Miklós Palkovits

A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.

Norepinephrine and dopamine content of hypothalamic nuclei of the rat

Abstract The concentrations of norepinephrine and dopamine of 27 isolated rat hypothalamic nuclei or nuclear subdivisions have been determined using an enzymatic-isotopic assay. The hypothalamic nuclei contain 3–20 times more NE and DA than is measured in the cortex. They are distributed unevenly throughout the hypothalamus and individual hypothalamic nuclei are heterogenous as regards their content of the amines. The dopamine content of the median eminence is among the highest measured in the brain (65 ng/mg protein). The norepinephrine content is about half that of dopamine. All of the hypothalamic nuclei contain dopamine. It is highly concentrated in the rostral subdivisions of the arcuate nucleus, the paraventricular and dorsomedial nuclei, the retrochiasmatic area, the medial posterior subdivision of the ventromedial nucleus and in the medial forebrain bundle at the posterior hypothalamic level. The highest concentrations of norepinephrine were found in the paraventricular and dorsomedial nuclei and in the retrochiasmatic area. The rostal subdivision of the arcuate nucleus and the periventricular and preoptic suprachiasmatic nuclei are also rich in norepinephrine. The posterior hypothalamus (premammillary nuclei, caudal subdivision of the arcurate nucleus, posterior hypothalamic nucleus) contains norepinephrine and dopamine in relatively low concentrations.

Regional distribution of substance P in the brain of the rat

Using a sensitive radioimmunoassay we have studied the regional distribution of substance P. The level of substance P is higher in the mesencephalon, hypothalamus and preoptic area than in other regions of the brain. Substance P is found in especially high concentrations in the reticular part of the substantia nigra and the interpeduncular nucleus. It is present in large amounts in several septal, preoptic and hypothalamic nuclei as well.

Neuroanatomy of Central Cardiovascular Control. Nucleus Tractus Solitarii: Afferent and Efferent Neuronal Connections in Relation to the Baroreceptor Reflex Arc

Publisher Summary In this chapter, the neuroanatomy of the baroreceptor reflex arc is reviewed and relevant new data are presented. The baroreceptor reflex arc consists of a multisynaptic neuronal chain. Primary neurones have perikarya in the nodose ganglion, and they connect the peripheral baroreceptor sites with the nucleus tractus solitarii (NTS) via fibres in the 9th and 10th cranial nerves. The first synapse in the baroreceptor reflex arc and also the origin of the secondary neurones are located in the caudal and partly in the commissural parts of the NTS. Neuroanatomical topography of the NTS and neighboring medullary nuclei in the rat is presented and detailed. The fibres of the secondary neurones terminate in various medullary nuclei and probably reach—directly or by multisynaptic pathways—the higher regions that may modulate the baroreceptor reflex arc. The chapter presents a discussion on the possible site of these modulatory centers in the hypothalamus and in the brain stem and of the loop of the descending fibres from these regions to the medullary and spinal baroreceptor neurones. The efferent preganglionic neurones of the baroreceptor reflex arc are located in the medulla oblongata and in the intermedio-lateral nucleus of the spinal cord. The chapter outlines the possible interrelationship between the modulatory biogenic amine-containing neurones and the baroreceptor reflex arc.

Thyrotropin-Releasing Hormone in Specific Nuclei of Rat Brain

The regional distribution of thyrotropin-releasing hormone (TRH) in rat brain was studied. The greatest concentration of TRH was found in the median eminence. High concentrations were also found in several hypothalamic nuclei. Outside the hypothalamus, relatively large amounts of TRH were found in the septal and preoptic areas.

Catecholaminergic Systems in Stress: Structural and Molecular Genetic Approaches

Stressful stimuli evoke complex endocrine, autonomic, and behavioral responses that are extremely variable and specific depending on the type and nature of the stressors. We first provide a short overview of physiology, biochemistry, and molecular genetics of sympatho-adrenomedullary, sympatho-neural, and brain catecholaminergic systems. Important processes of catecholamine biosynthesis, storage, release, secretion, uptake, reuptake, degradation, and transporters in acutely or chronically stressed organisms are described. We emphasize the structural variability of catecholamine systems and the molecular genetics of enzymes involved in biosynthesis and degradation of catecholamines and transporters. Characterization of enzyme gene promoters, transcriptional and posttranscriptional mechanisms, transcription factors, gene expression and protein translation, as well as different phases of stress-activated transcription and quantitative determination of mRNA levels in stressed organisms are discussed. Data from catecholamine enzyme gene knockout mice are shown. Interaction of catecholaminergic systems with other neurotransmitter and hormonal systems are discussed. We describe the effects of homotypic and heterotypic stressors, adaptation and maladaptation of the organism, and the specificity of stressors (physical, emotional, metabolic, etc.) on activation of catecholaminergic systems at all levels from plasma catecholamines to gene expression of catecholamine enzymes. We also discuss cross-adaptation and the effect of novel heterotypic stressors on organisms adapted to long-term monotypic stressors. The extra-adrenal nonneuronal adrenergic system is described. Stress-related central neuronal regulatory circuits and central organization of responses to various stressors are presented with selected examples of regulatory molecular mechanisms. Data summarized here indicate that catecholaminergic systems are activated in different ways following exposure to distinct stressful stimuli.

Isolation and measurement of the endogenous cannabinoid receptor agonist, anandamide, in brain and peripheral tissues of human and rat

Anandamide (arachidonylethanolamide) is a novel lipid neurotransmitter first isolated from porcine brain which has been shown to be a functional agonist for the cannabinoid CB1 and CB2 receptors. Anandamide has never been isolated from human brain or peripheral tissues and its role in human physiology has not been examined. Anandamide was measured by LC/MS/MS and was found in human and rat hippocampus (and human parahippocampal cortex), striatum, and cerebellum, brain areas known to express high levels of CB1 cannabinoid receptors. Significant levels of anandamide were also found in the thalamus which expresses low levels of CB1 receptors. Anandamide was also found in human and rat spleen which expresses high levels of the CB2 cannabinoid receptor. Small amounts of anandamide were also detected in human heart and rat skin. Only trace quantities were detected in pooled human serum, plasma, and CSF. The distribution of anandamide in human brain and spleen supports its potential role as an endogenous agonist in central and peripheral tissues. The low levels found in serum, plasma, and CSF suggest that it is metabolized in tissues where it is synthesized, and that its action is probably not hormonal in nature.

Dysregulation in the Suicide Brain: mRNA Expression of Corticotropin-Releasing Hormone Receptors and GABAA Receptor Subunits in Frontal Cortical Brain Region

Corticotropin-releasing hormone (CRH) and GABA have been implicated in depression, and there is reason to believe that GABA may influence CRH functioning. The levels of CRH, and mRNA for CRH-binding protein, CRH1, and CRH2 receptors, as well as various GABAA receptor subunits (α1, α2, α3, α4, α5, δ, and γ2), were determined in several frontal cortical brain regions of depressed suicide victims and nondepressed individuals who had not died by suicide. Relative to the comparison group, CRH levels were elevated in frontopolar and dorsomedial prefrontal cortex, but not in the ventrolateral prefrontal cortex of suicide victims. Conversely, using quantitative PCR analyses, it was observed that, in frontopolar cortex, mRNA for CRH1, but not CRH2, receptors were reduced in suicide brains, possibly secondary to the high levels of CRH activity. In addition, mRNA of the α1, α3, α4, and δ receptor subunits was reduced in the frontopolar region of suicide victims. Interestingly, a partial analysis of the GABAA receptor functional genome revealed high cross-correlations between subunit expression in cortical regions of nondepressed individuals, suggesting a high degree of coordinated gene regulation. However, in suicide brains, this regulation was perturbed, independent of overall subunit abundance. These findings raise the possibility that the CRH and GABAA receptor subunit changes, or the disturbed coordination between these GABAA receptor subunits, contribute to depression and/or suicidality or are secondary to the illness/distress associated with it.

Physiological role of a novel neuropeptide, apelin, and its receptor in the rat brain

Apelin, a peptide recently isolated from bovine stomach tissue extracts, has been identified as the endogenous ligand of the human orphan APJ receptor. We established a stable Chinese hamster ovary (CHO) cell line expressing a gene encoding the rat apelin receptor fused to the enhanced green fluorescent protein, to investigate internalization and the pharmacological profile of the apelin receptor. Stimulation of this receptor by the apelin fragments K17F (Lys1‐Phe‐Arg‐Arg‐Gln‐Arg‐Pro‐Arg‐Leu‐Ser‐His‐Lys‐Gly‐Pro‐Met‐Pro‐Phe17) and pE13F (pGlu5‐Arg‐Pro‐Arg‐Leu‐Ser‐His‐Lys‐Gly‐Pro‐Met‐Pro‐Phe17) resulted in a dose‐dependent inhibition of forskolin‐induced cAMP production and promoted its internalization. In contrast, the apelin fragments R10F (Arg8‐Leu‐Ser‐His‐Lys‐Gly‐Pro‐Met‐Pro‐Phe17) and G5F (Gly13‐Pro‐Met‐Pro‐Phe17) were inactive. The physiological role of apelin and its receptor was then investigated by showing for the first time in rodent brain: (i) detection of apelin neurons in the supraoptic and paraventricular nuclei by immunohistochemistry with a specific polyclonal anti‐apelin K17F antibody; (ii) detection of apelin receptor mRNA in supraoptic vasopressinergic neurons by in situ hybridization and immunohistochemistry; and (iii) a decrease in vasopressin release following intracerebroventricular injection of K17F, or pE13F, but not R10F. Thus, apelin locally synthesized in the supraoptic nucleus could exert a direct inhibitory action on vasopressinergic neuron activity via the apelin receptors synthesized in these cells. Furthermore, central injection of pE13F significantly decreased water intake in dehydrated normotensive rats but did not affect blood pressure. Together, these results suggest that neuronal apelin plays an important role in the central control of body fluid homeostasis.

Dopamine Biosynthesis Is Selectively Abolished in Substantia Nigra/Ventral Tegmental Area but Not in Hypothalamic Neurons in Mice with Targeted Disruption of the Nurr1 Gene

To ascertain the function of an orphan nuclear receptor Nurr1, a transcription factor belonging to a large gene family that includes receptors for steroids, retinoids, and thyroid hormone, we generated Nurr1-null mice by homologous recombination. Mice, heterozygous for a single mutated Nurr1 allele, appear normal, whereas mice homozygous for the null allele die within 24 h after birth. Dopamine (DA) was absent in the substantia nigra (SN) and ventral tegmental area (VTA) of Nurr1-null mice, consistent with absent tyrosine hydroxylase (TH), L-aromatic amino acid decarboxylase, and other DA neuron markers. TH immunoreactivity and mRNA expression in hypothalamic, olfactory, and lower brain stem regions were unaffected. L-Dihydroxyphenylalanine treatments, whether given to the pregnant dams or to the newborns, failed to rescue the Nurr1-null mice. We were unable to discern differences between null and wild-type mice in the cellularity, presence of neurons, or axonal projections to the SN and VTA. These findings provide evidence for a new mechanism of DA depletion in vivo and suggest a unique role for Nurr1 in fetal development and/or postnatal survival.