Knockout of histamine receptor H3 alters adaptation to sudden darkness and monoamine levels in the zebrafish.

AIM: Histamine receptor H3 (HRH3) has substantial neuropharmacological potential. Currently, knockout models of this receptor have been investigated only in mice. We characterized the expression of this receptor in the zebrafish and generated a zebrafish HRH3 knockout line. Using this model, we studied the role of HRH3 in important behaviours. We also analysed the effect of HRH3 knockout on monoaminergic systems, which has not been thoroughly studied in any animal model. METHODS: Generation of a mutant zebrafish line using the CRISPR-Cas9 system. Analysis of locomotor and social behaviour. Expression of HRH3 was characterized using in situ hybridization. Analysis of monoamine networks using HPLC, immunohistochemistry and quantitative PCR. RESULTS: We found that HRH3 knockout zebrafish larvae showed a shorter period of increased locomotion after a sudden onset of darkness, while the knockout larvae had a wild-type-like acute response to sudden darkness. Adult knockout fish showed decreased swimming velocity, although locomotor activity of knockout larvae was unaltered. Additionally, levels of dopamine and serotonin were significantly decreased in the knockout fish, while monoamine-related gene expression and immunohistochemistry patterns were unchanged. CONCLUSIONS: Our results show that HRH3 knockout larvae adapt faster to sudden darkness, suggesting a role for this receptor in regulating responses to changes in the environment. The decreased levels of dopamine and serotonin provide the first direct evidence that knockout of HRH3 alters these systems.

Narcolepsy patients' blood-based miRNA expression profiling: miRNA expression differences with Pandemrix vaccination.

OBJECTIVES: Narcolepsy is a neurological sleep disorder characterized by excessive daytime sleepiness and nighttime sleep disturbance. Among children and adolescents vaccinated with Pandemrix vaccine in Finland and Sweden, the number of narcolepsy cases increased. Our aim was to identify miRNAs involved in narcolepsy and their association with Pandemrix vaccination. MATERIALS AND METHODS: We performed global miRNA proofing by miRNA microarrays followed by RT-PCR verification on 20 narcolepsy patients (Pandemrix-associated and Pandemrix-non-associated) and 17 controls (vaccinated and non-vaccinated). RESULTS: Between all narcolepsy patients and controls, 11 miRNAs were differentially expressed; 17 miRNAs showed significantly differential expression between Pandemrix-non-associated narcolepsy patients and non-vaccinated healthy controls. MiR-188-5p and miR-4499 were over-expressed in narcolepsy patients vs healthy controls. Two miRNAs, miR-1470 and miR-4455, were under-expressed in Pandemrix-associated narcolepsy patients vs Pandemrix-non-associated narcolepsy patients. CONCLUSIONS: We identified miRNA expression patterns in narcolepsy patients that linked them to mRNA targets known to be involved in brain-related pathways or brain disorders.

Interactions of the orexin/hypocretin neurones and the histaminergic system.

Histaminergic and orexin/hypocretin systems are components in the brain wake-promoting system. Both are affected in the sleep disorder narcolepsy, but the role of histamine in narcolepsy is unclear. The histaminergic neurones are activated by the orexin/hypocretin system in rodents, and the development of the orexin/hypocretin neurones is bidirectionally regulated by the histaminergic system in zebrafish. This review summarizes the current knowledge of the interactions of these two systems in normal and pathological conditions in humans and different animal models.

The tyrosine hydroxylase 2 (TH2) system in zebrafish brain and stress activation of hypothalamic cells.

Two tyrosine hydroxylases (TH1 and TH2) are found in teleost fish, but no antibodies are available for TH2 protein to analyze the detailed structure of the system. We generated antibodies targeting TH2 and used them to characterize the TH2-producing cells in larval and adult zebrafish brain. The rabbit antisera reliably detected two bands corresponding to TH1 and TH2 close to 55 kDa in brain homogenates. The antisera detected neurons in brain nuclei which express th1 and th2 mRNA; knockdown of th2 expression by morpholino oligonucleotide injection abolished both the th2 mRNA signal and immunoreactivity with the rabbit antisera in TH2 cells. Double staining of samples with the rabbit antiserum made against TH2 and a monoclonal antibody which detects only TH1 allowed identification of cell groups expressing either one of the proteins. Cell groups in preoptic area, anterior, intermediate, and posterior part of the paraventricular organ contained neurons stained with the new TH2 antisera but not with the characterized monoclonal TH1 antibody. Neurons immunoreactive for TH2 and 5-HT were distinct. In situ hybridization for the mRNA of the immediate early gene c-fos combined with TH1/TH2 immunohistochemistry was used to characterize the cells of the zebrafish brain reacting to handling stress and a noxious chemical stimulus. Strong upregulation of c-fos expression was detected in hypothalamic nuclei containing TH2 cells, but few of the c-fos-expressing cells were positive for TH2, suggesting that these stressors do not directly activate a large proportion of TH2 cells.

A zebrafish model of PINK1 deficiency reveals key pathway dysfunction including HIF signaling.

The PTEN induced putative kinase 1 (PINK1) gene is mutated in patients with hereditary early onset Parkinson's disease (PD). The targets of PINK1 and the mechanisms in PD are still not fully understood. Here, we carried out a high-throughput and unbiased microarray study to identify novel functions and pathways for PINK1. In larval zebrafish, the function of pink1 was inhibited using splice-site morpholino oligonucleotides and the samples were hybridized on a two-color gene expression array. We found 177 significantly altered genes in pink1 morphants compared with the uninjected wildtype controls (log fold change values from -1.6 to +0.9). The five most prominent pathways based on critical biological processes and key toxicological responses were hypoxia-inducible factor (HIF) signaling, TGF-ß signaling, mitochondrial dysfunction, RAR activation, and biogenesis of mitochondria. Furthermore, we verified that potentially important genes such as hif1α, catalase, SOD3, and atp1a2a were downregulated in pink1 morphants, whereas genes such as fech, pax2a, and notch1a were upregulated. Some of these genes have been found to play important roles in HIF signaling pathways. The pink1 morphants were found to have heart dysfunction, increased erythropoiesis, increased expression of vascular endothelial growth factors, and increased ROS. Our findings suggest that a lack of pink1 in zebrafish alters many vital and critical pathways in addition to the HIF signaling pathway.

Histamine is required for H3 receptor-mediated alcohol reward inhibition, but not for alcohol consumption or stimulation.

BACKGROUND AND PURPOSE: Conflicting data have been published on whether histamine is inhibitory to the rewarding effects of abused drugs. The purpose of this study was to clarify the role of neuronal histamine and, in particular, H3 receptors in alcohol dependence-related behaviours, which represent the addictive effects of alcohol. EXPERIMENTAL APPROACH: Alcohol-induced conditioned place preference (alcohol-CPP) was used to measure alcohol reward. Alcohol-induced locomotor stimulation, alcohol consumption and kinetics were also assessed. mRNA levels were quantified using radioactive in situ hybridization. KEY RESULTS: Low doses of H3 receptor antagonists, JNJ-10181457 and JNJ-39220675, inhibited alcohol reward in wild-type (WT) mice. However, these H3 receptor antagonists did not inhibit alcohol reward in histidine decarboxylase knock-out (HDC KO) mice and a lack of histamine did not alter alcohol consumption. Thus H3 receptor antagonists inhibited alcohol reward in a histamine-dependent manner. Furthermore, WT and HDC KO mice were similarly stimulated by alcohol. The expression levels of dopamine D1 and D2 receptors, STEP61 and DARPP-32 mRNA in striatal subregions were unaltered in HDC KO mice. No differences were seen in alcohol kinetics in HDC KO compared to WT control animals. In addition, JNJ-39220675 had no effect on alcohol kinetics in WT mice. CONCLUSIONS AND IMPLICATIONS: These data suggest that histamine is required for the H3 receptor-mediated inhibition of alcohol-CPP and support the hypothesis that the brain histaminergic system has an inhibitory role in alcohol reward. Increasing neuronal histamine release via H3 receptor blockade could therefore be a novel way of treating alcohol dependence.

Expression of histamine receptor genes Hrh3 and Hrh4 in rat brain endothelial cells.

BACKGROUND AND PURPOSE: Brain vascular endothelial cells express histamine H1 and H2 receptors, which regulate brain capillary permeability. We investigated whether H3 and H4 receptors are also expressed in these cells and may thus play a role in permeability regulation. EXPERIMENTAL APPROACH: An immortalized rat brain endothelial cell line RBE4 was used to assess the presence of H3 and H4 receptors. Reverse transcription-PCR (RT-PCR) and sequencing were used to identify the receptor mRNAs. The receptors were stimulated with histamine and immepip, and specific inverse agonists/antagonists ciproxifan and JNJ 7777120 were used to block H3 and H4 receptors, respectively. KEY RESULTS: RT-PCR of mRNA extracted from cultured immortalized RBE4 cells revealed two rat H4 receptor gene (Hrh4) transcripts, one full-length (coding sequence 1173 bp), and one with a 164 bp deletion. Also, two rat H3 receptor gene (Hrh3) isoform mRNAs were expressed in RBE4 cells, and sequencing showed they were the full-length H3 receptor and the 144 bp deletion form. Both histamine and immepip (H3 and H4 receptor agonists) activated the Erk1/2 MAPK pathway in the RBE4 cells and in vivo in brain blood vessels by activating H4 receptors, as the H4 receptor-specific inverse agonists/antagonist JNJ 7777120, but not ciproxifan, H3 receptor antagonist, dose-dependently blocked this effect in RBE4 cells. CONCLUSIONS AND IMPLICATIONS: Both Hrh3 and Hrh4 receptors are expressed in rat brain endothelial cells, and activation of the histamine H4 receptor activates the Erk1/2 cascade. H3 and H4 receptors in endothelial cells are potentially important for regulation of blood-brain barrier permeability, including trafficking of immunocompetent cells.

MANF regulates dopaminergic neuron development in larval zebrafish.

Mesencephalic astrocyte derived neurotrophic factor (MANF) is recognized as a dopaminergic neurotrophic factor, which can protect dopaminergic neurons from neurotoxic damage. However, little is known about the function of MANF during the vertebrate development. Here, we report that MANF expression is widespread during embryonic development and in adult organs analyzed by qPCR and in situ hybridization in zebrafish. Knockdown of MANF expression with antisense splice-blocking morpholino oligonucleotides resulted in no apparent abnormal phenotype. Nevertheless, the dopamine level of MANF morphants was lower than that of the wild type larvae, the expression levels of the two tyrosine hydroxylase gene transcripts were decreased and a decrease in neuron number in certain groups of th1 and th2 cells in the diencephalon region in MANF morphants was observed. These defects were rescued by injection of exogenous manf mRNA. Strikingly, manf mRNA could partly restore the decrease of th1 positive cells in Nr4a2-deficient larvae. These results suggest that MANF is involved in the regulation of the development of dopaminergic system in zebrafish.

Galanin gene expression and effects of its knock-down on the development of the nervous system in larval zebrafish.

Despite the known importance of galanin in the nervous system of vertebrates, the galanin gene structure and expression and the consequences of galanin deficiency in developing zebrafish are unknown. We cloned the galanin gene and analyzed its expression by using in situ hybridization, PCR, and immunocytochemistry throughout the early development of zebrafish until the end of the first week of life. The single zebrafish galanin gene encoded for a single amidated galanin peptide and a galanin message-associated peptide. Two forms resulting from alternative processing were identified. Galanin mRNA was maternally expressed and found in developing fish throughout early development. In situ hybridization showed the first positive neurons in three groups in the brain at 28 hours postfertilization. At 2 days postfertilization, three prosencephalic neuron groups were seen in the preoptic area and in rostral and caudal periventricular hypothalamus. In addition, two other groups of weakly stained neurons were visible, one in the midbrain and another in the hindbrain. Translation inhibition of galanin mRNA with morpholino oligonucleotides caused complete disappearance of galanin immunoreactivity in the brain until 7 dpf and did not induce known cascades of nonspecific pathways or morphological abnormalities. A minor disturbance of sensory ganglia was found. Galanin knockdown did not alter the expression of tyrosine hydroxylases 1 and 2, choline acetyltransferase, histidine decarboxylase, or orexin mRNA. The results suggest that galanin does not regulate the development of these key markers of specific neurons, although galanin-expressing fibers were in a close spatial proximity to several neurons of these neuronal populations.

Human kisspeptins activate neuropeptide FF2 receptor.

We studied the possible activation of a neuropeptide FF2 receptor (NPFF2R) by kisspeptins, neuropeptides derived from the mouse and human metastin or Kiss-1 precursor. The hypothesis was that the human kisspeptins, which share the C-terminal dipeptide RF-NH(2) with NPFF, might activate the NPFF2R, as has previously been shown for two related peptides, prolactin-releasing peptide and RF-amide-related peptide. Using two-electrode voltage clamp of Xenopus oocytes, we found that 100 nM NPFF strongly activated the human NPFF2R expressed together with rat GIRK1/4 inward rectifier potassium channels, and that 100 nM hKisspeptin-13 and hKisspeptin-8 had about 25% relative efficacy to that of NPFF. The current response induced by hKisspeptin-13 was proportional to its concentration (1-500 nM). The corresponding mouse peptides resulted in low activation only. When hNPFF2R was expressed in Chinese hamster ovary (CHO) cells, NPFF and its stable analog (1DMe)Y8Fa induced guanosine 5'-(gamma-[(35)S]thio)-triphosphate (GTP-gamma-[(35)S]) binding with EC(50) values of 13+/-4 and 16+/-4 nM, respectively. hKisspeptin-13 induced the binding with an EC(50) value of 110+/-50 nM, whereas mKisspeptin-13 induced very modestly activation with an EC(50) value>2 microM. The results suggest that, besides regulation of reproduction, kisspeptins have a potential to mediate physiological effects on, for example autonomic regulation and nociception in man via the NPFF2R pathways.

The comparative neuroanatomy and neurochemistry of zebrafish CNS systems of relevance to human neuropsychiatric diseases.

Modulatory neurotransmitters which signal through G protein-coupled receptors control brain functions which deteriorate in degenerative brain diseases. During the past decade many of these systems have been mapped in the zebrafish brain. The main architecture of the systems in zebrafish brain resembles that of the mammals, despite differences in the development of the telencephalon and mesodiencephalon. Modulatory neurotransmitters systems which degenerate in human diseases include dopamine, noradrenaline, serotonin, histamine, acetylcholine and orexin/hypocretin. Although the number of G protein-coupled receptors in zebrafish is clearly larger than in mammals, many receptors have similar expression patterns, binding and signaling properties as in mammals. Distinct differences between mammals and zebrafish include duplication of the tyrosine hydroxylase gene in zebrafish, and presence of one instead of two monoamine oxidase genes. Zebrafish are sensitive to neurotoxins including MPTP, and exposure to this neurotoxin induces a decline in dopamine content and number of detectable tyrosine hydroxylase immunoreactive neurons in distinct nuclei. Sensitivity to important neurotoxins, many available genetic methods, rapid development and large-scale quantitative behavioral methods in addition to advanced quantitative anatomical methods render zebrafish an optimal organism for studies on disease mechanisms.

Hypocretin/orexin in fish physiology with emphasis on zebrafish.

One hypocretin/orexin (hcrt) gene has been identified in several fish species. The first pufferfish gene was identified in 2002 and the zebrafish gene was cloned in 2004. Its structure is very similar to that of mammals, and it encodes for two active peptides with C-termini similar to those of mammals. The gene is expressed in the brain in only one hypothalamic nucleus, which sends projections to the telencephalon, diencephalon, mesencephalon and rhombencephalon. The terminal fibres are found in close contact with many aminergic cell groups, including those of raphe serotonergic, locus coeruleus noradrenergic, several dopaminergic cell groups and the sole histaminergic hypothalamic cluster. One receptor corresponding to mammalian hcrt 2 receptor has been identified in fish. Overexpression of hcrt in zebrafish has been reported to consolidate wakefulness and inhibit rest. On the other hand, fish lacking the hcrt receptor show short and fragmented sleep instead of sleepiness and cataplexy. Food deprivation increases hcrt mRNA expression in zebrafish brain, and intracerebroventricular hcrt peptides stimulate food consumption and feeding behaviour in goldfish. Hcrt peptides thus have important roles in fish physiology. Many genetic and functional methods available render fish, especially zebrafish, a suitable organism to study new aspects of hcrt physiology in vertebrates.

Altered histamine H3 receptor radioligand binding in post-mortem brain samples from subjects with psychiatric diseases.

BACKGROUND AND PURPOSE: Histamine is a modulatory neurotransmitter in the brain. Auto- and hetero-histamine H3 receptors are present in human brain and are potential targets of antipsychotics. These receptors may also display disease-related abnormalities in psychiatric disorders. Here we have assessed how histamine H3 receptors in human brain may be affected in schizophrenia, bipolar disorder, major depression. EXPERIMENTAL APPROACH: Histamine H3 receptor radioligand binding assays were applied to frozen post-mortem prefrontal and temporal cortical sections and anterior hippocampal sections from subjects with schizophrenia, bipolar disorder, major depression and matched controls. KEY RESULTS: Compared with the controls, increased H3 receptor radioligand binding was found in dorsolateral prefrontal cortex of schizophrenic subjects (especially the ones who were treated with atypical antipsychotics), and bipolar subjects with psychotic symptoms. No differences in H3 receptor radioligand binding were found in the temporal cortex. In hippocampal formation of control subjects, H3 receptor radioligand binding was prominent in dentate gyrus, subiculum, entorhinal cortex and parasubiculum. Decreased H3 binding was found in the CA4 area of bipolar subjects. Decreased H3 binding in CA2 and presubiculum of medication-free bipolar subjects was also seen. CONCLUSIONS AND IMPLICATIONS: The results suggest that histamine H3 receptors in the prefrontal cortex take part in the modulation of cognition, which is impaired in schizophrenic subjects and bipolar subjects with psychotic symptoms. Histamine H3 receptors probably regulate connections between hippocampus and various cortical and subcortical regions and could also be involved in the neuropathology of schizophrenia and bipolar disorder.

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.

MPTP and MPP+ target specific aminergic cell populations in larval zebrafish.

Larval zebrafish offers a good model to approach brain disease mechanisms, as structural abnormalities of their small brains can be correlated to quantifiable behavior. In this study, the structural alterations in one diencephalic dopaminergic nucleus induced by 1-methyl-4-phenylpyridinium (MPP+), a toxin inducing Parkinson's disease in humans, and those found in several neuronal groups after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the pretoxin, were associated with decreased swimming speed. Detailed cell counts of dopaminergic groups indicated a transient decline of tyrosine hydroxylase expressing neurons up to about 50% after MPTP. The MPTP effect was partly sensitive to monoamine oxidase inhibitor deprenyl. Detailed analysis of the developing catecholaminergic cell groups suggests that the cell groups emerged at their final positions and no obvious significant migration from the original positions was seen. One 5-HT neuron group was also affected by MPTP treatment, whereas other groups remained intact, suggesting that the effect is selective. New nomenclature for developing catecholaminergic cell groups corresponding to adult groups is introduced. The diencephalic cell population consisting of groups 5,6 and 11 was sensitive to both MPTP and MPP+ and in this respect resembles mammalian substantia nigra. The results suggest that MPTP and MPP+ induce a transient functional deficit and motility disorder in larval zebrafish.

Postischemic regulation of central histamine receptors.

This study characterizes changes occurring in the central histaminergic system associated with ischemia-reperfusion pathology in the rat. Specifically, after a postocclusion time period of 48 h, we have analyzed histamine H(1) receptor mRNA expression, histamine H(2) receptor protein amount and binding densities, and histamine H(3) receptor mRNA expression and binding densities in brain regions that have been suggested to be selectively vulnerable to transient global ischemia, i.e. hippocampus, thalamus, caudate-putamen, and cerebral cortex. We found an increase in H(1) receptor mRNA expression in the caudate-putamen: given that ischemia reduces glucose uptake and H(1) receptor activation has been shown to decrease this effect, an increase of expression levels may result in mitigating tissue damage due to energy failure observed in ischemia. A decrease in H(2) receptor binding densities in the caudate-putamen was also observed; the ischemia-induced decrease in H(2) receptor protein was also detectable by Western blot analysis. This phenomenon may underlie the previously reported ischemia induced striatal dopamine release. H(3) receptor mRNA expression was increased in the caudate putamen of the postischemic brain but was decreased in the globus pallidus and the thalamus; in association with this, H(3) receptor binding densities were increased in the cortex, caudate-putamen, globus pallidus, and hippocampus. The upregulation of H(3) receptor ligand binding may be involved in the previously reported continuous neuronal histamine release. Our data suggest that central histamine receptor expression and ligand binding are altered in brain ischemia in distinct areas, and may participate in neuroprotection and/or ischemia-associated neuronal damage.

RFamide-related peptides signal through the neuropeptide FF receptor and regulate pain-related responses in the rat.

The mammalian RFamide-related peptide RFRP1 was found to signal through the neuropeptide FF 2 receptor expressed in Xenopus oocytes. The peptide induced a dose-dependent outward current, which was dependent on the simultaneous expression of GIRK1 and GIRK4 potassium channels. In neuropathic rats, RFRP1 administered intrathecally induced tactile antiallodynia and thermal antinociception, whereas in the solitary tract nucleus it produced only mechanical antihyperalgesia. Expression of the RFamide-related peptide mRNA in the rat CNS was distinctly different from that of neuropeptide FF. Most notably, the gene was not expressed in the hindbrain or spinal cord at detectable levels. However, there was a prominent group of RFamide-related peptide mRNA-expressing neurons in the central hypothalamus, in the area in and between the dorsomedial and ventromedial nuclei. The results suggest that RFamide-related peptides are potentially involved in pain regulation through a hypothalamo-medullary projection system, and possibly via action on neuropeptide FF 2 receptors. In neuropathic animals, the pain suppressive effect of RFamide-related peptide varies depending on the submodality of noxious test stimulation and the site of RFamide-related peptide administration.

The laminar histamine receptor system in human prefrontal cortex suggests multiple levels of histaminergic regulation.

Human prefrontal cortex is essential for high brain functions and its activity is modulated by multiple neurotransmitters, including histamine. However, the histamine receptors in this brain area have not been systematically studied so far. In situ hybridization and receptor binding autoradiography were employed to map and quantify the mRNA expression and receptor binding of three of the four histamine receptors (H(1), H(2), H(3)). mRNA expression and receptor binding of these three histamine receptors displayed characteristic laminar distribution patterns. Both H(1) and H(3) receptor mRNAs were mainly expressed in the deeper layers (H(1) in laminae V and VI; H(3) in lamina V), where most of the corticothalamic projections originate, whereas H(2) receptor mRNA was primarily expressed in the superficial layer II. Receptor ligand binding of these three histamine receptors displayed relatively even distribution patterns throughout the gray matter. However, higher densities of H(1) and H(3) receptor radioligand binding sites were seen in the middle layers III and IV that receive abundant thalamic inputs and where some of the apical dendrites of the deep-layer pyramidal neurons terminate, whereas higher density of H(2) receptor radioligand binding sites was seen in the superficial layers I-III. The results, together with data on histaminergic regulation of thalamic oscillations suggest that histamine regulates both cortico-cortical and thalamo-cortical circuits. As histamine receptors are also abundant in thalamus, histamine may be involved also in human diseases of the thalamocortical system.

Traumatic brain injury results in mast cell increase and changes in regulation of central histamine receptors.

Experimental fluid-percussion models produce brain injury by rapidly injecting saline into the closed cranium of rats. In this study our purpose was to determine how the central histaminergic system, which controls excitability and neurotransmitter release through G-protein coupled receptors, is affected by the pathophysiology of traumatic brain injury. We found that mast cell infiltration, as a result of the trauma, occurred primarily in the injured cortex and did not proceed beyond the fimbria of the hippocampus. In comparing injured animals with controls we found that H3 receptor binding densities are significantly decreased bilaterally in the cortex but are significantly increased bilaterally in the thalamus. H3 receptor binding densities may well be affected by mast cell secretion of mediators (i.e. histamine, heparin, leukotrienes), evidenced by detection of a cosecreted enzyme (mast cell tryptase) in the extracellular region. Moreover, we detected significant decreases in H1 and H3 receptor mRNA as well as Cu/Zn-dependent superoxide dismutase (SOD) mRNA in the thalamic region closest to the trauma. These significant decreases delineate the extent of cellular damage because of trauma and may underlie sustained cognitive and motor deficits displayed by these animals.

Neuropeptide FF, but not prolactin-releasing peptide, mRNA is differentially regulated in the hypothalamic and medullary neurons after salt loading.

Hypothalamic paraventricular and supraoptic nuclei are involved in the body fluid homeostasis. Especially vasopressin peptide and mRNA levels are regulated by hypo- and hyperosmolar stimuli. Other neuropeptides such as dynorphin, galanin and neuropeptide FF are coregulated with vasopressin. In this study neuropeptide FF and another RF-amide peptide, the prolactin-releasing peptide mRNA levels were studied by quantitative in situ hybridization after chronic salt loading, a laboratory model of chronic dehydration. The neuropeptide FF mRNA expressing cells virtually disappeared from the hypothalamic supraoptic and paraventricular nuclei after salt loading, suggesting that hyperosmolar stress downregulated the NPFF gene transcription. The neuropeptide FF mRNA signal levels were returned to control levels after the rehydration period of 7 days. No changes were observed in those medullary nuclei that express neuropeptide FF mRNA. No significant changes were observed in the hypothalamic or medullary prolactin-releasing peptide mRNA levels. Neuropeptide FF mRNA is drastically downregulated in the hypothalamic magnocellular neurons after salt loading. Other neuropeptides studied in this model are concomitantly coregulated with vasopressin: i.e. their peptide levels are downregulated and mRNA levels are upregulated which is in contrast to neuropeptide FF regulation. It can thus be concluded that neuropeptide FF is not regulated through the vasopressin regulatory system but via an independent pathway. The detailed mechanisms underlying the downregulation of neuropeptide FF mRNA in neurons remain to be clarified.