Judit Baffi

Dual oxidases represent novel hydrogen peroxide sources supporting mucosal surface host defense

Lactoperoxidase (LPO) is an enzyme with antimicrobial properties present in saliva, milk, tears, and airway secretions. Although the formation of microbicidal oxidants by LPO has been recognized for some time, the source of hydrogen peroxide (H2O2) for LPO‐catalyzed reactions remains unknown. Reactive oxygen species produced by the phagocyte NADPH oxidase (phox) play a critical role in host defense against pathogens; however, analogous oxidant‐generating systems in other tissues have not been associated with antimicrobial activity. Several homologues of gp91phox, the catalytic core of this enzyme, were described recently; dual oxidase (Duox)1/thyroid oxidase 1 and Duox2/thyroid oxidase 2 were identified in the thyroid gland and characterized as H2O2 donors for thyroxin biosynthesis. We examined Duox1 and Duox2 expression in secretory glands and on mucosal surfaces and give evidence for their presence and activity in salivary glands, rectum, trachea, and bronchium. Epithelial cells in salivary excretory ducts and rectal glands express Duox2, whereas tracheal and bronchial epithelial cells express Duox1. Furthermore, we detected Duox1‐dependent H2O2 release by cultured human bronchial epithelial cells. Our observations suggest that Duox1 and Duox2 are novel H2O2 sources that can support LPO‐mediated antimicrobial defense mechanisms on mucosal surfaces.

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.

Stress-induced changes in messenger RNA levels of N-methyl-d-aspartate and AMPA receptor subunits in selected regions of the rat hippocampus and hypothalamus

The postsynaptic AMPA/kainate and N-methyl-D-aspartate-selective glutamate receptors are formed by several different subunits and the overall subunit composition of the receptor appears to determine its physiological and pharmacological properties. Although glutamatergic mechanisms have been implicated in various forms of hippocampal stress responses, the impact of stress on glutamate receptor subunit composition has not yet been elucidated. We have used cell-by-cell quantitative in situ hybridization to assess stress-induced changes in transcript levels of N-methyl-D-aspartate and AMPA receptor subunit genes in subdivisions of the rat hippocampus and hypothalamus that are implicated in the stress response. We found that 24 h after a single immobilization stress there was a significant increase in the cellular level of NR1 subunit messenger RNA (about 35-45% above control values) in hippocampal CA3 and CA1 pyramidal cells as well as in neurons of the hypothalamic supraoptic and paraventricular nuclei. Moreover, in the CA3 area we have detected a concomitant increase (50% above controls) in the level of NR2B subunit messenger RNA, while the expression of NR2A subunit gene did not change after stress. Stress induced a selective decrease in the level of AMPA receptor subunit glutamate receptor A messenger RNA in neurons of both the CA3 and CA1 areas (18 and 24%, respectively, below control values). These results suggest that the regulation of specific subunit messenger RNAs of the N-methyl-D-aspartate and AMPA receptors may be involved in altered hippocampal and hypothalamic responsiveness to glutamate and thus could play a critical role in stress-induced changes in their function.

Neuropeptides in the human superior cervical ganglion

Superior cervical ganglia from 7 human cadavers (3-7 h post mortem) were immunostained for tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and 14 different neuropeptides. The results show that ganglionic cells contain TH, DBH, neuropeptide Y (NPY), somatostatin, vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP). These substances were present predominantly within large ganglionic cells. Inside the ganglion, the number and topographical distribution of various types of immunoreactive cells differed from one another. NPY and CGRP immunoreactivities were found in some TH-positive cells, but that co-localization never exceeded the 30% of the TH cells. Leu-enkephalin showed a weak immunoreactivity, which was restricted to fibers or varicosities. Neuropeptides like substance P, dynorphin A and B, cholecystokinin, galanin, corticotropin-releasing factor, thyrotropin-releasing hormone, angiotensin II and neurotensin showed no immunoreactivity in the human superior cervical ganglion.

Fine Topography of Brain Areas Activated by Cold Stress

Neuronal activity in response to acute cold exposure was mapped in the central nervous system of adult rats using Fos immunostaining. A single, 3-hour exposure to cold elicited strong Fos-like immunoreactivity in the medial preoptic nucleus that is known as the thermoregulatory center of the brain. By this technique, pontine and medullary thermosensitive areas have been first localized and outlined anatomically. The medullary thermosensitive neurons occupy well-demarcated areas immediately ventral and dorsal to the spinal trigeminal nucleus, termed peritrigeminal and paratrigeminal nuclei, respectively. Cold-sensitive neurons were present in the dorsal part of the pontine reticular formation. Topographically, this area corresponds to the ‘pontine thermoregulatory area’, named on the basis of neurophysiological observations. In addition, thermosensitive neurons were found in the rostral thalamus and zona incerta. Several cell groups that showed strong Fos-like immunoreactivity in our previous pain-related stress experiments were also activated by cold exposure. The midline thalamic, hypothalamic dorsomedial, supramamillary and lateral parabrachial nuclei were targets of cold stress-induced noxious stimuli. Fos-positive neurons established specific topographical patterns in the paraventricular, arcuate, central amygdaloid nuclei, and the nucleus of the solitary tract. The possible involvement of central noradrenergic neurons in stress response to acute cold exposure was investigated by double immunostaining for tyrosine hydroxylase (TH) and Fos. None of the tyrosine hydroxylase positive neurons in the brain stem established Fos-like immunoreactivity, suggesting that the central noradrenergic system may have a minor, if any, role in cold-induced stress responses. Based on the topographical distribution of Fos-activated neurons, this study suggests that in addition to the hypothalamo-pituitary-adrenal axis, some other stress effector systems may play an important role in the maintenance of homeostasis during cold stress.

Nigrostriatal innervation is preserved in Nurr1-null mice, although dopaminergic neuron precursors are arrested from terminal differentiation.

Various factors, including the orphan nuclear receptor Nurr1, have been implicated in dopamine biosynthesis, but many of the specific events involved in this process have to be determined. Using genetic manipulations in mice, the obligatory role for Nurr1 in dopamine (DA) biosynthesis has been documented; however, the mechanism remains unclear. DA biosynthetic enzymes, transporters and receptors are absent in the substantia nigra (SN) and the ventral tegmental area (VTA) of Nurr1-null neonates. The current study establishes that the loss of Nurr1 function does not affect the normal ventralization of neuroepithelial cells to the ventral midbrain, their differentiation into neurons, and their topographical pattern in the SN and VTA. Futhermore, the absence of Nurr1 does not affect the survival of these DA precursor cells in the ventral midbrain, as determined by quantitative analysis of cells, expressing the general neuronal nuclear marker (NeuN) and the TUNEL assay for apoptosis. These neurons express cholecystokinin (CCK), a co-transmitter of dopaminergic neurons in this area. The untranslated exon 1-2 of the Nurr1 gene, which remains intact after homologous recombination, revealed the presence of dopaminergic precursors in the ventral midbrain of the Nurr1-null mice. In addition, these neurons establish their nigrostriatal projections, as shown by axonal transport of a fluorescent tracer, DiI. These results provide evidence that Nurr1 is essential for terminal differentiation of the dopaminergic neurons in the ventral midbrain but does not affect the early steps of their neurogenesis, migration, survival and striatal projections. Our findings suggest that activation of Nurr1 might be therapeutically useful in Parkinsons disease.

Differential expression of tyrosine hydroxylase in catecholaminergic neurons of neonatal wild-type and nurr1-deficient mice

The orphan nuclear receptor Nurr1 is a transcription factor that belongs to the steroid/thyroid hormone receptor superfamily and is expressed in many regions of the brain. To determine the physiological role of Nurr1, we previously generated mice with a null mutation in the Nurr1 gene. Nurr1-null mice appear to develop normally but die within 12 h after birth. Subsequent analysis revealed the absence of neurotransmitter dopamine and tyrosine hydroxylase immunoreactivity in the central dopaminergic area of newborn pups. Herein, using in situ hybridization histochemistry, we show that Nurr1 is expressed only in subset of catecholamine producing neurons (A2 partly, A8-A10 and A11 catecholaminergic cell groups), and is excluded from the norepinephrine producing neurons (A1, A2, A5-A6 catecholaminergic cell groups). Nurr1 was not expressed in the dopamine synthesizing cell groups (A12-A16 catecholaminergic cell groups) of the diencephalon and the olfactory bulb. As previously shown and confirmed in this study, tyrosine hydroxylase immunoreactivity was absent in the substantia nigra and ventral tegmental area of Nurr1-deficient mice. However, the loss of Nurr1 expression in A2 and A11 dopaminergic neurons did not affect their tyrosine hydroxylase immunoreactivity. This study begins to dissect cues necessary for understanding the complex regulation of the catecholaminergic biosynthetic pathway with regard to local, chemical and developmental changes in the brain.

Stressor-specific increase of vasopressin mRNA in paraventricular hypophysiotrophic neurons

Cellular levels of vasopressin (VP) and corticotropin-releasing factor (CRF) RNA transcripts were determined in hypophysiotrophic neurons after open-field and immobilization stress using quantitative in situ hybridization. We found that 8 min open-field stress is sufficient to produce a significant up-regulation of CRF mRNA, without any concomitant changes in the level of VP mRNA. In contrast, 8 min immobilization stress resulted in an increased labeling density of both CRF and VP mRNAs. These results suggest that the level of CRF and VP transcripts in parvicellular hypophysiotrophic neurons is differentially regulated in a stressor-specific manner.

Anxiolytic homophthalazines increase Fos-like immunoreactivity in selected brain areas of the rat

Nerisopam, an anxiolytic and antipsychotic homophthalazine induces rapid, intense expression of Fos-like immunoreactivity in the rostral, dorsomedial and lateral parts of the striatum in the rat. Fos-positive cells also occurred in the globus pallidus, the olfactory tubercle and in the accumbens nucleus (in the cone and shell portions) but the substantia nigra, the entopeduncular and the subthalamic nuclei were virtually Fos-negative. 5 h after nerisopam application, however, cells in the reticular zone of the substantia nigra showed Fos-like immunopositivity. After a daily application of nerisopam for two weeks, relatively weak Fos-like immunoreactivity was observed in the striatum and the subthalamic nucleus but not in the globus pallidus. Unilateral surgical transection of the striato-nigral pathway, which depleted tyrosine hydroxylase immunostaining in the ipsilateral striatum did not influence nerisopam-induced Fos-like immunoreactivity in the striatal neurons, either ipsi- or contralateral to the knife cut. Our results suggest that the striatal neurons are the primary targets of this anxiolytic and antipsychotic drug in the central nervous system.

Brain catecholamine systems in stress.

Publisher Summary In the study discussed in this chapter, immunohistochemical, in situ hybridization histochemistry, tract-tracing, and in vivo microdialysis techniques have been used to investigate the participation of the central catecholaminergic (CA) systems in the organization of responses to an acute neurogenic (pain) stress: subcutaneous injection of 0.5 ml of 4% formalin into the hind paw of rats. This single injection produces a local pain that develops immediately after injection and results in four- to fivefold increase in corticosterone and adrenocorticotropic hormone (ACTH) levels in the plasma and a similar increase in norepinephrine levels in the paraventricular nucleus (PVN). Formalin also induced a rapid expression of c-fos and corticotropin-releasing hormones (CRHs) mRNA in the PVN. The possible crossover of the nociceptive spinal afferents to the medullary CA neurons and the crossover of the ascending CA neurons to the PVN were investigated after spinal and medullary surgical hemisections. Hemisection between the spinal cord and the medulla oblongata did not influence formalin-induced elevation of plasma corticosterone and ACTH levels, and strong c-fos immunoreactivity was observed in the medullary CA cell groups and in the PVN bilaterally, as in the sham-operated animals. Hemisection between the medullary CA cell groups and the PVN (unilateral knife cut at the pons-medulla oblongata border) decreased CRH immunoreactivity and CRH gene expression in the PVN ipsilateral to the transection but remained unchanged contralateral to the knife cut. Although, unilateral pontomedullary hemisection reduce TH and CRH immunoreactivity and CRH gene expression in the PVN, it does not influence the high plasma corticosterone and ACTH levels in response to formalin stress. However, acute immobilization stress in hemisected animals is able to induce CRH expression. These findings suggest that in brain stem-hemisected animals, the remaining intact ascending pathways are sufficient to allow normal, or almost normal, activation of the hypothalamopituitary-adrenal axis during stress.