Targeting Catecholaminergic Systems in Transgenic Rats With a CAV-2 Vector Harboring a Cre-Dependent DREADD Cassette.

Techniques that allow the manipulation of specific neural circuits have greatly increased in the past few years. DREADDs (Designer receptors exclusively activated by designer drugs) provide an elegant way to manipulate individual brain structures and/or neural circuits, including neuromodulatory pathways. Considerable efforts have been made to increase cell-type specificity of DREADD expression while decreasing possible limitations due to multiple viral vectors injections. In line with this, a retrograde canine adenovirus type 2 (CAV-2) vector carrying a Cre-dependent DREADD cassette has been recently developed. In combination with Cre-driver transgenic animals, the vector allows one to target neuromodulatory pathways with cell-type specificity. In the present study, we specifically targeted catecholaminergic pathways by injecting the vector in knock-in rat line containing Cre recombinase cassette under the control of the tyrosine hydroxylase promoter. We assessed the efficacy of infection of the nigrostriatal pathway and the catecholaminergic pathways ascending to the orbitofrontal cortex (OFC) and found cell-type-specific DREADD expression.

Protracted motivational dopamine-related deficits following adolescence sugar overconsumption.

Adolescence represents a critical period characterized by major neurobiological changes. Chronic stimulation of the reward system during adolescence might constitute an important factor of vulnerability to pathological development. Increasing evidences suggest that adolescent overconsumption of sweet palatable foods impact reward-based processes. However, the neurobiological bases of these deficits remain poorly understood. Previous studies have demonstrated motivational deficits for palatable foods after sweet diet exposure during adolescence that might involve the dopamine (DA) system, a central actor in incentive processes. In the present study, the impact of adolescent sugar overconsumption on the sensitivity of the DA system was tested using pharmacological (Experiment 1) and receptor expression approaches (Experiment 2). Adolescent rats received free and continuous access to 5% sucrose solution from post-natal day 30-46. At adulthood, the functionality of the DA system in motivational processes was tested using systemic injections of specific DA receptors D1R or D2R agonists and antagonists during a motivation-dependent progressive ratio task (Experiment 1). Sucrose-exposed rats showed a lower motivation for saccharin and a decreased sensitivity to the effects of both D1R and D2R stimulation and blockade. In Experiment 2, Sucrose-exposed animals presented a lower expression of both D1R and D2R in the nucleus accumbens, a central brain region for incentive processes, but not in dorsal striatum or prefrontal cortex. These findings highlight the impact of sucrose overconsumption during adolescence on DA system that may support deficits in reward-related disorders.

Adolescent stimulation of D2 receptors alters the maturation of dopamine-dependent goal-directed behavior.

Adolescence is a period of high sensitivity to drugs and rewards, characterized by the immaturity of decision-making abilities. A chronic stimulation of reward systems during this period might constitute a factor of vulnerability to the development of psychiatric disorders. However, the long-term consequences of such an exposure have seldom been explored. Here, we investigate at the adult age the effects of chronic dopamine (DA) stimulation during adolescence on both the maturation of DA systems and the cognitive processes underlying goal-directed actions. We first demonstrate that chronic stimulation of D2 receptors by quinpirole during adolescence alters the development of DA systems. This treatment has particularly prominent effects on the mesocortical DA pathway where it decreases DA fibers density, DA concentration, and DA receptors expression. Furthermore, we show that quinpirole-treated rats exhibit specific impairments in instrumental goal-directed behavior, as they fail to adapt their action when action-outcome relationships change in a contingency degradation procedure. These results therefore highlight the vulnerability of DA system and prefrontal areas to prolonged stimulation during adolescence, and its potential long-term impact on cognitive functions.

Parallel maturation of goal-directed behavior and dopaminergic systems during adolescence.

Adolescence is a crucial developmental period characterized by specific behaviors reflecting the immaturity of decision-making abilities. However, the maturation of precise cognitive processes and their neurobiological correlates at this period remain poorly understood. Here, we investigate whether a differential developmental time course of dopamine (DA) pathways during late adolescence could explain the emergence of particular executive and motivational components of goal-directed behavior. First, using a contingency degradation protocol, we demonstrate that adolescent rats display a specific deficit when the causal relationship between their actions and their consequences is changed. When the rats become adults, this deficit disappears. In contrast, actions of adolescents remain sensitive to outcome devaluation or to the influence of a pavlovian-conditioned stimulus. This aspect of cognitive maturation parallels a delayed development of the DA system, especially the mesocortical pathway involved in action adaptation to rule changes. Unlike in striatal and nucleus accumbens regions, DA fibers and DA tissue content continue to increase in the medial prefrontal cortex from juvenile to adult age. Moreover, a sustained overexpression of DA receptors is observed in the prefrontal region until the end of adolescence. These findings highlight the relationship between the emergence of specific cognitive processes, in particular the adaptation to changes in action consequences, and the delayed maturation of the mesocortical DA pathway. Similar developmental processes in humans could contribute to the adolescent vulnerability to the emergence of several psychiatric disorders characterized by decision-making deficits.

Transient role of the rat prelimbic cortex in goal-directed behaviour.

Lesion studies show that goal-directed actions mediated by action-outcome (A-O) associations and habits mediated by stimulus-response (S-R) associations can be dissociated during instrumental training, with the prelimbic region of the medial prefrontal cortex being involved in the former and the infralimbic region in the latter. The present work further investigates the role of the prelimbic region in acquisition vs. expression of goal-directed instrumental behaviour, using reversible neuronal inactivation and outcome devaluation procedures. In a first experiment, inactivating the prelimbic cortex at the time of testing did not alter the sensitivity to devaluation, indicating that this region was not essential for the expression of A-O associations. In a second experiment, the prelimbic cortex was inactivated throughout the training phase. At the time of testing the performance was insensitive to devaluation, indicating that the acquired response was not goal-directed but mediated by an S-R association. These data challenge the view that the habit system replaces the goal-directed system as training progresses. They show that the prelimbic cortex plays a transient but crucial role in the acquisition of goal-directed responding and that the A-O and S-R systems can operate in a competitive fashion early in training.

A role for medial prefrontal dopaminergic innervation in instrumental conditioning.

To investigate the involvement of dopaminergic projections to the prelimbic and infralimbic cortex in the control of goal-directed responses, a first experiment examined the effect of pretraining 6-OHDA lesions of these cortices. We used outcome devaluation and contingency degradation procedures to separately assess the representation of the outcome as a goal or the encoding of the contingency between the action and its outcome. All groups acquired the instrumental response at a normal rate, indicating that dopaminergic activity in the medial prefrontal cortex is not necessary for the acquisition of instrumental learning. Sham-operated animals showed sensitivity to both outcome devaluation and contingency degradation. Animals with dopaminergic lesions of the prelimbic cortex, but not the infralimbic cortex, failed to adapt their instrumental response to changes in contingency, whereas their response remained sensitive to outcome devaluation. In a second experiment, aimed at determining whether dopamine was specifically needed during contingency changes, we performed microinfusions of the dopamine D(1)/D(2) receptor antagonist flupenthixol in the prelimbic cortex only before contingency degradation sessions. Animals with infusions of flupenthixol failed to adapt their response to changes in contingency, thus replicating the deficit of animals with dopaminergic lesions in Experiment 1. These results demonstrate that dissociable neurobiological mechanisms support action-outcome relationships and goal representation, dopamine signaling in the prelimbic cortex being necessary for the former but not the latter.

Oxytocin-containing neurons in the hypothalamic parvicellular paraventricular nucleus of the jerboa: no plasticity related to acute immobilization.

OBJECTIVES AND METHODS: The presence of oxytocin (OT) and its putative participation to the phenotypic plasticity of CRH neurones in the stressed jerboa was investigated. We analysed by immunocytochemistry the OT expression within the hypothalamic parvicellular paraventricular nucleus (pPVN) of male jerboas submitted to an acute immobilization (30 min). RESULTS: OT presence was clearly demonstrated in the pPVN of the jerboa and no significant difference in the number of OT immunolabeled cells was observed whatever the experimental conditions. Interestingly, CRH-immunoreactive neurons coexpressed OT within cell bodies and terminals in a similar way both in control and stressed animals. The level of coexpression was regionally heterogeneous and was not sensitive to the stress immobilization. CONCLUSION: The present data reveal for the first time the occurrence of OT in hypothalamic pPVN neurons of the jerboa. Moreover, this OT expression level does not change upon an acute immobilization stress. These new data, coupled together with our previous work in the jerboa, incontestably establish a clear dichotomy between a stress-responsive CRH/CCK system and a stress non-responsive OT/VP system in the pPVN.

Gonadotropin-releasing hormone neuron requirements for puberty, ovulation, and fertility.

The absolute requirement for reproduction implies that the hypothalamo-pituitary-gonadal axis, controlling fertility, is an evolutionary robust mechanism. The GnRH neurons of the hypothalamus represent the key cell type within the body dictating fertility. However, the level of functional redundancy within the GnRH neuron population is unknown. As a result of a fortuitous transgene insertion event, GNR23 mice exhibit a marked allele-dependent reduction in GnRH neuron number within their brain. Wild-type mice have approximately 600 GnRH neurons, compared with approximately 200 (34%) and approximately 70 (12%) in GNR23(+/-) and GNR23(-/-) mice, respectively. Using these mice, we examined the minimal GnRH neuron requirements for fertility. Male GNR23(-/-) mice exhibited normal fertility. In contrast, female GNR23(-/-) mice were markedly subfertile, failing to produce normal litters, have estrous cycles, or ovulate. The failure of ovulation resulted from an inability of the few existing GnRH neurons to generate the LH surge. This was not the case, however, for the first cycle at puberty that appeared normal. Together, these observations demonstrate that 12% of the GnRH neuron population is sufficient for pulsatile gonadotropin secretion and puberty onset, whereas between 12 and 34% are required for cyclical control in adult female mice. This indicates that substantial redundancy exists within the GnRH neuronal population and suggests that the great majority of GnRH neurons must be dysfunctional before fertility is affected.

Disruption of ephrin signaling associates with disordered axophilic migration of the gonadotropin-releasing hormone neurons.

Ephrin signaling is involved in repulsive and attractive interactions mediating axon guidance and cell-boundary formation in the developing nervous system. As a result of a fortuitous transgene integration event, we have identified here a potential role for EphA5 in the axophilic migration of gonadotropin-releasing hormone (GnRH) neurons from the nasal placode into the brain along ephrin-expressing vomeronasal axons. Transgene integration in the GNR23 mouse line resulted in a 26 kb deletion in chromosome 5, approximately 67 kb 3' to Epha5. This induced a profound, region-specific upregulation of EphA5 mRNA and protein expression in the developing mouse brain. The GnRH neurons in GNR23 mice overexpressed EphA5 from embryonic day 11, whereas ephrin A3 and A5 mRNA levels in olfactory neurons were unchanged. The GnRH neurons were found to be slow in commencing their migration from the olfactory placode and also to form abnormal clusters of cells on the olfactory axons, prohibiting their migration out of the nose. As a result, adult hemizygous mice had only 40% of the normal complement of GnRH neurons in the brain, whereas homozygous mice had <15%. This resulted in infertility in adult female homozygous GNR23 mice, suggesting that some cases of human hypogonadotropic hypogonadism may result from ephrin-related mutations. These data provide evidence for a role of EphA-ephrin signaling in the axophilic migration of the GnRH neurons during embryogenesis.

Paraventricular nucleus neurons producing neurotensin after lipopolysaccharide treatment project to the median eminence.

Inflammation consists in secretion of cytokines that stimulate the hypothalamo-pituitary-adrenal (HPA) axis to release the anti-inflammatory corticosterone. Upstream in this axis are corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus (PVN) whose multipeptidergic phenotype changes: both corticotropin-releasing hormone mRNAs and neurotensin mRNAs are up-regulated. Combining in situ hybridization with a retrograde neuronal marker, we demonstrated that neurotensin-containing neurons in the paraventricular nucleus project to the median eminence.