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.

Transduction profiles in minipig following MRI guided delivery of AAV-5 into thalamic and corona radiata areas.

BACKGROUND: As a step towards clinical use of AAV-mediated gene therapy, brains of large animals are used to settle delivery parameters as most brain connections, and relative sizes in large animals and primates, are reasonably common. Prior to application in the clinic, approaches that have shown to be successful in rodent models are tested in larger animal species, such as dogs, non-human primates, and in this case, minipigs. NEW METHOD: We evaluated alternate delivery routes to target the basal ganglia by injections into the more superficial corona radiata, and, deeper into the brain, the thalamus. Anatomically known connections can be used to predict the expression of the transgene following infusion of AAV5. For optimal control over delivery of the vector with regards to anatomical location in the brain and spread in the tissue, we have used magnetic resonance image-guided convection-enhanced diffusion delivery. RESULTS: While the transduction of the cortex was observed, only partial transduction of the basal ganglia was achieved via the corona radiata. Thalamic administration, on the other hand, resulted in widespread transduction from the midbrain to the frontal cortex COMPARISON WITH EXISTING METHODS: Compared to other methods, such as delivery directly to the striatum, thalamic injection may provide an alternative when for instance, injection into the basal ganglia directly is not feasible. CONCLUSIONS: The study results suggest that thalamic administration of AAV5 has significant potential for indications where the transduction of specific areas of the brain is required.

Antioxidant treatment ameliorates prefrontal hypomyelination and cognitive deficits in a rat model of schizophrenia.

Cognitive dysfunction in schizophrenia (SZ) is thought to arise from neurodevelopmental abnormalities that include interneuron hypomyelination in the prefrontal cortex (PFC). Here we report that RNA-sequencing of the medial (m)PFC of the APO-SUS rat model with SZ-relevant cognitive inflexibility revealed antioxidant metabolism as the most-enriched differentially expressed pathway. Antioxidant-related gene expression was altered throughout postnatal development and preceded hypomyelination. Furthermore, reduced glutathione levels and increased mitochondria numbers were observed in the mPFC. Strikingly, chronic treatment with the glutathione precursor N-acetylcysteine (NAC) from postnatal days 5-90 restored not only antioxidant-related mRNA expression and mitochondria numbers, but also myelin-related mRNA expression and mPFC-dependent cognitive dysfunction, while blood glutathione levels remained unaffected. The promyelinating effect of NAC was at least partly due to a positive effect on oligodendrocyte lineage progression. Together, our findings highlight that oxidative stress may contribute to cognitive symptoms in the APO-SUS rat model of SZ and encourage antioxidant therapy in early phases of SZ.

Transduction patterns in the CNS following various routes of AAV-5-mediated gene delivery.

Various administration routes of adeno-associated virus (AAV)-based gene therapy have been examined to target the central nervous system to answer the question what the most optimal delivery route is for treatment of the brain with certain indications. In this study, we evaluated AAV5 vector system for its capability to target the central nervous system via intrastriatal, intrathalamic or intracerebroventricular delivery routes in rats. AAV5 is an ideal candidate for gene therapy because of its relatively low level of existing neutralizing antibodies compared to other serotypes, and its broad tissue and cell tropism. Intrastriatal administration of AAV5-GFP resulted in centralized localized vector distribution and expression in the frontal part of the brain. Intrathalamic injection showed transduction and gradient expression from the rostral brain into lumbar spinal cord, while intracerebroventricular administration led to a more evenly, albeit relatively superficially distributed, transduction and expression throughout the central nervous system. To visualize the differences between localized and intra-cerebral spinal fluid administration routes, we compared intrastriatal to intracerebroventricular and intrathecal administration of AAV5-GFP. Together, our results demonstrate that for efficient transgene expression, various administration routes can be applied.

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.

Integrated molecular landscape of amyotrophic lateral sclerosis provides insights into disease etiology.

Amyotrophic lateral sclerosis (ALS) is a severe, progressive and ultimately fatal motor neuron disease caused by a combination of genetic and environmental factors, but its underlying mechanisms are largely unknown. To gain insight into the etiology of ALS, we here conducted genetic network and literature analyses of the top-ranked findings from six genome-wide association studies of sporadic ALS (involving 3589 cases and 8577 controls) as well as genes implicated in ALS etiology through other evidence, including familial ALS candidate gene association studies. We integrated these findings into a molecular landscape of ALS that allowed the identification of three main processes that interact with each other and are crucial to maintain axonal functionality, especially of the long axons of motor neurons, i.e. (1) Rho-GTPase signaling; (2) signaling involving the three regulatory molecules estradiol, folate, and methionine; and (3) ribonucleoprotein granule functioning and axonal transport. Interestingly, estradiol signaling is functionally involved in all three cascades and as such an important mediator of the molecular ALS landscape. Furthermore, epidemiological findings together with an analysis of possible gender effects in our own cohort of sporadic ALS patients indicated that estradiol may be a protective factor, especially for bulbar-onset ALS. Taken together, our molecular landscape of ALS suggests that abnormalities within three interconnected molecular processes involved in the functioning and maintenance of motor neuron axons are important in the etiology of ALS. Moreover, estradiol appears to be an important modulator of the ALS landscape, providing important clues for the development of novel disease-modifying treatments.

Oxidative stress, prefrontal cortex hypomyelination and cognitive symptoms in schizophrenia.

Schizophrenia (SZ) is a neurodevelopmental disorder with a broad symptomatology, including cognitive symptoms that are thought to arise from the prefrontal cortex (PFC). The neurobiological aetiology of these symptoms remains elusive, yet both impaired redox control and PFC dysconnectivity have been recently implicated. PFC dysconnectivity has been linked to white matter, oligodendrocyte (OL) and myelin abnormalities in SZ patients. Myelin is produced by mature OLs, and OL precursor cells (OPCs) are exceptionally susceptible to oxidative stress. Here we propose a hypothesis for the aetiology of cognitive symptomatology in SZ: the redox-induced prefrontal OPC-dysfunctioning hypothesis. We pose that the combination of genetic and environmental factors causes oxidative stress marked by a build-up of reactive oxygen species that, during late adolescence, impair OPC signal transduction processes that are necessary for OPC proliferation and differentiation, and involve AMP-activated protein kinase, Akt-mTOR-P70S6K and peroxisome proliferator receptor alpha signalling. OPC dysfunctioning coincides with the relatively late onset of PFC myelination, causing hypomyelination and disruption of connectivity in this brain area. The resulting cognitive deficits arise in parallel with SZ onset. Hence, our hypothesis provides a novel neurobiological framework for the aetiology of SZ cognitive symptoms. Future research addressing our hypothesis could have important implications for the development of new (combined) antioxidant- and promyelination-based strategies to treat the cognitive symptoms in SZ.

Integrated molecular landscape of Parkinson's disease.

Parkinson's disease is caused by a complex interplay of genetic and environmental factors. Although a number of independent molecular pathways and processes have been associated with familial Parkinson's disease, a common mechanism underlying especially sporadic Parkinson's disease is still largely unknown. In order to gain further insight into the etiology of Parkinson's disease, we here conducted genetic network and literature analyses to integrate the top-ranked findings from thirteen published genome-wide association studies of Parkinson's disease (involving 13.094 cases and 47.148 controls) and other genes implicated in (familial) Parkinson's disease, into a molecular interaction landscape. The molecular Parkinson's disease landscape harbors four main biological processes-oxidative stress response, endosomal-lysosomal functioning, endoplasmic reticulum stress response, and immune response activation-that interact with each other and regulate dopaminergic neuron function and death, the pathological hallmark of Parkinson's disease. Interestingly, lipids and lipoproteins are functionally involved in and influenced by all these processes, and affect dopaminergic neuron-specific signaling cascades. Furthermore, we validate the Parkinson's disease -lipid relationship by genome-wide association studies data-based polygenic risk score analyses that indicate a shared genetic risk between lipid/lipoprotein traits and Parkinson's disease. Taken together, our findings provide novel insights into the molecular pathways underlying the etiology of (sporadic) Parkinson's disease and highlight a key role for lipids and lipoproteins in Parkinson's disease pathogenesis, providing important clues for the development of disease-modifying treatments of Parkinson's disease.

Elevated microRNA-181c and microRNA-30d levels in the enlarged amygdala of the valproic acid rat model of autism.

Autism spectrum disorders are severe neurodevelopmental disorders, marked by impairments in reciprocal social interaction, delays in early language and communication, and the presence of restrictive, repetitive and stereotyped behaviors. Accumulating evidence suggests that dysfunction of the amygdala may be partially responsible for the impairment of social behavior that is a hallmark feature of ASD. Our studies suggest that a valproic acid (VPA) rat model of ASD exhibits an enlargement of the amygdala as compared to controls rats, similar to that observed in adolescent ASD individuals. Since recent research suggests that altered neuronal development and morphology, as seen in ASD, may result from a common post-transcriptional process that is under tight regulation by microRNAs (miRs), we examined genome-wide transcriptomics expression in the amygdala of rats prenatally exposed to VPA, and detected elevated miR-181c and miR-30d expression levels as well as dysregulated expression of their cognate mRNA targets encoding proteins involved in neuronal system development. Furthermore, selective suppression of miR-181c function attenuates neurite outgrowth and branching, and results in reduced synaptic density in primary amygdalar neurons in vitro. Collectively, these results implicate the small non-coding miR-181c in neuronal morphology, and provide a framework of understanding how dysregulation of a neurodevelopmentally relevant miR in the amygdala may contribute to the pathophysiology of ASD.

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.

MicroRNA networks direct neuronal development and plasticity.

MicroRNAs (miRNAs) constitute a class of small, non-coding RNAs that act as post-transcriptional regulators of gene expression. In neurons, the functions of individual miRNAs are just beginning to emerge, and recent studies have elucidated roles for neural miRNAs at various stages of neuronal development and maturation, including neurite outgrowth, dendritogenesis, and spine formation. Notably, miRNAs regulate mRNA translation locally in the axosomal and synaptodendritic compartments, and thereby contribute to the dynamic spatial organization of axonal and dendritic structures and their function. Given the critical role for miRNAs in regulating early brain development and in mediating synaptic plasticity later in life, it is tempting to speculate that the pathology of neurological disorders is affected by altered expression or functioning of miRNAs. Here we provide an overview of recently identified mechanisms of neuronal development and plasticity involving miRNAs, and the consequences of miRNA dysregulation.

Using transgenic animal models in neuroendocrine research: lessons from Xenopus laevis.

Transgenic animals are commonly employed to explore the function of individual proteins. Transgenic animal models include the mouse, the zebrafish, and the South African clawed toad Xenopus laevis. In contrast to mice and zebrafish, with Xenopus transgenesis DNA integration is mostly achieved in the one-cell stage. Moreover, Xenopus (as well as zebrafish) eggs are relatively large, the embryos are transparent, a large offspring is generated, and maintenance of the offspring is easy. In our transgenic studies in Xenopus, we focus on the well-characterized neuroendocrine melanotrope cells of the pituitary pars intermedia that are regulated during the process of adaptation of Xenopus to a changing environment. When the animal is placed on a black background, the melanotrope cells produce and process large amounts of the prohormone proopiomelanocortin (POMC). We apply stable melanotrope-specific transgenesis that is achieved by mixing a Xenopus POMC-promoter/transgene construct with sperm nuclei and injecting this mixture into unfertilized eggs. Since in the melanotrope cells the POMC promoter is much more active in black-adapted animals, the level of transgene expression can be manipulated by placing the animal on either a black or a white background. In this paper we review the possibilities of the Xenopus melanotrope-specific transgenic approach. Following a brief overview of the functioning of Xenopus melanotrope cells, stable melanotrope-specific transgenesis is discussed and our transgenic studies on brain-derived neurotrophic factor and secretory pathway components are described as examples of the transgenic approach in a physiological context and close to the in vivo situation.

Identification of proSAAS homologs in lower vertebrates: conservation of hydrophobic helices and convertase-inhibiting sequences.

The prohormone convertases (PCs) 1/3 and 2 accomplish the major proteolytic cleavage events in neuroendocrine tissues; each of these convertases has a small associated binding protein that inhibits convertase action in the secretory pathway. The proSAAS protein binds to PC1/3, whereas the 7B2 protein binds to PC2. However, both convertase-binding proteins are more widely expressed than their cognate enzymes, suggesting that they may perform other functions as well. All known mammalian proSAASs are over 85% conserved; thus, identifying functionally important segments has been impossible. Here, we report the first identification of nonmammalian proSAAS molecules, from Xenopus and zebrafish (Danio rerio). Although these two proteins show an overall amino acid sequence identity of only 29 and 30% with mouse proSAAS, two 14-16 residue hydrophobic segments (predicted to form alpha-helices) and two, nine through 11 residue sequences containing basic convertase cleavage sites are highly conserved; therefore, these sequences may be of functional importance. Confidence that these nonmammalian molecules represent authentic proSAAS is supported by the finding that both inhibit mouse PC1/3 with nanomolar inhibition constants; human furin was not inhibited. In vitro, the two proteins were cleaved by PC2 and furin to three or more peptide products. Both zebrafish and Xenopus proSAAS exhibited neural and endocrine distributions, as assessed by in situ and PCR experiments, respectively. In summary, the identification of proSAAS molecules in lower vertebrates provides clues as to functional regions within this widely expressed neuroendocrine protein.

Genetic and environmental factors in complex neurodevelopmental disorders.

Complex neurodevelopmental disorders, such as schizophrenia, autism, attention deficit (hyperactivity) disorder, (manic) depressive illness and addiction, are thought to result from an interaction between genetic and environmental factors. Association studies on candidate genes and genome-wide linkage analyses have identified many susceptibility chromosomal regions and genes, but considerable efforts to replicate association have been surprisingly often disappointing. Here, we summarize the current knowledge of the genetic contribution to complex neurodevelopmental disorders, focusing on the findings from association and linkage studies. Furthermore, the contribution of the interaction of the genetic with environmental and epigenetic factors to the aetiology of complex neurodevelopmental disorders as well as suggestions for future research are discussed.

Ontogenic reduction of Aph-1b mRNA and gamma-secretase activity in rats with a complex neurodevelopmental phenotype.

Selectively bred apomorphine susceptible (APO-SUS) rats display a complex behavioral phenotype remarkably similar to that of human neurodevelopmental disorders, such as schizophrenia. We recently found that the APO-SUS rats have only one or two Aph-1b gene copies (I/I and II/II rats, respectively), whereas their phenotypic counterpart has three copies (III/III). Aph-1b is a component of the gamma-secretase enzyme complex that is involved in multiple (neuro)developmental signaling pathways. Nevertheless, surprisingly little is known about gamma-secretase expression during development. Here, we performed a longitudinal quantitative PCR study in embryos and the hippocampus of I/I, II/II and III/III rats, and found gene-dosage dependent differences in Aph-1b, but not Aph-1a, mRNA expression throughout pre- and post-natal development. On the basis of the developmental mRNA profiles, we assigned relative activities to the various Aph-1a and -1b gene promoters. Furthermore, in the three rat lines, we observed both tissue-specific and temporal alterations in gamma-secretase cleavage activity towards one of its best-known substrates, the amyloid-beta precursor protein APP. We conclude that the low levels of Aph-1b mRNA and gamma-secretase activity observed in the I/I and II/II rats during the entire developmental period may well underlie their complex phenotype.

A fast method to study the secretory activity of neuroendocrine cells at the ultrastructural level.

Cryo field emission scanning electron microscopy (cryo-FE-SEM) is a versatile technique that allows the investigation of the three-dimensional organization of cells at the ultrastructural level over a wide range of magnifications. Unfortunately, cryopreparation of the specimens for this technique remains cumbersome, in particular because ice crystal formation must be prevented during freezing. Here we report that a light prefixation with glutaraldehyde and incubation in glycerol as cryoprotectant or a high-pressure freezing approach are both excellent procedures for cryopreparation of animal cells to be used in combination with cryo-FE-SEM. Using the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis as a physiologically inducible neuroendocrine system, we compared the ultrastructural characteristics of inactive and hyperactive neuroendocrine cells. The overall quality of the ultrastructural images was comparable for the two cryopreparation procedures, although some fine structures were better conserved using high-pressure freezing. Melanotrope cells in a secretory inactive state contained numerous storage granules and a poorly developed endoplasmic reticulum (ER), while large amounts of rough ER were present in hyperactive cells. Thus, the cryo-FE-SEM approach described here allows a fast ultrastructural study on the secretory activity of neuroendocrine cells.

Isolation and characterization of the Xenopus laevis orthologs of the human papillary renal cell carcinoma-associated genes PRCC and MAD2L2 (MAD2B).

Recently we found that the human papillary renal cell carcinoma-associated protein PRCC interacts with the cell cycle control protein Mad2B, and translocates this protein to the nucleus where it exerts its mitotic checkpoint function. Here we have successfully isolated Xenopus laevis Mad2B and PRCC cDNAs. The full-length xMad2B cDNA encodes a 211 amino acid protein that is highly homologous to human Mad2B, thus pointing to an important function for this protein in higher eukaryotes. The full-length xPRCC cDNA encodes a 544 amino acid protein. Remarkably, this protein contains an amino-terminal region distinct from that in mouse and human, whereas the C-terminal region is highly conserved. Northern blot and RT-PCR analyses revealed a relatively low expression of both xMad2B and xPRCC in most tissues examined. However, an abundant expression was observed in testis and oocyte, indicating a role in meiotic division processes. Coimmunoprecipitation and immunofluorescence analyses revealed that, despite its distinct amino terminus, the xPRCC-protein is still capable of interacting with xMad2B and of shuttling this protein to the nucleus. Therefore, the well-established animal model Xenopus laevis can be used as a powerful system to study in detail the role of xPRCC and xMad2B in the intricate processes of cell cycle control.

Phlorizin treatment prevents the decrease in plasma insulin levels but not the progressive histopathological changes in the pancreatic islets during aging of Zucker diabetic fatty rats.

A hallmark of Type 2 diabetes mellitus (T2DM) is chronic hyperglycemia, which is thought to play a role in pancreatic beta-cell failure. Here we investigated whether treatment of Zucker diabetic fatty (ZDF) rats, an animal model for T2DM, with the renal glucose transport inhibitor phlorizin could prevent alterations in the pancreatic islets. ZDF rats were treated with phlorizin or vehicle for 13 weeks starting with 6-week-old rats and before the onset of hyperglycemia. During the treatment, blood glucose levels in sham-treated ZDF rats increased rapidly from 7.7 +/- 0.3 to 24.8 +/- 0.6 mmol/l, whereas those in phlorizin-treated ZDF rats increased only slightly, but significantly, from 7.0 +/- 0.2 to 8.9 +/- 0.6 mmol/l. Phlorizin prevented the decrease in plasma insulin levels and caused a higher increase in body weight of the ZDF rats. Compared to 6-week-old untreated ZDF rats, in 19-week-old sham- and phlorizin-treated ZDF rats similar changes were found in islet architecture (more irregular boundaries and a disrupted mantle of peripheral islet cells) and in the mitochondria at the ultrastructural level (swelling of the matrix and disruption of the cristae). Using reverse transcriptase-polymerase chain reaction, no differences in mRNA expression levels were found for insulin, islet amyloid polypeptide (IAPP), and the prohormone convertase (PC) 1 and PC2 between 6-week-old untreated ZDF rats and 19-week-old sham- and phlorizin-treated ZDF rats. However, immunohistochemistry revealed similar decreases in insulin and IAPP protein expression in 19-week-old sham- and phlorizin-treated ZDF rats compared to those in 6-week-old untreated ZDF rats. These observations indicate that during aging of ZDF rats phlorizin treatment does not prevent the decreases in insulin and IAPP protein expression and the progressive histopathological changes in the pancreatic islets. Therefore, it is highly unlikely that these changes are caused by chronic hyperglycemia.

NPY-mRNA expressions in the nucleus accumbens, caudate putamen and cerebral cortex of apomorphine-susceptible and apomorphine-unsusceptible rats.

Using the apomorphine-induced stereotyped gnawing response as a selection criterion, two distinct groups of rats can be distinguished, apomorphine-susceptible (APO-SUS) and apomorphine-unsusceptible (APO-UNSUS) rats. These two lines differ in several components of both striatal and extrastriatal areas. This study deals with the expression of neuropeptide Y (NPY)mRNA-expressing neurons in the nucleus accumbens, caudate putamen and cerebral cortex of both rat lines, using non-radioactive in situ hybridisation. The morphology of the neurons in the three regions is similar, viz. oblong, rectangular or triangular, with two or three processes. The neurons are homogeneously distributed in all regions, and in the nucleus accumbens they are particularly numerous ventrally to the anterior commissure. Using automated image analysis, the mean numerical density of NPYmRNA-positive neurons per brain region and the mean NPYmRNA expression level per neuron per brain region were determined. No differences appear in the numerical densities of NPYmRNA-containing neurons in the nucleus accumbens, caudate putamen and cortex between APO-SUS and APO-UNSUS rats. However, distinct differences between the rat lines are present in the level of NPYmRNA expression per neuron in the nucleus accumbens and in the caudate putamen, showing that NPY contributes to the differential neurochemical make-up of these rat lines that is responsible for their obvious differences in behaviour, physiology and immune competence.

Automated nanoflow liquid chromatography-tandem mass spectrometry for a differential display proteomic study on Xenopus laevis neuroendocrine cells.

Many proteomic projects based on a comparison of protein profiles displayed on two-dimensional polyacrylamide gel electrophoresis rely on the identification of these proteins using peptide mass fingerprinting on a matrix-assisted laser desorption/ionization mass spectrometer after tryptic digestion. However, this approach is limited to an organism of which genomic information is largely available, i.e. when the total genome sequence is known. For other organisms, mass spectrometric sequence analysis is necessary for protein identification. We established a nano-LC-MS-MS system based on a quadrupole time-of-flight mass spectrometer, which allows automated sequence analysis of tryptic digestion mixtures from single gel spots. This system is applied in a differential-display proteomic study to identify differentially expressed proteins in the neuroendocrine cells of the neurointermediate pituitary of black- and white-background adapted Xenopus laevis.