Pertti Panula

The role of histamine and the tuberomamillary nucleus in the nervous system

The histaminergic system in the brain is a phylogenetically old group of neurons that project to most of the central nervous system. It holds a key position in the regulation of basic body functions, including the sleep–waking cycle, energy and endocrine homeostasis, synaptic plasticity and learning. Four histamine receptors have now been cloned, and three of them are widely distributed in the mammalian brain. Here, we will discuss the localization, biochemistry and physiological functions of the components of the histaminergic system.

Histamine-immunoreactive nerve fibers in the rat brain

A new immunohistochemical method that utilizes carbodiimide as a tissue fixative was applied to study the distribution of histamine-immunoreactive neuronal fibers and terminals in the rat brain. Immunoreactive fibers were observed in almost all major regions of the brain. They were most numerous in the different hypothalamic nuclei. Dense networks of immunoreactive fibers were also seen in the medial septum, nucleus of the diagonal band and ventral tegmental area. A moderate density of fibers was seen throughout the cerebral cortex, in some parts of the olfactory bulb and tubercle, bed nucleus of the stria terminalis, amygdala, basal parts of the hippocampus, inferior and superior colliculi, substantia nigra, lateral and medial parabrachial nucleus, and the nucleus of the solitary tract. Few histamine-immunoreactive fibers were seen in most parts of the caudate putamen, most thalamic nuclei, most pontine and ventral medullary nuclei. Histamine-immunoreactive neuronal cell bodies were found exclusively in the tuberomammillary nucleus, in agreement with previous reports. The results provide evidence for a widespread distribution of histamine-containing nerve fibers and terminals in the rat brain. Although immunohistochemical localization of histamine does not give direct evidence of a functional role of histamine in any brain area, this distribution suggests involvement in functions of the limbic system including the septal nuclei, hypothalamus and amygdala. The relatively dense histamine-immunoreactive fiber networks in the colliculi and dorsal cochlear nucleus indicate that this amine may play a role in visual functions and hearing. The paucity of immunoreactive fibers in the pontine and medullary areas suggests that the caudal projections originating from the tuberomammillary complex are minor ones compared to the major rostral projections. Several fiber projections originating from the tuberomammillary complex could be deduced from serial frontal, sagittal and horizontal sections. They contained fibers that crossed the midline at several levels of the brain. The results provide information on the target areas of the histaminergic neurons and form a basis for the examination of cellular contracts between the histaminergic neurons and other cells.

Comparative anatomy of the histaminergic and other aminergic systems in zebrafish (Danio rerio).

The histaminergic system and its relationships to the other aminergic transmitter systems in the brain of the zebrafish were studied by using confocal microscopy and immunohistochemistry on brain whole‐mounts and sections. All monoaminergic systems displayed extensive, widespread fiber systems that innervated all major brain areas, often in a complementary manner. The ventrocaudal hypothalamus contained all monoamine neurons except noradrenaline cells. Histamine (HA), tyrosine hydroxylase (TH), and serotonin (5‐HT) ‐containing neurons were all found around the posterior recess (PR) of the caudal hypothalamus. TH‐ and 5‐HT‐containing neurons were found in the periventricular cell layer of PR, whereas the HA‐containing neurons were in the surrounding cell layer as a distinct boundary. Histaminergic neurons, which send widespread ascending and descending fibers, were all confined to the ventrocaudal hypothalamus. Histaminergic neurons were medium in size (∼12 μm) with varicose ascending and descending ipsilateral and contralateral fiber projections. Histamine was stored in vesicles in two types of neurons and fibers. A close relationship between HA fibers and serotonergic raphe neurons and noradrenergic locus coeruleus neurons was evident. Putative synaptic contacts were occasionally detected between HA and TH or 5‐HT neurons. These results indicate that reciprocal contacts between monoaminergic systems are abundant and complex. The results also provide evidence of homologies to mammalian systems and allow identification of several previously uncharacterized systems in zebrafish mutants. J. Comp. Neurol. 440:342–377, 2001.

The Orexin/Hypocretin System in Zebrafish Is Connected to the Aminergic and Cholinergic Systems

The orexin/hypocretin (ORX) system is involved in physiological processes such as feeding, energy metabolism, and the control of sleep and wakefulness. The ORX system may drive the aminergic and cholinergic activities that control sleep and wakefulness states because of the ORX fiber projections to the aminergic and cholinergic cell clusters. The biological mechanisms and relevance of the interactions between these neurotransmitter systems are poorly understood. We studied these systems in zebrafish, a model organism in which it is possible to simultaneously study these systems and their interactions. We cloned a zebrafish prepro-ORX gene that encodes for the two functional neuropeptides orexin-A (ORX-A) and orexin-B (ORX-B). The prepro-ORX gene of the zebrafish consisted of one exon in contrast to mammals. The sequence of the ORX-A peptide of the zebrafish was less conserved than the ORX-B peptide compared with other vertebrates. By using in situ hybridization and immunohistochemistry, we found that the organization of the ORX system of zebrafish was similar to the ORX system in mammals, including a hypothalamic cell cluster and widespread fiber projections. The ORX system of the zebrafish showed a unique characteristic with an additional putatively ORX-containing cell group. The ORX system innervated several aminergic nuclei, raphe, locus ceruleus, the mesopontine-like area, dopaminergic clusters, and histaminergic neurons. A reciprocal relationship was found between the ORX system and several aminergic systems. Our results suggest that the architecture of these neurotransmitter systems is conserved in vertebrates and that these neurotransmitter systems in zebrafish may be involved in regulation of states of wakefulness and energy homeostasis by similar mechanisms as those in mammals.

A histamine-containing neuronal system in human brain

A well-organized network of varicose fibers was revealed throughout the frontal and temporal cortex of adult humans with specific antisera against histamine. The densest network of fibers was seen in lamina I, where varicose fibers were seen to run in parallel to the overlying pia mater. Electron microscopic immunohistochemistry revealed histamine-immunostaining in granules in a small number of nerve fibers and varicosities. Hypothalamic samples obtained from autopsy brains of adult humans revealed numerous histamine-immunoreactive nerve cell bodies in the posterior basal hypothalamus in and around the tuberomammillary nucleus. The results suggest that a histaminergic neuronal system reminiscent of that described in rodents is present in human brain.

Histamine in endocrine cells in the stomach

SummaryAntibodies to histamine were used to examine the localization of the amine in cells of the stomach and upper small intestine of a great variety of species, including cartilaginous and bony fish, amphibia, reptiles (lizard), birds (chicken) and a large number of mammals. In all species gastric histamine was localized in endocrine cells (invariably found in the epithelium) and mast cells (usually with an extra-epithelial localization). The endocrine cells were identified as such by immunostaining with antibodies to chromogranin A and the mast cells were identified by toluidine blue staining. Histamine-immunoreactive endocrine cells were found almost exclusively in the acid-producing part of the stomach; only rarely were such cells observed in the pyloric gland area. They were fairly numerous in the gastric mucosa of the two subclasses of fish as well as in the amphibia and reptile species studied. Here, the majority of the histamine-immunoreactive endocrine cells seemed to have contact with the gastric lumen (open type cells) and were located in the surface epithelium (certain fish only) or together with mucous neck cells at the bottom of the pits. In the chicken, histamine-immunoreactive endocrine cells were numerous and located peripherally in the deep compound glands. They were without contact with the lumen (closed type) and had long basal extensions (“paracrine” appearance), running close to the base of the oxyntico-peptic cells. In mammals, the number of histamine-immunoreactive endocrine cells in the stomach varied greatly. They were particularly numerous in the rat and notably few in the dog, monkey and man. In all mammals, the histamine-immunoreactive endocrine cells were of the closed type and located basally in the oxyntic glands. They often had a “paracrine” appearance with long basal processes. Histamine-storing mast cells, finally, were few in both subclasses of fish as well as in the amphibian species and in the lizard. They were fairly numerous in chicken proventriculus (beneath the surface epithelium), few in the oxyntic mucosa of mouse, rat and hamster, moderate in number in hedge-hog, guinea-pig, rabbit, pig and monkey, and numerous in cat, dog and man. In the oxyntic mucosa of the latter three species mast cells sometimes seemed to have an intraepithelial localization which made their distinction from endocrine cells difficult. In newborn cats (1–3 days old) in human foetuses (17–24 weeks gestational age) mast cells were relatively few in the gastric mucosa and the histamine-containing endocrine cells were easier to demonstrate as a consequence. Patients with achlorhydria (and pernicious anemia) or suffering from hypergastrinemia due to gastrinoma had a greatly increased number of histamine-storing endocrine cells in the oxyntic mucosa compared with normal individuals.

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.

Carbodiimide as a tissue fixative in histamine immunohistochemistry and its application in developmental neurobiology.

The object of this study was to develop an immunohistochemical method that could be used to study neuronal histamine, especially in nerve fibers and terminals where most previous methods have not been applicable. Three new antisera were produced in rabbits against conjugated histamine, and the fixative used in conjugation, 1-ethyl-3(3-diamethylaminopropyl)-carbodiimide (EDCDI), was used in tissue fixation and compared to paraformaldehyde. Specificity of the antisera was established with dot-blot tests on nitrocellulose, with blocking controls and affinity-purified antibodies. EDCDI appeared to be superior to paraformaldehyde as a fixative, and histamine-immunoreactive nerve cells were visualized in developing rat brain during late fetal development from embryonal day 12. By the second postnatal week, the distribution of histamine-immunoreactive neurons in rat brain had reached the adult pattern and immunoreactive nerve fibers were seen in many areas. Posterior hypothalamic neurons from newborn rat in vitro showed strong immunoreactivity for histamine and developed long varicose fibers, which covered the culture dish by the end of the fourth week in vitro. Fixation with EDCDI also allowed detection of histamine in gastric enterochromaffin-like cells and mast cells in rat. The results suggest that the histamine-containing neuron system in rat brain develops during the late fetal and early postnatal periods, and that immunoreactive neurons develop long fibers both in vivo and in vitro.

BODIPY-cholesterol: a new tool to visualize sterol trafficking in living cells and organisms.

Analysis of sterol distribution and transport in living cells has been hampered by the lack of bright, photostable fluorescent sterol derivatives that closely resemble cholesterol. In this study, we employed atomistic simulations and experiments to characterize a cholesterol compound with fluorescent boron dipyrromethene difluoride linked to sterol carbon‐24 (BODIPY‐cholesterol). This probe packed in the membrane and behaved similarly to cholesterol both in normal and in cholesterol‐storage disease cells and with trace amounts allowed the visualization of sterol movement in living systems. Upon injection into the yolk sac, BODIPY‐cholesterol did not disturb zebrafish development and was targeted to sterol‐enriched brain regions in live fish. We conclude that this new probe closely mimics the membrane partitioning and trafficking of cholesterol and, because of its excellent fluorescent properties, enables the direct monitoring of sterol movement by time‐lapse imaging using trace amounts of the probe. This is, to our knowledge, the first cholesterol probe that fulfills these prerequisites.

Neuronal location of the bombesin-like immunoreactivity in the central nervous system of the rat

The immunohistochemical distribution of bombesin-like immunoreactivity in the central nervous system of the rat was revealed using a rabbit antibody against [Glu7]bombesin(6-14). In radioimmunoassay, the antibody had minimal cross reactivity with substance P thereby enhancing the significance of histochemical controls proving that the immunoreactivity detected was related to bombesin but not to substance P. Bombesin-immunoreactive neurons were detected in several brain structures including the hypothalamus, interpeduncular nucleus, central grey, dorsolateral tegmental nucleus, dorsal parabrachial nucleus, nucleus of the solitary tract and trigeminal complex. In the spinal cord, intense immunoreactivity was found in the superficial layers of the posterior horn. Since in this area the reaction diminished after rhizotomy the location of the peptide in afferent neurons was considered. In the anterior horn the bombesin-like immunoreactivity located in nerve terminal-like structures was unchanged after rhizotomy suggesting that the cell bodies were located in CNS.