HGprt deficiency disrupts dopaminergic circuit development in a genetic mouse model of Lesch-Nyhan disease.

In Lesch-Nyhan disease (LND), deficiency of the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase (HGprt) leads to a characteristic neurobehavioral phenotype dominated by dystonia, cognitive deficits and incapacitating self-injurious behavior. It has been known for decades that LND is associated with dysfunction of midbrain dopamine neurons, without overt structural brain abnormalities. Emerging post mortem and in vitro evidence supports the hypothesis that the dopaminergic dysfunction in LND is of developmental origin, but specific pathogenic mechanisms have not been revealed. In the current study, HGprt deficiency causes specific neurodevelopmental abnormalities in mice during embryogenesis, particularly affecting proliferation and migration of developing midbrain dopamine (mDA) neurons. In mutant embryos at E14.5, proliferation was increased, accompanied by a decrease in cell cycle exit and the distribution and orientation of dividing cells suggested a premature deviation from their migratory route. An abnormally structured radial glia-like scaffold supporting this mDA neuronal migration might lie at the basis of these abnormalities. Consequently, these abnormalities were associated with an increase in area occupied by TH+ cells and an abnormal mDA subpopulation organization at E18.5. Finally, dopaminergic innervation was disorganized in prefrontal and decreased in HGprt deficient primary motor and somatosensory cortices. These data provide direct in vivo evidence for a neurodevelopmental nature of the brain disorder in LND. Future studies should not only focus the specific molecular mechanisms underlying the reported neurodevelopmental abnormalities, but also on optimal timing of therapeutic interventions to rescue the DA neuron defects, which may also be relevant for other neurodevelopmental disorders.

Perinatal exposure of rats to the HIV drug efavirenz affects medial prefrontal cortex cytoarchitecture.

Efavirenz (EFV) is used for antiretroviral treatment of HIV infection, and successfully inhibits viral replication and mother-to-child transmission of HIV during pregnancy and childbirth. Unfortunately, the drug induces neuropsychiatric symptoms such as anxiety and depressed mood and potentially affects cognitive performance. EFV acts on, among others, the serotonin transporter and serotonin receptors that are expressed in the developing brain. Yet, how perinatal EFV exposure affects brain cytoarchitecture remains unclear. Here, we exposed pregnant and lactating rats to EFV, and examined in the medial prefrontal cortex (mPFC) of their adult offspring the effects of the maternal EFV exposure on cortical architecture. We observed a significant decrease in the number of cells, mainly mature neurons, in the infra/prelimbic and cingulate cortices of adult offspring. Next, we found an altered cortical cytoarchitecture characterized by a significant reduction in deep- and superficial-layer cells. This was accompanied by a sharp increase in programmed cell death, as we identified a significantly higher number of cleaved Caspase-3-positive cells. Finally, the serotonergic and dopaminergic innervation of the mPFC subdomains was increased. Thus, the perinatal exposure to EFV provoked in the mPFC of adult offspring cell death, significant changes in cytoarchitecture, and disturbances in serotonergic and dopaminergic innervation. Our results are important in the light of EFV treatment of HIV-positive pregnant women, and its effect on brain development and cognitive behavior.

Cadherin-13, a risk gene for ADHD and comorbid disorders, impacts GABAergic function in hippocampus and cognition.

Cadherin-13 (CDH13), a unique glycosylphosphatidylinositol-anchored member of the cadherin family of cell adhesion molecules, has been identified as a risk gene for attention-deficit/hyperactivity disorder (ADHD) and various comorbid neurodevelopmental and psychiatric conditions, including depression, substance abuse, autism spectrum disorder and violent behavior, while the mechanism whereby CDH13 dysfunction influences pathogenesis of neuropsychiatric disorders remains elusive. Here we explored the potential role of CDH13 in the inhibitory modulation of brain activity by investigating synaptic function of GABAergic interneurons. Cellular and subcellular distribution of CDH13 was analyzed in the murine hippocampus and a mouse model with a targeted inactivation of Cdh13 was generated to evaluate how CDH13 modulates synaptic activity of hippocampal interneurons and behavioral domains related to psychopathologic (endo)phenotypes. We show that CDH13 expression in the cornu ammonis (CA) region of the hippocampus is confined to distinct classes of interneurons. Specifically, CDH13 is expressed by numerous parvalbumin and somatostatin-expressing interneurons located in the stratum oriens, where it localizes to both the soma and the presynaptic compartment. Cdh13(-/-) mice show an increase in basal inhibitory, but not excitatory, synaptic transmission in CA1 pyramidal neurons. Associated with these alterations in hippocampal function, Cdh13(-/-) mice display deficits in learning and memory. Taken together, our results indicate that CDH13 is a negative regulator of inhibitory synapses in the hippocampus, and provide insights into how CDH13 dysfunction may contribute to the excitatory/inhibitory imbalance observed in neurodevelopmental disorders, such as ADHD and autism.

Molecular underpinnings of prefrontal cortex development in rodents provide insights into the etiology of neurodevelopmental disorders.

The prefrontal cortex (PFC), seat of the highest-order cognitive functions, constitutes a conglomerate of highly specialized brain areas and has been implicated to have a role in the onset and installation of various neurodevelopmental disorders. The development of a properly functioning PFC is directed by transcription factors, guidance cues and other regulatory molecules and requires the intricate and temporal orchestration of a number of developmental processes. Disturbance or failure of any of these processes causing neurodevelopmental abnormalities within the PFC may contribute to several of the cognitive deficits seen in patients with neurodevelopmental disorders. In this review, we elaborate on the specific processes underlying prefrontal development, such as induction and patterning of the prefrontal area, proliferation, migration and axonal guidance of medial prefrontal progenitors, and their eventual efferent and afferent connections. We furthermore integrate for the first time the available knowledge from genome-wide studies that have revealed genes linked to neurodevelopmental disorders with experimental molecular evidence in rodents. The integrated data suggest that the pathogenic variants in the neurodevelopmental disorder-associated genes induce prefrontal cytoarchitectonical impairments. This enhances our understanding of the molecular mechanisms of prefrontal (mis)development underlying the four major neurodevelopmental disorders in humans, that is, intellectual disability, autism spectrum disorders, attention deficit hyperactivity disorder and schizophrenia, and may thus provide clues for the development of novel therapies.

Differential distribution and regulation of expression of synaptosomal-associated protein of 25 kDa isoforms in the Xenopus pituitary gland and brain.

Synaptosomal-associated protein of 25 kDa (SNAP-25) regulates various membrane fusion processes including exocytosis by endocrine and neural cells. To increase our understanding of the occurrence and regulation of SNAP-25 isoforms, we identified and characterized SNAP-25a and SNAP-25b mRNAs in the pituitary gland and brain of the amphibian Xenopus laevis. The proteins are strongly conserved and are resistant to botulinum neurotoxin A but not to botulinum neurotoxin E, as shown by Western blotting. The spatial distribution of the two SNAP-25 isoforms was assessed with in situ hybridization. Both SNAP-25a mRNA and SNAP-25b mRNA reside in cells in the pituitary distal lobe and, particularly, in the endocrine melanotrope cells in the pituitary intermediate lobe. The melanotrope cells are involved in the background adaptation process of the skin by releasing alpha-melanophore-stimulating hormone. Quantitation of the respective in situ hybridization signals in the Xenopus pars intermedia indicated a differential response, SNAP-25b mRNA being more highly expressed in black-adapted animals than SNAP-25a mRNA, and more than in white-adapted toads. This differential upregulation was also studied by real-time reverse transcriptase polymerase chain reaction, showing that in the intermediate pituitary lobe, both isoforms are physiologically controlled by the background light intensity stimulus, but with different intensities; in black-adapted animals SNAP-25b mRNA is upregulated by 3.33 times compared with white-adapted animals, but SNAP-25a only by 1.96 times. As to neural tissue, in situ hybridization showed that both isoforms coexist throughout the brain, sometimes with similar strengths, but in various areas either SNAP-25a mRNA or SNAP-25b mRNA expression is prevalent. It is speculated that each of the SNAP-25 isoforms in the Xenopus pituitary and brain has a distinct function in cellular fusion processes including secretion, and that their occurrence and regulation depend on the type of secreted neurotransmitter/hormone and/or the activity state of the cell.

Multiple control and dynamic response of the Xenopus melanotrope cell.

Some amphibian brain-melanotrope cell systems are used to study how neuronal and (neuro)endocrine mechanisms convert environmental signals into physiological responses. Pituitary melanotropes release alpha-melanophore-stimulating hormone (alpha-MSH), which controls skin color in response to background light stimuli. Xenopus laevis suprachiasmatic neurons receive optic input and inhibit melanotrope activity by releasing neuropeptide Y (NPY), dopamine (DA) and gamma-aminobutyric acid (GABA) when animals are placed on a light background. Under this condition, they strengthen their synaptic contacts with the melanotropes and enhance their secretory machinery by upregulating exocytosis-related proteins (e.g. SNAP-25). The inhibitory transmitters converge on the adenylyl cyclase system, regulating Ca(2+) channel activity. Other messengers like thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH, from the magnocellular nucleus), noradrenalin (from the locus coeruleus), serotonin (from the raphe nucleus) and acetylcholine (from the melanotropes themselves) stimulate melanotrope activity. Ca(2+) enters the cell and the resulting Ca(2+) oscillations trigger alpha-MSH secretion. These intracellular Ca(2+) dynamics can be described by a mathematical model. The oscillations travel as a wave through the cytoplasm and enter the nucleus where they may induce the expression of genes involved in biosynthesis and processing (7B2, PC2) of pro-opiomelanocortin (POMC) and release (SNAP-25, munc18) of its end-products. We propose that various environmental factors (e.g. light and temperature) act via distinct brain centers in order to release various neuronal messengers that act on the melanotrope to control distinct subcellular events (e.g. hormone biosynthesis, processing and release) by specifically shaping the pattern of melanotrope Ca(2+) oscillations.

Dynamics and plasticity of peptidergic control centres in the retino-brain-pituitary system of Xenopus laevis.

This review deals particularly with the recent literature on the structural and functional aspects of the retino-brain-pituitary system that controls the physiological process of background adaptation in the aquatic toad Xenopus laevis. Taking together the large amount of multidisciplinary data, a consistent picture emerges of a highly plastic system that efficiently responds to changes in the environmental light condition by releasing POMC-derived peptides, such as the peptide alpha-melanophore-stimulating hormone (alpha-MSH), into the circulation. This plasticity is exhibited by both the central nervous system and the pituitary pars intermedia, at the level of molecules, subcellular structures, synapses, and cells. Signal transduction in the pars intermedia of the pituitary gland of Xenopus laevis appears to be a complex event, involving various environmental factors (e.g., light and temperature) that act via distinct brain centres and neuronal messengers converging on the melanotrope cells. In the melanotropes, these messages are translated by specific receptors and second messenger systems, in particular via Ca(2+) oscillations, controlling main secretory events such as gene transcription, POMC-precursor translation and processing, posttranslational peptide modifications, and release of a bouquet of POMC-derived peptides. In conclusion, the Xenopus hypothalamo-hypophyseal system involved in background adaptation reveals how neuronal plasticity at the molecular, cellular and organismal levels, enable an organism to respond adequately to the continuously changing environmental factors demanding physiological adaptation.

Physiological control of Xunc18 expression in neuroendocrine melanotrope cells of Xenopus laevis.

In mammals, the brain-specific protein munc18-1 regulates synaptic vesicle exocytosis at the synaptic junction, in a step before vesicle fusion. We hypothesize that the rate of biosynthesis of munc18-1 messenger RNA (mRNA) and the amount of munc18-1 present in neurons and neuroendocrine cells are related to the physiologically controlled state of activity. To test this hypothesis, the homolog of munc18-1 in the clawed toad Xenopus laevis, xunc18, was studied in the brain and in the neuroendocrine melanotrope cells in the intermediate lobe of the pituitary gland, at both the mRNA and the protein level. In toads adapted to a black background, the melanotropes release the peptide alpha-melanophore-stimulating hormone (alpha-MSH), which induces darkening of the skin, whereas in animals adapted to a white background the cells hardly release but store alpha-MSH, making the animal's skin look pale. The intermediate pituitary lobe of black-adapted animals revealed a strong hybridization reaction with the xunc18 mRNA probe, whereas a much weaker hybridization was observed in the intermediate lobe of white-adapted animals (optical density black: 3.4 +/- 0.2 vs. white: 0.8 +/- 0.1; P < 0.02). Immunocytochemically, Xenopus munc18-like protein has been detected throughout the brain, in identified neuronal perikarya as well as in axon tracts. Western blot analysis and immunocytochemistry further demonstrated the presence of xunc18 in the neural, intermediate and distal lobe of the pituitary gland. Xunc18 protein was furthermore determined in immunoblots of homogenates of melanotropes dissociated from the pituitary gland. In melanotropes of toads adapted to a black background, the integrated optical density of the xunc18 immunosignal was 2.7 +/- 0.5 times higher than in cells of white-adapted toads (P < 0.0001). It is concluded that, in Xenopus melanotrope cells, the amounts of both xunc18 mRNA and xunc18 protein are up-regulated in conjunction with the induction of exocytosis of alpha-MSH as a result of a physiological stimulation (environmental light condition). We propose that xunc18 is involved in physiologically controlled exocytotic secretion of neuroendocrine messengers.

Localization and physiological regulation of the exocytosis protein SNAP-25 in the brain and pituitary gland of Xenopus laevis.

In mammals, the synaptosomal-associated protein of 25 kDa, SNAP-25, is generally thought to play a role in synaptic exocytosis of neuronal messengers. Using a polyclonal antiserum against rat SNAP-25, we have shown the presence of a SNAP-25-like protein in the brain of the South-African clawed toad Xenopus laevis by Western blotting and immunocytochemistry. Xenopus SNAP-25 is ubiquitously present throughout the brain, where its distribution in various identified neuronal perikarya and axon tracts is described. Western blot analysis and immunocytochemistry also demonstrated the presence of SNAP-25 in the neural, intermediate and distal lobes of the pituitary gland. Intensity line plots of confocal laser scanning microscope images of isolated melanotropes indicated that SNAP-25 is produced and processed in the rough endoplasmatic reticulum and Golgi apparatus, and is associated with the plasma membrane. Immunoelectron microscopy substantiated the idea that SNAP-25 is present in the plasma membrane but also showed a close association of SNAP-25 with the bounding membrane of peptide-containing secretory granules in both the neurohemal axon terminals in the neural lobe and the endocrine melanotropes in the intermediate lobe. Quantitative Western blotting revealed that adapting Xenopus to a dark background has a clear stimulatory effect on the expression of SNAP-25 in the neural lobe and in the melanotrope cells. This background light intensity-dependent stimulation of SNAP-25 expression was confirmed by the demonstration of increased immunofluorescence recorded by confocal laser scanning microscopy of individual melanotropes of black background-adapted toads. On the basis of this study on Xenopus laevis, we conclude that SNAP-25 (i) plays a substantial role in the secretion of a wide variety of neuronal messengers; (ii) functions in the central nervous system but also in neurohormonal and endocrine systems; (iii) acts at the plasma membrane but possibly also at the membrane of synaptic vesicles and peptide-containing secretory granules; (iv) acts not only locally (as in synapses), but at various sites of the plasma membrane (as in the endocrine melanotrope cell); and (v) can be upregulated in its expression by physiological stimuli that increase the extent of the molecular machinery involved in exocytosis.

Gonadotrophs but not somatotrophs carry gonadotrophin-releasing hormone receptors: receptor localisation, intracellular calcium, and gonadotrophin and GH release.

Gonadotrophs are the primary target cells for GnRH in the pituitary. However, during a limited period of neonatal life in the rat, lactotrophs and somatotrophs respond to GnRH as well. Also, in the adults of a number of teleost fishes (e.g. carp, goldfish, and tilapia but not trout), GnRH is a potent GH secretagogue. In studying hypophysiotrophic actions of the two forms of GnRH present in the African catfish (Clarias gariepinus), chicken GnRH-II ([His5,Trp7,Tyr8]GnRH; cGnRH-II) and catfish GnRH ([His5,Asn8]GnRH; cfGnRH), we have investigated the effects of GnRH on catfish gonadotrophs and somatotrophs. GnRH binding was examined by incubating dispersed pituitary cells attached to coverslips with 125I-labelled [D-Arg6,Trp7,Leu8,Pro9-Net]GnRH (sGnRHa), a salmon GnRH analogue with high affinity for the GnRH receptor. Following fixation and immunohistochemistry using antisera against catfish LH and GH, 125I-labelled sGnRHa was localised autoradiographically and silver grains were quantified on gonadotrophs and somatotrophs. Specific binding of 125I-labelled sGnRHa was restricted to gonadotrophs. Both cfGnRH and cGnRH-II dose-dependently inhibited 125I-labelled sGnRHa binding to gonadotrophs. To substantiate the localisation of functional GnRH receptors, the effects of cfGnRH and cGnRH-II on the cytosolic free calcium concentration ([Ca2+]i) were examined in Fura-2-loaded somatotrophs and gonadotrophs. GnRH-induced increases in [Ca2+]i appeared to be confined to gonadotrophs, in which both endogenous GnRHs caused a single and transient increase in [Ca2+]i. The amplitude of this [Ca2+]i transient depended on the GnRH dose and correlated well with the GnRHs' effect on LH release. In vivo experiments demonstrated that GnRH treatments which markedly elevated plasma LH levels had no effect on plasma GH levels, while a dopamine agonist (apomorphine) significantly elevated plasma GH levels. We conclude that the two endogenous forms of GnRH in the African catfish are not directly involved in the regulation of the release of GH, suggesting that GnRHs cannot be considered as GH secretagogues in teleosts in general.