Chronic Distress in Male Mice Impairs Motivation Compromising Both Effort and Reward Processing With Altered Anterior Insular Cortex and Basolateral Amygdala Neural Activation.

In humans and mammals, effort-based decision-making for monetary or food rewards paradigms contributes to the study of adaptive goal-directed behaviours acquired through reinforcement learning. Chronic distress modelled by repeated exposure to glucocorticoids in rodents induces suboptimal decision-making under uncertainty by impinging on instrumental acquisition and prompting negative valence behaviours. In order to further disentangle the motivational tenets of adaptive decision-making, this study addressed the consequences of enduring distress on relevant effort and reward-processing dimensions. Experimentally, appetitive and consummatory components of motivation were evaluated in adult C57BL/6JRj male mice experiencing chronic distress induced by oral corticosterone (CORT), using multiple complementary discrete behavioural tests. Behavioural data (from novelty suppressed feeding, operant effort-based choice, free feeding, and sucrose preference tasks) collectively show that behavioural initiation, effort allocation, and hedonic appreciation and valuation are altered in mice exposed to several weeks of oral CORT treatment. Additionally, data analysis from FosB immunohistochemical processing of postmortem brain samples highlights CORT-dependent dampening of neural activation in the anterior insular cortex (aIC) and basolateral amygdala (BLA), key telencephalic brain regions involved in appetitive and consummatory motivational processing. Combined, these results suggest that chronic distress-induced irregular aIC and BLA neural activations with reduced effort production and attenuated reward value processing during reinforcement-based instrumental learning could result in maladaptive decision-making under uncertainty. The current study further illustrates how effort and reward processing contribute to adjust the motivational threshold triggering goal-directed behaviours in versatile environments.

Chronic exposure to glucocorticoids induces suboptimal decision-making in mice.

Anxio-depressive symptoms as well as severe cognitive dysfunction including aberrant decision-making (DM) are documented in neuropsychiatric patients with hypercortisolaemia. Yet, the influence of the hypothalamo-pituitary-adrenal (HPA) axis on DM processes remains poorly understood. As a tractable mean to approach this human condition, adult male C57BL/6JRj mice were chronically treated with corticosterone (CORT) prior to behavioural, physiological and neurobiological evaluation. The behavioural data indicate that chronic CORT delays the acquisition of contingencies required to orient responding towards optimal DM performance in a mouse Gambling Task (mGT). Specifically, CORT-treated animals show a longer exploration and a delayed onset of the optimal DM performance. Remarkably, the proportion of individuals performing suboptimally in the mGT is increased in the CORT condition. This variability seems to be better accounted for by variations in sensitivity to negative rather than to positive outcome. Besides, CORT-treated animals perform worse than control animals in a spatial working memory (WM) paradigm and in a motor learning task. Finally, Western blotting neurobiological analyses show that chronic CORT downregulates glucocorticoid receptor expression in the medial Prefrontal Cortex (mPFC). Besides, corticotropin-releasing factor signalling in the mPFC of CORT individuals negatively correlates with their DM performance. Collectively, this study describes how chronic exposure to glucocorticoids induces suboptimal DM under uncertainty in a mGT, hampers WM and motor learning processes, thus affecting specific emotional, motor, cognitive and neurobiological endophenotypic dimensions relevant for precision medicine in biological psychiatry.

A basal ganglia-like cortical-amygdalar-hypothalamic network mediates feeding behavior.

The insular cortex (INS) is extensively connected to the central nucleus of the amygdala (CEA), and both regions send convergent projections into the caudal lateral hypothalamus (LHA) encompassing the parasubthalamic nucleus (PSTN). However, the organization of the network between these structures has not been clearly delineated in the literature, although there has been an upsurge in functional studies related to these structures, especially with regard to the cognitive and psychopathological control of feeding. We conducted tract-tracing experiments from the INS and observed a pathway to the PSTN region that runs parallel to the canonical hyperdirect pathway from the isocortex to the subthalamic nucleus (STN) adjacent to the PSTN. In addition, an indirect pathway with a relay in the central amygdala was also observed that is similar in its structure to the classic indirect pathway of the basal ganglia that also targets the STN. C-Fos experiments showed that the PSTN complex reacts to neophobia and sickness induced by lipopolysaccharide or cisplatin. Chemogenetic (designer receptors exclusively activated by designer drugs [DREADD]) inhibition of tachykininergic neurons (Tac1) in the PSTN revealed that this nucleus gates a stop "no-eat" signal to refrain from feeding when the animal is subjected to sickness or exposed to a previously unknown source of food. Therefore, our anatomical findings in rats and mice indicate that the INS-PSTN network is organized in a similar manner as the hyperdirect and indirect basal ganglia circuitry. Functionally, the PSTN is involved in gating feeding behavior, which is conceptually homologous to the motor no-go response of the adjacent STN.

Modelling decision-making under uncertainty: A direct comparison study between human and mouse gambling data.

Decision-making is a conserved evolutionary process enabling us to choose one option among several alternatives, and relies on reward and cognitive control systems. The Iowa Gambling Task allows the assessment of human decision-making under uncertainty by presenting four card decks with various cost-benefit probabilities. Participants seek to maximise their monetary gain by developing long-term optimal-choice strategies. Animal versions have been adapted with nutritional rewards, but interspecies data comparisons are scarce. Our study directly compares the non-pathological decision-making performance between humans and wild-type C57BL/6 mice. Human participants completed an electronic Iowa Gambling Task version, while mice a maze-based adaptation with four arms baited in a probabilistic way. Our data shows closely matching performance between both species with similar patterns of choice behaviours. However, mice showed a faster learning rate than humans. Moreover, both populations were clustered into good, intermediate and poor decision-making categories with similar proportions. Remarkably, mice characterised as good decision-makers behaved the same as humans of the same category, but slight differences among species are evident for the other two subpopulations. Overall, our direct comparative study confirms the good face validity of the rodent gambling task. Extended behavioural characterisation and pathological animal models should help strengthen its construct validity and disentangle the determinants in animals and humans decision-making.

Morphofunctional Organization of the Connections From the Medial and Intermediate Parts of the Central Nucleus of the Amygdala Into Distinct Divisions of the Lateral Hypothalamic Area in the Rat.

Projections from the central nucleus of the amygdala (CEA) into the lateral hypothalamic area (LHA) show a very complex pattern. After injection of an anterograde tracer (Phaseolus vulgaris leucoagglutinin-PHAL) into the medial and intermediate parts of the CEA, we observed that labeled axons converged onto the caudal lateral LHA but provided distinct patterns in rostral tuberal regions. These projections were compared to that of neurons containing the peptides "melanin-concentrating hormone" (MCH) or hypocretin (Hcrt). Because the distribution of these neurons is stereotyped, it was possible to characterize distinct divisions into the LHA. Some of them in the rostral tuberal LHA [the dorsal (LHAd) and suprafornical regions (LHAs)] received a distinct innervation by projections that originated from neurons in respectively anterior or posterior regions of the medial part (CEAm) or from the intermediate part (CEAi) of the central nucleus. Therefore, this work illustrates that projections from the CEAm and CEAi converge into the caudal lateral LHA but diverge into the rostral tuberal LHA.

Characterization of McDonald's intermediate part of the Central nucleus of the amygdala in the rat.

The actual organization of the central nucleus of the amygdala (CEA) in the rat is mostly based on cytoarchitecture and the distribution of several cell types, as described by McDonald in 1982. Four divisions were identified by this author. However, since this original work, one of these divisions, the intermediate part, has not been consistently recognized based on Nissl-stained material. In the present study, we observed that a compact condensation of retrogradely labeled cells is found in the CEA after fluorogold injection in the anterior region of the tuberal lateral hypothalamic area (LHA) in the rat. We then searched for neurochemical markers of this cell condensation and found that it is quite specifically labeled for calbindin (Cb), but also contains calretinin (Cr), tyrosine hydroxylase (TH) and methionine-enkephalin (Met-Enk) immunohistochemical signals. These neurochemical features are specific to this cell group which, therefore, is distinct from the other parts of the CEA. We then performed cholera toxin injections in the mouse LHA to identify this cell group in this species. We found that neurons exist in the medial and rostral CEAl that project into the LHA but they have a less tight organization than in the rat.

Parasubthalamic and calbindin nuclei in the posterior lateral hypothalamus are the major hypothalamic targets for projections from the central and anterior basomedial nuclei of the amygdala.

The parasubthalamic nucleus (PSTN) and the ventrally adjacent calbindin nucleus (CbN) form a nuclear complex in the posterior lateral hypothalamic area (LHA), recently characterized as connected with the central nucleus of the amygdala (CEA). The aim of the present work is to analyze in detail the projections from the amygdala into the PSTN/CbN, also focusing on pathways into the LHA. After fluorogold injections into the PSTN/CbN, the medial part of the CEA (CEAm) appears to be the main supplier of projections from the CEA. Other amygdalar nuclei contribute to the innervation of the PSTN/CbN complex, including the anterior part of the basomedial nucleus (BMAa). Injections of the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHAL), into the CEAm and BMAa revealed that projections from the CEAm follow two pathways into the LHA: a dorsal pathway formed by axons that also innervate the paraventricular hypothalamic nucleus, the anterior perifornical LHA and the PSTN, and a ventral pathway that runs laterally adjacent to the ventrolateral hypothalamic tract (vlt) and ends in the CbN. By contrast, the BMAa and other telencephalic structures, such as the fundus striatum project to the CbN via the ventral pathway. Confirming the microscopic observation, a semi-quantitative analysis of the density of these projections showed that the PSTN and the CbN are the major hypothalamic targets for the projections from the CEAm and the BMAa, respectively. PSTN and CbN receive these projections through distinct dorsal and ventral routes in the LHA. The ventral pathway forms a differentiated tract, named here the ventrolateral amygdalo-hypothalamic tract (vlah), that is distinct from, but runs adjacent to, the vlt. Both the vlt and the vlah had been previously described as forming an olfactory path into the LHA. These results help to better characterize the CbN within the PSTN/CbN complex and are discussed in terms of the functional organization of the network involving the PSTN and the CbN as well as the CEA and the BMAa.

The MCH neuron population as a model for the development and evolution of the lateral and dorsal hypothalamus.

The LHA contains neurons producing melanin-concentrating hormone (MCH) or hypocretin (Hcrt) that have emerged as being more conspicuous and representative of the posterior LHA. In this review, we focus on MCH neurons and show that they have unique qualities. Their distribution is conserved in the posterior hypothalamus of all vertebrates and their ontogenetic differentiation is very precocious in the rodent embryo. In mammals, interspecific differences in their medio-lateral distribution suggest that the LHA differentiation may follow species specific strategies. These characteristics make a very valuable tool of MCH neurons to study the development as well as the phylogenetical origin and differentiation of the LHA.

A premammillary lateral hypothalamic nuclear complex responds to hedonic but not aversive tastes in the male rat.

The lateral hypothalamic area (LHA) has two major roles: arousal/waking and food intake controls. Here, it is shown that a premammillary part of the LHA is neurochemically and cytoarchitectonically distinct from the tuberal LHA in male rats. This part contains nuclear masses, namely the parasubthalamic nucleus and the calbindin nucleus, involved in pathways that predict its participation in the control of food intake. Analyzing c-Fos expression in experiments related to feeding behavior, this region responded specifically to the ingestion of palatable nutriments.

Anatomical organization of MCH connections with the pallidum and dorsal striatum in the rat.

Neurons producing the melanin-concentrating hormone (MCH) are distributed in the posterior hypothalamus, but project massively throughout the forebrain. Many aspects regarding the anatomical organization of these projections are still obscure. The present study has two goals: first to characterize the topographical organization of neurons projecting into the cholinergic basal forebrain (globus pallidus, medial septal complex), and second to verify if MCH neurons may indirectly influence the dorsal striatum (caudoputamen) by innervating afferent sources to this structure. In the first series of experiments, the retrograde tracer fluorogold was injected into multiple sites in the pallidal and medial septal regions and the distribution of retrogradely labeled neurons were analyzed in the posterior lateral hypothalamus. In the second series of experiments, fluorogold was injected into the caudoputamen, and the innervation by MCH axons of retrogradely labeled cells was analyzed. Our results revealed that the MCH system is able to interact with the basal nuclei in several different ways. First, MCH neurons provide topographic inputs to the globus pallidus, medial septal complex, and substantia innominata. Second, striatal projecting neurons in the cortex, thalamus, and substantia nigra presumably receive only sparse inputs from MCH neurons. Third, the subthalamic nucleus is heavily innervated by MCH projections, thus, presumably serves as one important intermediate station to mediate MCH influence on other parts of the basal nuclei.

Melanin-concentrating hormone expression in the rat hypothalamus is not affected in an experiment of prenatal alcohol exposure.

Alcohol consumption during pregnancy can cause a "fetal alcoholic syndrome" (FAS) in the progeny. This syndrome is characterized by important brain defects often associated to a decreased expression of the morphogenic protein sonic hedgehog (Shh). The goal of this study was to verify if a FAS could modify the differentiation of hypothalamic neurons producing MCH. Indeed, the expression of this peptide and neurons producing it are dependent of a Shh controlled genetic cascade in the embryo. To address this question, female rats received a 15% ethanol solution to drink during pregnancy and lactation. Higher abortion rate and smaller pups at birth confirmed that descendants were affected by this experimental condition. MCH expression was analyzed by RT-qPCR and immunohistochemistry in embryos taken at E11 and E13, or in pups and young adults born from control and alcoholic mothers. MCH expression level, number of MCH neurons or ratio of MCH sub-populations were not modified by our experimental conditions. However, Shh expression was significantly lover at E11 and we also observed that hindbrain serotonergic neurons were affected as reported in the literature. These findings as well as other data from the literature suggest that protective mechanisms are involved to maintain peptide expressions and differentiation of some specific neuron populations in the ventral diencephalon in surviving embryos exposed to ethanol during pregnancy.

Characterization of a mammalian prosencephalic functional plan.

Hypothalamic organizational concepts have greatly evolved as the primary hypothalamic pathways have been systematically investigated. In the present review, we describe how the hypothalamus arises from a molecularly heterogeneous region of the embryonic neural tube but is first differentiated as a primary neuronal cell cord (earliest mantle layer). This structure defines two axes that align onto two fundamental components: a longitudinal tractus postopticus(tpoc)/retinian component and a transverse supraoptic tract(sot)/olfactory component. We then discuss how these two axonal tracts guide the formation of all major tracts that connect the telencephalon with the hypothalamus/ventral midbrain, highlighting the existence of an early basic plan in the functional organization of the prosencephalic connectome.

Development of posterior hypothalamic neurons enlightens a switch in the prosencephalic basic plan.

In rats and mice, ascending and descending axons from neurons producing melanin-concentrating hormone (MCH) reach the cerebral cortex and spinal cord. However, these ascending and descending projections originate from distinct sub-populations expressing or not "Cocaine-and-Amphetamine-Regulated-Transcript" (CART) peptide. Using a BrdU approach, MCH cell bodies are among the very first generated in the hypothalamus, within a longitudinal cell cord made of earliest delaminating neuroblasts in the diencephalon and extending from the chiasmatic region to the ventral midbrain. This region also specifically expresses the regulatory genes Sonic hedgehog (Shh) and Nkx2.2. First MCH axons run through the tractus postopticus (tpoc) which gathers pioneer axons from the cell cord and courses parallel to the Shh/Nkx2.2 expression domain. Subsequently generated MCH neurons and ascending MCH axons differentiate while neurogenesis and mantle layer differentiation are generalized in the prosencephalon, including telencephalon. Ascending MCH axons follow dopaminergic axons of the mesotelencephalic tract, both being an initial component of the medial forebrain bundle (mfb). Netrin1 and Slit2 proteins that are involved in the establishment of the tpoc and mfb, respectively attract or repulse MCH axons.We conclude that first generated MCH neurons develop in a diencephalic segment of a longitudinal Shh/Nkx2.2 domain. This region can be seen as a prosencephalic segment of a medial neurogenic column extending from the chiasmatic region through the ventral neural tube. However, as the telencephalon expends, it exerts a trophic action and the mfb expands, inducing a switch in the longitudinal axial organization of the prosencephalon.

In vitro interactions between bone marrow stromal cells and hippocampal slice cultures.

Bone marrow stromal cells (BMSCs) are capable of differentiating into various cell types including brain cells. Several groups have also demonstrated trophic effects of MSC grafts in experimental ischemia models. However, the underlying molecular mechanisms of these effects are not fully understood. We developed an "in vitro graft model" which consisted in a coculture of GFP-expressing BMSCs and hippocampal organotypic slice cultures. Total marrow cells (MCs) or BMSCs after one (BMSC(1P)) or five passages (BMSC(5P)) were transplanted on hippocampal slices. During the 10 days of our experiments, MCs and BMSC(1P) migrated toward the tissue, but their total number remained constant. Conversely, the number of BMSC(5P) decreased over the 10 days of the experiment, and no migration could be detected. Using immunohistochemistry, we observed that the hippocampal slices induced the expression of neural antigens in very few grafted cells, but MCs and BMSC(1P) improved the conservation of the hippocampal slice culture. Similar experiments using BMSC(5P) did not produce any significant change. We conclude that the number of passages greatly influence BMSCs survival rate, migration and neuroprotective capacities.

Identification and expression of a new splicing variant of FAD-sulfhydryl oxidase in adult rat brain.

Flavoproteins of the quiescin/sulfhydryl oxidase (QSOX) family catalyze oxidation of peptide and protein thiols to disulfides with the reduction of oxygen to hydrogen peroxide. We report here the molecular cloning of a new putative sulfhydryl oxidase cDNA, rQSOX-L (GenBank Accession no ), from adult rat brain and its expression studied by RT-PCR, Northern and Western blots in rat tissues. DNA-sequencing demonstrated the existence of two cDNAs in rat cortex, corresponding to a long transcript (rQSOX-L) and a short transcript (rQSOX-S) which differed by 851 nucleotides due to alternative splicing. The new transcript, rQSOX-L (3356 nucleotides), was specifically expressed in brain, hypophysis, heart, testis and seminal vesicle. The distribution of this variant is not homogeneous in the different tissues studied and suggests a complex gene regulation. The full-length rQSOX-L cDNA has an open reading frame of 2250-bp encoding a protein of 750 amino acids that contains a signal peptide sequence, a protein-disulfide-isomerase-type thioredoxin and ERV1-ALR domains and a long form specific C-terminal extension. The rQSOX-L protein is highly homologous to members of the sulfhydryl oxidase/Quiescin family and contains particularly two potential sites for N-glycosylation. This protein isoform was specifically detected in rat brain tissues in opposition to the low molecular form that was ubiquitous. Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis of the immunoprecipitate tryptic fragments allowed the identification of rQSOX-L protein.

Cell-specific localization of the sulphydryl oxidase QSOX in rat peripheral tissues.

Rat quiescin/sulphydryl oxidase (rQSOX) introduces disulphide bridges into peptides and proteins with the reduction of molecular oxygen to hydrogen peroxide. Its occurrence has been previously highlighted in a wide range of organs by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analyses, methods that have provided information concerning its expression in whole organs but that do not reveal the cell types expressing this enzyme. In this report, in addition to RT-PCR and Western blot experiments, the cell-specific localization of rQSOX has been investigated in a wide range of male and female adult rat tissues by using in situ hybridization and immunohistochemistry. Labelling was detected in most organs and systems including the immune, endocrine and reproductive systems, the respiratory, digestive and urinary tracts and the skin. No labelling was observed in the heart, blood vessel endothelium, liver or smooth and skeletal muscles. rQSOX expression was mainly localized in epithelial cells specialized in secretion, strengthening the hypothesis that QSOX enzymes play an important role in the mechanism of secretion, notably in the folding of secreted proteins. The intracellular patterns of immunolabelling indicate that the protein usually follows the secretory pathway, which is in accordance with its secreted nature and its presumed involvement in the elaboration of the extracellular matrix. In seminiferous tubules, where a high level of expression was noticed, QSOX might play an important physiological role in sperm function and serve as a marker for the diagnosis of male infertility.

Expression of the sulfhydryl oxidase ALR (Augmenter of Liver Regeneration) in adult rat brain.

Mammalian Augmenter of Liver Regeneration protein (ALR) was first identified as a secondary growth factor involved in liver regeneration. Its sulfhydryl oxidase activity and involvement in iron homeostasis have been recently demonstrated. ALR is expressed in a broad range of peripheral organs, and initial experiments gave also evidence for the occurrence of this protein in brain. In the present study, we investigated in detail the expression of ALR in rat brain sections and determined its cellular and subcellular localizations using biomolecular and immunohistochemical procedures. As shown by Northern blot, ALR is differentially expressed throughout the rat brain, with the highest mRNA levels in the cerebellum and diencephalon. High protein levels were also detected in the brain and cerebellum by Western blot. ALR immunoreactivity was found in neurons and glial cells throughout brain rostrocaudal extent. Labeled astrocytes were particularly abundant in the white matter, and immunoreactive neurons were observed in several regions including the olfactory bulb, isocortex, hippocampal formation, amygdala, thalamus, hypothalamus, some nuclei of the brainstem and cerebellum. In neurons, immunoelectron microscopy showed the protein in the nucleus and mainly in mitochondria. These subcellular localizations may correlate with the occurrence of two ALR protein isoforms in the brain. In the central nervous system, the enzyme might be of importance in heavy metal homeostasis whose dysregulation can induce neurodegenerative disorders.

Ontogenesis of the sulfhydryl oxidase QSOX expression in rat brain.

The spatiotemporal pattern of distribution of the sulfhydryl oxidase QSOX throughout ontogeny was mapped in rat brain using immunohistochemistry. The enzyme was detected on embryonic day (E) 12 in the dawning mantle layer, but the adult-like pattern was acquired postnatally around day 30 (P30). Throughout ontogenesis, rQSOX was detected in immature and mature neurons, but not in glial cells. The rQSOX developmental pattern can be divided into four periods: on E12 the enzyme was detected in the brainstem, more precisely in motoneurons; later (E16), rQSOX-positive cells were also observed in the forebrain, in the caudoputamen, and the subventricular zone. During late embryogenesis (E18-20), the amount of rQSOX cells considerably increased throughout the brain; they initially appeared in the hippocampus, then in the isocortex. From birth onwards, complex modifications of the rQSOX distribution occurred leading to the adult pattern by P30. Although rQSOX exhibits an overall increasing spatiotemporal pattern of distribution, different expression strategies were distinguished depending on the cell type or brain area. By comparing the rQSOX ontogeny with data on neurogenesis and brain histogenesis, we hypothesize that the enzyme could play a role in guiding migrating cells, their settling, and neuronal maturation, e.g., during outgrowth and synaptogenesis.