Beneficial effects of prolonged 2-phenylethyl alcohol inhalation on chronic distress-induced anxio-depressive-like phenotype in female mice.

Chronic distress-induced hypothalamic-pituitary-adrenal axis deregulations have been associated with the development of neuropsychiatric disorders such as anxiety and depression. Currently available drugs treating such pathological conditions have limited efficacy and diverse side effects, revealing the need of new safer strategies. Aromatic plant-based compounds are largely used in herbal medicine due to their therapeutic properties on mood, physiology, and general well-being. The purpose of this study was to investigate the effects of 2-phenylethyl alcohol (PEA), one of the pharmacologically active constituents of rose essential oil, on chronic corticosterone (CORT)-induced behavioral and neurobiological changes in female mice. Animals followed a prolonged PEA inhalation exposure (30 min per day) for 15 consecutive days prior to behavioral evaluation with open-field, forced swim and novelty-suppressed feeding tests. CORT treatment induced an anxio-depressive-like phenotype, evidenced by a reduced locomotor activity in the open-field, and an increased latency to feed in the novelty-suppressed feeding paradigms. To elucidate the neural correlates of our behavioral results, immunohistochemistry was further performed to provide a global map of neural activity based on cerebral cFos expression. The altered feeding behavior was accompanied by a significant decrease in the number of cFos-positive cells in the olfactory bulb, and altered functional brain connectivity as shown by cross-correlation-based network analysis. CORT-induced behavioral and neurobiological alterations were reversed by prolonged PEA inhalation, suggesting a therapeutic action that allows regulating the activity of neural circuits involved in sensory, emotional and feeding behaviors. These findings might contribute to better understand the therapeutic potential of PEA on anxio-depressive symptoms.

The distribution of Dlx1-2 and glutamic acid decarboxylase in the embryonic and adult hypothalamus reveals three differentiated LHA subdivisions in rodents.

The lateral hypothalamus (LHA) is still a poorly understood brain region. Based on published Dlx and Gad gene expression patterns in the embryonic and adult hypothalamus respectively, three large areas are identified in the LHA. A central tuberal LHA region is already well described as it contains neurons producing the peptides melanin-concentrating hormone or hypocretin. This region is rich in GABAergic neurons and is specified by Dlx gene expression in the rodent embryo. Rostrally and caudally bordering the tuberal LHA, two Dlx-GAD-GABA poor regions are then easily delineated. The three regions show different organizational schema. The tuberal region is reticularly organized, connected with the cerebral cortex and the spinal cord, and its embryonic development occurs along the tractus postopticus. The region anterior to it is associated with the stria medullaris in both embryonic and adult subjects. The posterior LHA region is made of differentiated nuclei and includes the subthalamic nucleus. Therefore, the LHA is divided into three distinct parts: in addition to the well-known tuberal LHA, caudal and anterior LHA regions exist that have specific anatomical and functional characteristics. The hypothalamus is made up of several dozens of nuclei or areas that are more or less well differentiated and whose boundaries and arrangements are drawn differently according to authors and atlases (Allen Institute, 2004; Paxinos and Franklin, 2019; Paxinos and Watson, 2013; Swanson, 2004). The dominant hypothesis for more than 50 years is that these structures are distributed within three antero-posterior areas (anterior, tuberal, posterior) and more or less three longitudinal zones (lateral, medial and periventricular) (Fig. 1). In addition to these regions, several adjacent territories are often associated to the hypothalamus. The preoptic area is functionally related to the hypothalamus, but it is better seen as a telencephalic structure based on developmental data (Croizier et al., 2015; Puelles and Rubenstein, 2015). Lately, the zona incerta and the subthalamic nucleus (STN) have also been associated to the hypothalamus on the basis of their connections and development for the STN (Altman and Bayer, 1986; Barbier and Risold, 2021; Swaab et al., 2021). However, the zona incerta is still included in the 'pre-thalamus' or "ventral thalamus" in the embryo (Puelles and Rubenstein, 2015). Thus, the boundaries of the hypothalamus remain blurred around what we can call a 'core' made of the anterior to posterior regions (Brooks, 1988). In addition, unlike other large brain regions that are characterized early on by a molecular signature, i.e. by the embryonic expression of specific molecular markers, data illustrating the distribution of dozens of transcription factors involved in brain patterning and cell lineage specification confirmed the extremely heterogeneous and mosaic nature of the anterior and posterior regions of the hypothalamus (Alvarez-Bolado, 2019; Puelles et al., 2013; Puelles and Rubenstein, 2015). The rich nuclear organization of the medial and periventricular zones of the hypothalamus is consistent with the mosaic expression of developmental genes. The LHA, however, is often perceived as much more homogeneous in its cytoarchitectural organization. At the same time, there is little information regarding the expression of developmental genes in the anterior and posterior territories of the LHA. Most studies focus on the tuberal LHA which expresses many of these genes. Admittedly, even in the adult hypothalamus, the internal boundaries of the LHA are difficult to identify and the same is true in the embryo. Developmental data alone are insufficient to achieve a better understanding of the LHA anatomical organization and for this region as for medial and periventricular zones, a coherence must be established between development and adult anatomical organization. Among the most useful neurochemical markers to identify large regions of the forebrain, those involved in the identification of GABAergic and glutamatergic neurons have proven to be particularly efficient. Indeed, GABAergic neurons are not ubiquitously distributed. Large regions of the forebrain are rich in such cells, including the basal telencephalon, but others contain few or no GABAergic cells and are rich in glutamatergic neurons instead (for example the dorsal thalamus that is free of GABA-neurons in rodents). The same applies for the hypothalamus: several structures of the hypothalamus are free of GABAergic neurons, as, for example, the mammillary nuclei (Hahn et al., 2019). Recently, we also identified a GABA-poor posterior LHA territory that includes the (STN), and is localized caudal to the GABA-rich tuberal LHA (Barbier et al., 2020; Barbier and Risold, 2021; Chometton et al., 2016b). Therefore, the LHA seems partitioned into GABA-rich/GABA-poor regions. However, to define or confirm distinct neuroanatomical entities, these regions must have a specific embryological origin, and show specific hodological patterns and functions. Hence, the purpose of this short review is to identify divisions of the LHA based on developmental and neurochemical criteria. Such an analysis seems to us relevant in order to allow later functional studies on regions whose boundaries will be based on objective criteria.

Understanding the Significance of the Hypothalamic Nature of the Subthalamic Nucleus.

The subthalamic nucleus (STN) is an essential component of the basal ganglia and has long been considered to be a part of the ventral thalamus. However, recent neurodevelopmental data indicated that this nucleus is of hypothalamic origin which is now commonly acknowledged. In this work, we aimed to verify whether the inclusion of the STN in the hypothalamus could influence the way we understand and conduct research on the organization of the whole ventral and posterior diencephalon. Developmental and neurochemical data indicate that the STN is part of a larger glutamatergic posterior hypothalamic region that includes the premammillary and mammillary nuclei. The main anatomic characteristic common to this region involves the convergent cortical and pallidal projections that it receives, which is based on the model of the hyperdirect and indirect pathways to the STN. This whole posterior hypothalamic region is then integrated into distinct functional networks that interact with the ventral mesencephalon to adjust behavior depending on external and internal contexts.

Projections from the dorsomedial division of the bed nucleus of the stria terminalis to hypothalamic nuclei in the mouse.

As stressful environment is a potent modulator of feeding, we seek in the present work to decipher the neuroanatomical basis for an interplay between stress and feeding behaviors. For this, we combined anterograde and retrograde tracing with immunohistochemical approaches to investigate the patterns of projections between the dorsomedial division of the bed nucleus of the stria terminalis (BNST), well connected to the amygdala, and hypothalamic structures such as the paraventricular (PVH) and dorsomedial (DMH), the arcuate (ARH) nuclei and the lateral hypothalamic areas (LHA) known to control feeding and motivated behaviors. We particularly focused our study on afferences to proopiomelanocortin (POMC), agouti-related peptide (AgRP), melanin-concentrating-hormone (MCH) and orexin (ORX) neurons characteristics of the ARH and the LHA, respectively. We found light to intense innervation of all these hypothalamic nuclei. We particularly showed an innervation of POMC, AgRP, MCH and ORX neurons by the dorsomedial and dorsolateral divisions of the BNST. Therefore, these results lay the foundation for a better understanding of the neuroanatomical basis of the stress-related feeding behaviors.

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.

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.

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.

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.

A comparative analysis shows morphofunctional differences between the rat and mouse melanin-concentrating hormone systems.

Sub-populations of neurons producing melanin-concentrating hormone (MCH) are characterized by distinct projection patterns, birthdates and CART/NK3 expression in rat. Evidence for such sub-populations has not been reported in other species. However, given that genetically engineered mouse lines are now commonly used as experimental models, a better characterization of the anatomy and morphofunctionnal organization of MCH system in this species is then necessary. Combining multiple immunohistochemistry experiments with in situ hybridization, tract tracing or BrdU injections, evidence supporting the hypothesis that rat and mouse MCH systems are not identical was obtained: sub-populations of MCH neurons also exist in mouse, but their relative abundance is different. Furthermore, divergences in the distribution of MCH axons were observed, in particular in the ventromedial hypothalamus. These differences suggest that rat and mouse MCH neurons are differentially involved in anatomical networks that control feeding and the sleep/wake cycle.

The fetal hypothalamus has the potential to generate cells with a gonadotropin releasing hormone (GnRH) phenotype.

BACKGROUND: Neurospheres (NS) are colonies of neural stem and precursor cells capable of differentiating into the central nervous system (CNS) cell lineages upon appropriate culture conditions: neurons, and glial cells. NS were originally derived from the embryonic and adult mouse striatum subventricular zone. More recently, experimental evidence substantiated the isolation of NS from almost any region of the CNS, including the hypothalamus. METHODOLOGY/FINDINGS: Here we report a protocol that enables to generate large quantities of NS from both fetal and adult rat hypothalami. We found that either FGF-2 or EGF were capable of inducing NS formation from fetal hypothalamic cultures, but that only FGF-2 is effective in the adult cultures. The hypothalamic-derived NS are capable of differentiating into neurons and glial cells and most notably, as demonstrated by immunocytochemical detection with a specific anti-GnRH antibody, the fetal cultures contain cells that exhibit a GnRH phenotype upon differentiation. CONCLUSIONS/SIGNIFICANCE: This in vitro model should be useful to study the molecular mechanisms involved in GnRH neuronal differentiation.

The circumventricular organs: an atlas of comparative anatomy and vascularization.

The circumventricular organs are small sized structures lining the cavity of the third ventricle (neurohypophysis, vascular organ of the lamina terminalis, subfornical organ, pineal gland and subcommissural organ) and of the fourth ventricle (area postrema). Their particular location in relation to the ventricular cavities is to be noted: the subfornical organ, the subcommissural organ and the area postrema are situated at the confluence between ventricles while the neurohypophysis, the vascular organ of the lamina terminalis and the pineal gland line ventricular recesses. The main object of this work is to study the specific characteristics of the vascular architecture of these organs: their capillaries have a wall devoid of blood-brain barrier, as opposed to central capillaries. This particular arrangement allows direct exchange between the blood and the nervous tissue of these organs. This work is based on a unique set of histological preparations from 12 species of mammals and 5 species of birds, and is taking the form of an atlas.

Phospholipid transfer protein (PLTP) deficiency reduces brain vitamin E content and increases anxiety in mice.

Vitamin E supplementation constitutes a promising strategy in the prevention of neurodegenerative diseases. Here, we show that a phospholipid transfer protein (PLTP) is widely expressed in the brain where it appears to function as a transfer factor for alpha-tocopherol, the main isomer of vitamin E. PLTP deficiency results in significant depletion of brain alpha-tocopherol in both homozygous (-30.1%, P<0.0002) and heterozygous (-18.0%, P<0.05) PLTP knocked-out mice. Alpha-tocopherol depletion in PLTP-deficient homozygotes is associated with the elevation of lipofuscin (+25% and +450% increases in cortex and substantia nigra, respectively), cholesterol oxides (+54.5%, P<0.05), and cellular peroxides (+32.3%, P<0.01) in the brain. Complete PLTP deficiency in homozygotes is accompanied by increased anxiety as shown by fewer entries (8.3% vs. 44.4% in controls, P<0.01) and less time spent (1.7% vs. 41.3% in controls, P<0.05) in the open arms of an elevated plus-maze, in the absence of locomotor deterioration. Thus, the vitamin E transfer activity of PLTP appears to be a key process in preventing oxidative damage in the brain, and PLTP-deficient mice could be a new model of the contribution of oxidative brain injury in the etiology of neurodegenerative diseases.

Specific alteration of the expression of selected hypothalamic neuropeptides during acute and late mouse brain infection using a morbillivirus: relevance to the late-onset obesity?

Neurotropic viruses are involved in pathologies of the central nervous system, triggering transient or irreversible disorders, such as neurological diseases or homeostasis imbalance. In experimental animals, viruses have been shown to cause obesity, a complex disease depending on multiple factors, including genetic susceptibility and environmental components. Using a mouse model of virally induced obesity following brain infection by the Canine Distemper Virus (CDV), a morbillivirus closely related to the human measles virus, we investigated the modulation of expression of several hypothalamic neuropeptides known to intervene in the regulation of body weight and energy expenditure, both during the acute and late stages of infection. During the acute stage, while viral replication occurs, we found a dramatic decrease of expressions of neuropeptides, in particular neuropeptide Y, melanin-concentrating hormone (MCH), hypocretin, vasopressin and tachykinins, the magnitude of which seemed to be linked to the viral burden and the individual susceptibility. The effect of the virus, however, varied with the hypothalamic nucleus and neuropeptide involved, suggesting that certain circuits were affected while others remained intact. During the late stage of infection, marked recovery to the initial hypothalamic levels of peptide expression was seen in a number of lean animals, suggesting recovery of homeostasis equilibrium. Interestingly, some neuropeptidergic systems remained disturbed in mice exhibiting obese phenotype, arguing for their involvement in triggering/maintaining obesity. Even though our data could not fully explain the viral-induced obesity, they may be helpful in understanding the molecular events associated with obesity and in investigating therapeutic alternatives.