Michela Marinelli

Leptin Receptor Signaling in Midbrain Dopamine Neurons Regulates Feeding

The leptin hormone is critical for normal food intake and metabolism. While leptin receptor (Lepr) function has been well studied in the hypothalamus, the functional relevance of Lepr expression in the ventral tegmental area (VTA) has not been investigated. The VTA contains dopamine neurons that are important in modulating motivated behavior, addiction, and reward. Here, we show that VTA dopamine neurons express Lepr mRNA and respond to leptin with activation of an intracellular JAK-STAT pathway and a reduction in firing rate. Direct administration of leptin to the VTA caused decreased food intake while long-term RNAi-mediated knockdown of Lepr in the VTA led to increased food intake, locomotor activity, and sensitivity to highly palatable food. These data support a critical role for VTA Lepr in regulating feeding behavior and provide functional evidence for direct action of a peripheral metabolic signal on VTA dopamine neurons.

Formation of accumbens GluR2-lacking AMPA receptors mediates incubation of cocaine craving

Relapse to cocaine use after prolonged abstinence is an important clinical problem. This relapse is often induced by exposure to cues associated with cocaine use. To account for the persistent propensity for relapse, it has been suggested that cue-induced cocaine craving increases over the first several weeks of abstinence and remains high for extended periods. We and others identified an analogous phenomenon in rats that was termed ‘incubation of cocaine craving’: time-dependent increases in cue-induced cocaine-seeking over the first months after withdrawal from self-administered cocaine. Cocaine-seeking requires the activation of glutamate projections that excite receptors for α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in the nucleus accumbens. Here we show that the number of synaptic AMPA receptors in the accumbens is increased after prolonged withdrawal from cocaine self-administration by the addition of new AMPA receptors lacking glutamate receptor 2 (GluR2). Furthermore, we show that these new receptors mediate the incubation of cocaine craving. Our results indicate that GluR2-lacking AMPA receptors could be a new target for drug development for the treatment of cocaine addiction. We propose that after prolonged withdrawal from cocaine, increased numbers of synaptic AMPA receptors combined with the higher conductance of GluR2-lacking AMPA receptors causes increased reactivity of accumbens neurons to cocaine-related cues, leading to an intensification of drug craving and relapse.

The mesopontine rostromedial tegmental nucleus: A structure targeted by the lateral habenula that projects to the ventral tegmental area of Tsai and substantia nigra compacta

Prior studies revealed that aversive stimuli and psychostimulant drugs elicit Fos expression in neurons clustered above and behind the interpeduncular nucleus that project strongly to the ventral tegmental area (VTA) and substantia nigra (SN) compacta (C). Other reports suggest that these neurons modulate responses to aversive stimuli. We now designate the region containing them as the “mesopontine rostromedial tegmental nucleus” (RMTg) and report herein on its neuroanatomy. Dense μ‐opioid receptor and somatostatin immunoreactivity characterize the RMTg, as do neurons projecting to the VTA/SNC that are enriched in GAD67 mRNA. Strong inputs to the RMTg arise in the lateral habenula (LHb) and, to a lesser extent, the SN. Other inputs come from the frontal cortex, ventral striatopallidum, extended amygdala, septum, preoptic region, lateral, paraventricular and posterior hypothalamus, zona incerta, periaqueductal gray, intermediate layers of the contralateral superior colliculus, dorsal raphe, mesencephalic, pontine and medullary reticular formation, and the following nuclei: parafascicular, supramammillary, mammillary, ventral lateral geniculate, deep mesencephalic, red, pedunculopontine and laterodorsal tegmental, cuneiform, parabrachial, and deep cerebellar. The RMTg has meager outputs to the forebrain, mainly to the ventral pallidum, preoptic‐lateral hypothalamic continuum, and midline‐intralaminar thalamus, but much heavier outputs to the brainstem, including, most prominently, the VTA/SNC, as noted above, and to medial tegmentum, pedunculopontine and laterodorsal tegmental nuclei, dorsal raphe, and locus ceruleus and subceruleus. The RMTg may integrate multiple forebrain and brainstem inputs in relation to a dominant LHb input. Its outputs to neuromodulatory projection systems likely converge with direct LHb projections to those structures. J. Comp. Neurol. 513:566–596, 2009.

Enhanced Reactivity and Vulnerability to Cocaine Following Methylphenidate Treatment in Adolescent Rats

Treatment of attention deficit hyperactivity disorder with the psychostimulant drug methylphenidate (MP) has increased dramatically among schoolchildren. We tested whether repeated exposure to moderate doses of MP (5 and 10 mg/kg IP for 5 or 7 days) in adolescent rats increased reactivity to cocaine measured by motor responses (ambulations and rearing) to a cocaine challenge in adulthood. We later tested whether repeated exposure to a low dose of MP (2 mg/kg IP for 7 days) enhanced the psychomotor effects of cocaine, measured by different challenge doses (0–30 mg/kg) as well as to the reinforcing effects of cocaine, measured by self-administration of low-dose infusion (75 μg/kg, IV). We found that exposure to moderate doses of MP enhanced psychomotor responses to cocaine but exposure to a low dose only increased cocaine self-administration. These results suggest that adolescent exposure to low doses of MP in rats may increase the incentive value of low reinforcers, thereby rendering adult rats more susceptible to cocaine self-administration.

Stress and addiction: glucocorticoid receptor in dopaminoceptive neurons facilitates cocaine seeking

The glucocorticoid receptor is a ubiquitous transcription factor mediating adaptation to environmental challenges and stress. Selective Nr3c1 (the glucocorticoid receptor gene) ablation in mouse dopaminoceptive neurons expressing dopamine receptor 1a, but not in dopamine-releasing neurons, markedly decreased the motivation of mice to self-administer cocaine, dopamine cell firing and the control exerted by dopaminoceptive neurons on dopamine cell firing activity. In contrast, anxiety was unaffected, indicating that glucocorticoid receptors modify a number of behavioral disorders through different neuronal populations.

Progeny of mothers drinking corticosterone during lactation has lower stress-induced corticosterone secretion and better cognitive performance

In order to test the hypothesis that maternal corticosterone influences hypothalamus-pituitary-adrenal (HPA) system activity in the adult rat and behaviors related to it, we induced a moderate increase in maternal plasma level of corticosterone by adding the hormone to the drinking water of the dams (200 micrograms/ml) from the day after delivery to weaning. Our previous experiments have shown that this procedure produces plasma levels of the hormone in the range of those following a mild psychic stress (from 4.3 +/- 0.5 to 9.5 +/- 1.8 micrograms/100 ml in the dams, and from 0.7 +/- 0.1 to 1.2 +/- 0.2 micrograms/100 ml in the pups at 10 days of lactation). Adrenal weights were slightly and temporarily decreased by treatment in both mothers and offspring. Only the male progeny was investigated in this study. Corticosterone-nursed rats had significantly less corticosterone and ACTH in basal conditions and after a 2 min restraint stress at 3 months of age, and showed better performances at weaning and at 1, 2 and 3 months of life in the Morris water maze. Our results demonstrate that a moderate increase in maternal corticosterone during lactation influences the activity of HPA axis and improves spatial learning ability of the adult offspring.

Calcium-permeable AMPA receptors are present in nucleus accumbens synapses after prolonged withdrawal from cocaine self-administration but not experimenter-administered cocaine.

Repeated noncontingent cocaine injections, which lead to behavioral sensitization, increase AMPA receptor (AMPAR) transmission in the rodent nucleus accumbens (NAc) in a withdrawal-dependent manner. On withdrawal days (WD) 10–21, this is attributable to upregulation of GluA1A2-containing AMPARs. However, synaptic incorporation of GluA2-lacking/Ca2+-permeable AMPARs (CP-AMPARs) was observed after longer withdrawal (WD35) from repeated noncontingent cocaine injections in young mice (Mameli et al., 2009). CP-AMPARs had previously been observed in NAc synapses only after prolonged (WD30–WD47) withdrawal from extended-access cocaine self-administration. Our goal was to determine whether rats receiving repeated noncontingent cocaine injections during adulthood similarly exhibit CP-AMPARs in the NAc after prolonged withdrawal. For comparison, we began by evaluating CP-AMPARs on WD35–WD49 after extended-access cocaine self-administration. Confirming our previous results, whole-cell recordings revealed inwardly rectifying AMPAR EPSCs, a hallmark of CP-AMPARs. This was observed in both core and shell. Next, we conducted the same analysis in adult rats treated with eight daily noncontingent cocaine injections and recorded on WD35–WD49. AMPAR EPSCs in core and shell did not show inward rectification and were insensitive to 1-naphthylacetylspermine (a selective antagonist of CP-AMPARs). Locomotor sensitization could still be demonstrated after this long withdrawal period, although the upregulation of GluA1A2-containing AMPARs observed at earlier withdrawal times was no longer detected. In conclusion, in adult rats, accumulation of synaptic CP-AMPARs in the NAc occurs after prolonged withdrawal from extended-access cocaine self-administration but not after prolonged withdrawal from noncontingent cocaine injections.

Dopamine Scales Performance in the Absence of New Learning

Learning and motivation are integral in shaping an organisms adaptive behavior. The dopamine system has been implicated in both processes; however, dissociating the two, both experimentally and conceptually, has posed significant challenges. We have developed an animal model that dissociates expression or scaling of a learned behavior from learning itself. An inducible dopamine transporter (DAT) knockdown mouse line has been generated, which exhibits significantly slower reuptake of released dopamine and increased tonic firing of dopamine neurons without altering phasic burst firing. Mice were trained in experimental tasks prior to inducing a hyperdopaminergic tone and then retested. Elevated dopamine enhanced performance in goal-directed operant responses. These data demonstrate that alterations in dopaminergic tone can scale the performance of a previously learned behavior in the absence of new learning.

Group I mGluR Activation Reverses Cocaine-Induced Accumulation of Calcium-Permeable AMPA Receptors in Nucleus Accumbens Synapses via a Protein Kinase C-Dependent Mechanism

Following prolonged withdrawal from extended access cocaine self-administration in adult rats, high conductance Ca2+-permeable AMPA receptors (CP-AMPARs) accumulate in nucleus accumbens (NAc) synapses and mediate the expression of “incubated” cue-induced cocaine craving. Using patch-clamp recordings from NAc slices prepared after extended access cocaine self-administration and >45 d of withdrawal, we found that group I metabotropic glutamate receptor (mGluR) stimulation using 3,5-dihydroxyphenylglycine (DHPG; 50 μm) rapidly eliminates the postsynaptic CP-AMPAR contribution to NAc synaptic transmission. This is accompanied by facilitation of Ca2+-impermeable AMPAR (CI-AMPAR)-mediated transmission, suggesting that DHPG may promote an exchange between CP-AMPARs and CI-AMPARs. In saline controls, DHPG also reduced excitatory transmission but this occurred through a CB1 receptor-dependent presynaptic mechanism rather than an effect on postsynaptic AMPARs. Blockade of CB1 receptors had no significant effect on the alterations in AMPAR transmission produced by DHPG in the cocaine group. Interestingly, the effect of DHPG in the cocaine group was mediated by mGluR1 whereas its effect in the saline group was mediated by mGluR5. These results indicate that regulation of synaptic transmission in the NAc is profoundly altered after extended access cocaine self-administration and prolonged withdrawal. Furthermore, they suggest that activation of mGluR1 may represent a potential strategy for reducing cue-induced cocaine craving in abstinent cocaine addicts.

Prominent Activation of Brainstem and Pallidal Afferents of the Ventral Tegmental Area by Cocaine

Blockade of monoamine transporters by cocaine should not necessarily lead to certain observed consequences of cocaine administration, including increased firing of ventral mesencephalic dopamine (DA) neurons and accompanying impulse-stimulated release of DA in the forebrain and cortex. Accordingly, we hypothesize that the dopaminergic-activating effect of cocaine requires stimulation of the dopaminergic neurons by afferents of the ventral tegmental area (VTA). We sought to determine if afferents of the VTA are activated following cocaine administration. Rats were injected in the VTA with retrogradely transported Fluoro-Gold and, after 1 week, were allowed to self-administer cocaine or saline via jugular catheters for 2 h on 6 consecutive days. Other rats received a similar amount of investigator-administered cocaine through jugular catheters. Afterward, the rats were killed and the brains processed immunohistochemically for retrogradely transported tracer and Fos, the protein product of the neuronal activation-associated immediate early gene, c-fos. Forebrain neurons exhibiting both Fos and tracer immunoreactivity were enriched in both cocaine groups relative to the controls only in the globus pallidus and ventral pallidum, which, together, represented a minor part of total forebrain retrogradely labeled neurons. In contrast, both modes of cocaine administration strongly increased double-labeling relative to the controls in the brainstem, specifically in the caudal ventromedial mesencephalon and rostromedial pontine tegmentum. It is concluded that a previously unappreciated activation of pallidal and brainstem afferents may contribute to the modulation of dopaminergic neuronal activity following cocaine administration.