conclude that although gonadal hormones, and in particular estradiol, may modify the magnitude of the response to amphetamine and morphine, they appear not to be involved in those neurochemical and neuronal changes that occur during and following repeated drug exposures, and that underlie the enhanced sensitivity to the stimulant effects of a drug seen when such animals are compared with animals receiving the drug for the first time.

Long-lasting induction of astrocytic basic fibroblast growth factor by repeated injections of amphetamine: blockade by concurrent treatment with a glutamate antagonist.

Repeated administration of stimulant drugs leads to lasting changes in their behavioral and neurochemical effects. These changes are initiated by drug actions in the somatodendritic regions of midbrain dopaminergic neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) and continue to develop for a period of time after termination of drug treatment. Here we show that repeated administration of amphetamine (3.0 mg/kg, i.p.; three injections, once every other day) results in sustained increases in basic fibroblast growth factor immunoreactivity (bFGF-IR) in both VTA and SNc, 200-500% over that seen in saline-treated animals. Increases were observed 24 hr, 72 hr, 1 week and 1 month after the last drug injection. Because glutamate participates in the development of sensitization to stimulant drugs, we assessed the effect of the glutamate antagonist, kynurenic acid (KYN), on amphetamine-induced bFGF-IR. Coadministration of KYN prevented the increases in bFGF-IR in both VTA and SNc assessed 1 week after the amphetamine treatment. No changes in bFGF-IR were observed in the nucleus accumbens or dorsal striatum. bFGF-IR was found to be associated with astrocytes and not with dopaminergic neurons. These findings suggest that sustained enhancement of astrocytic bFGF expression in DA somatodendritic regions is a mechanism whereby stimulant drugs exert enduring effects on midbrain DA function. We hypothesize that increased glutamatergic activity elicited by amphetamine and other stimulant drugs places excessive demands on the functioning of DA neurons recruiting regulatory and neuroprotective processes that lead to enduring changes in DA neuron functioning and connectivity.

Sensitization of stress-induced feeding in rats repeatedly exposed to brief restraint: the role of corticosterone.

Groups of male Wistar rats lived in cages capable of monitoring feeding and drinking continuously at 0.1-s intervals, 24 h per day. Intact animals were subjected to 20 min of restraint stress or to brief handling (Brief Pick-Up), daily or every third day, 6 h after the beginning of the 12-h light period. In both studies, food-intake increased in the first hour after restraint, peaking between 15 and 45 min. Smaller increases were seen following Brief Pick-Up. More interestingly, the amount of food eaten increased across test sessions, indicating sensitization of the response to stress. Drinking also increased following stress, occurring before feeding and diminishing after the first 15 min. In adrenalectomized animals implanted with slow-release pellets to replace basal diurnal levels of corticosterone (ADX animals), sensitization of the feeding response to restraint stress developed across test sessions, although in these animals, the acute increase in food-intake following restraint stress was attenuated. ADX animals subjected only to Brief Pick-Up showed no increases in food-intake. Daily injections of 3.0 mg/kg corticosterone given to such ADX animals were unable to mimic the effects of restraint on either food-intake or drinking, nor did they augment the effects of restraint in ADX animals. We conclude that sensitization to the effects of brief restraint stress on food-intake can occur independently of a stress-induced rise in plasma corticosterone.

Reinstatement of heroin-reinforced behavior following long-term extinction: implications for the treatment of relapse to drug taking.

The effect of non-contingent priming injections of heroin on drug-reinforced behavior following long-term extinction was examined. Male rats were trained to lever press for 100µg/kg/infusion of intravenous heroin during four 6h sessions per day. The drug-reinforced behavior was extinguished by daily reductions in dose followed by at least 12 sessions of saline self-administration. A single non-contingent priming injection of heroin (100µg/kg, i.v.) resulted in the reinstatement of the drug-reinforced behavior, even though extinction conditions remained. The results indicate that the drug-induced reinstatement of previously extinguished drug-reinforced behavior is possible even after long periods of extinction. The implications of the present findings for the treatment of relapse to drug abuse in humans are discussed.