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.

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.

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.

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 (approximately 12 microm) 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.

Characterization of gamma-aminobutyrate type A receptors with atypical coupling between agonist and convulsant binding sites in discrete brain regions.

Gamma-ainobutyric acid type A (GABA(A)) receptor ionophore ligand t-[35S]butylbicyclophosphorothionate ([35S]TBPS) was used in an autoradiographic assay on brain cryostat sections to visualize and characterize atypical GABA-insensitive [35S]TBPS binding previously described in certain recombinant GABA(A) receptors and the cerebellar granule cell layer. Picrotoxinin-sensitive but 1-mM GABA-insensitive [35S]TBPS binding was present in the rat cerebellar granule cell layer, many thalamic nuclei, subiculum and the internal rim of the cerebral cortex, amounting in these regions up to 6% of the basal binding determined in the absence of exogenous GABA. Similar binding properties were detected also in human and chicken brain sections. Like the GABA-sensitive [35S]TBPS binding, GABA-insensitive binding was profoundly decreased by pentobarbital, pregnanolone, loreclezole and Mg2+. The binding was reversible and apparently dependent on Cl- ions. Localization of the GABA-insensitive [35S]TBPS binding was not identical to that of high-affinity [3H]muscimol binding and diazepam-insensitive [3H]Ro 15-4513 binding, two previously established receptor subtype-dependent binding heterogeneities in the rat brain. The present study reveals a component of the GABA-ionophore enriched in the thalamus and cerebellar granule cells, possibly representing poorly desensitized or desensitizing receptors.

Modulation of histamine H3 receptors in the brain of 6-hydroxydopamine-lesioned rats.

Parkinson's disease is a major neurological disorder that primarily affects the nigral dopaminergic cells. Nigral histamine innervation is altered in human postmortem Parkinson's disease brains. However, it is not known if the altered innervation is a consequence of dopamine deficiency. The aim of the present study was to investigate possible changes in the H3 receptor system in a well-characterized model of Parkinson's disease--the 6-hydroxydopamine (6-OHDA) lesioned rats. Histamine immunohistochemistry showed a minor increase of the fibre density index but we did not find any robust increase of histaminergic innervation in the ipsilateral substantia nigra on the lesioned side. In situ hybridization showed equal histidine decarboxylase mRNA expression on both sides in the posterior hypothalamus. H3 receptors were labelled with N-alpha-[3H]-methyl histamine dihydrochloride ([3H] NAMH). Upregulation of binding to H3 receptors was found in the substantia nigra and ventral aspects of striatum on the ipsilateral side. An increase of GTP-gamma-[35S] binding after H3 agonist activation was found in the striatum and substantia nigra on the lesioned side. In situ hybridization of H3 receptor mRNA demonstrated region-specific mRNA expression and an increase of H3 receptor mRNA in ipsilateral striatum. Thus, the histaminergic system is involved in the pathological process after 6-OHDA lesion of the rat brain at least through H3 receptor. On the later stages of the neurotoxic damage, less H3 receptors became functionally active. Increased H3 receptor mRNA expression and binding may, for example, modulate GABAergic neuronal activity in dopamine-depleted striatum.

Peptide GEGLSS-like immunoreactivity in the rat central nervous system.

A rabbit antiserum was raised against the N-terminal fragment peptide, GEGLSS (Gly-Glu-Gly-Leu-Ser-Ser) of bovine neuropeptide AF (NPAF, A18Famide). NPAF is an octadecapeptide isolated from the bovine brain together with neuropeptide FF (NPFF). GEGLSS-like immunoreactivity was localized with immunofluorescence technique in colchicine-treated rats in neuronal cell bodies of the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. A few neurons were also observed in the retrochiasmatic part of the SON. GEGLSS-like immunoreactivity was also localized to nerve terminals of the posterior pituitary. No GEGLSS-ir neuronal cell bodies were observed in the medial hypothalamus, in an area that contains NPFF-ir neurons. GEGLSS immunoreactivity was also seen in the fibers and terminals of nucleus of the solitary tract. We injected a retrograde tracer, fluorogold, to the posterior pituitary gland and visualized GEGLSS-ir neuronal cell bodies double-labeled with the tracer in SON, PVN, and SOR. The pituitary stalk transsection totally abolished the GEGLSS-ir structures from the posterior pituitary. Our results suggest that GEGLSS immunoreactivity in the rat brain has a more limited distribution than NPFF immunoreactivity. GEGLSS immunoreactivity was partially colocalized with arginine-vasopressin and oxytocin in neuronal cell bodies in the SON and PVN. Considering the fact that the known rat NPFF-NPAF precursor does not contain GEGLSS structure, the detected GEGLSS immunoreactivity may be derived from a previously unknown precursor.

Neuropeptide FF-containing efferent projections from the medial hypothalamus of rat: a Phaseolus vulgaris leucoagglutinin study.

Neuropeptide FF (FMRFamide-like peptide, morphine-modulating peptide) is an octapeptide isolated from the bovine brain. There is evidence that neuropeptide FF participates in the modulation of nociceptive mechanisms. Neuropeptide FF acts through its own receptors which are distinct from the opiate receptors. In the rat brain neuropeptide FF is found in two major cell populations. We have studied the efferent connections of the hypothalamic neuropeptide FF-containing cell group, which is located in the medial hypothalamus between the dorsomedial, ventromedial and periventricular hypothalamic nuclei. By using an anterograde tracing method (Phaseolus vulgaris leucoagglutinin) combined with double-staining immunohistochemistry we characterized the connections of this cell group with the limbic system, certain hypothalamic nuclei, periaqueductal gray and with the solitary tract nucleus. In the limbic system, the major targets were the lateral septal nucleus, bed nucleus of stria terminalis and certain subnuclei in the amygdala. These connections suggest that neuropeptide FF may act, in addition to its well-characterized action in the sensory system, in limbic functions. Efferent connections to the periaqueductal gray suggest that neuropeptide FF may modulate the opiate mediated analgesia at this site. Good correlation between our results and receptor autoradiography support the idea that the terminal areas which our results show are target areas of the neuropeptide FF-containing system.

Histamine-immunoreactive neurons in the brain of the teleost Gasterosteus aculeatus L. Correlation with hypothalamic tyrosine hydroxylase- and serotonin-immunoreactive neurons.

The distribution of putative histaminergic neurons in the brain of a teleost, the three-spined stickleback, was investigated by means of immunocytochemistry using specific antibodies against histamine (HA), and conventional microscopy as well as confocal laser scanning microscopy. Histamine-immunoreactive (HAir) neurons form discrete populations ventral to the nucleus of the posterior recess (NRP) and in the nucleus saccus vasculosus (NSV), which belong to the periventricular hypothalamic nuclei. The neuronal somata are subependymally located, and do not possess apical neurites contacting the cerebrospinal fluid. They give rise to both long-range and local axonal projections. The local projections give rise to a field of dense punctate immunoreaction dorsal to the NRP and lateral to the NSV. Long-range projections are comprised of ascending projections to the thalamus, habenula, preoptic area and dorsal telencephalon; and descending projections via the posterior tuberal nucleus, ventrally to the nucleus interpeduncularis, and dorsally into the central gray. HAir neurons occur together with serotoninergic cerebrospinal fluid-contacting (CSFc) neurons in the NRP, and with tyrosine hydroxylase-immunoreactive (THir) neurons in the NSV. Although HAir elements occur together with THir ones in many brain areas, direct contacts between the two neurotransmitter systems are rare. The putative histaminergic neurons in the brain of the three-spined stickleback constitute a very discrete neuronal system, with a major projection area in the dorsal telencephalon in a region which is considered homologous with the dorsal pallium of land vertebrates.

Distribution of histamine-, 5-hydroxytryptamine-, and tyrosine hydroxylase-immunoreactive neurons and nerve fibers in developing rat brain.

Although the general patterns of the developing histaminergic system in the rat brain are known, no comparative studies between the development of the brain histaminergic system and the development of other neuroactive substances have yet been published. Interestingly, separate immunohistochemical studies on the development of the 5-HT system and on the catecholaminergic system in the rat imply common features in the different aminergic systems. Therefore, the spatial distribution of histamine-immunoreactive (HA-ir) neurons and nerve fibers was compared to the distribution of 5-hydroxytryptamine (5-HT)-, and tyrosine hydroxylase-immunoreactive (TH-ir) ones in the developing rat brain between embryonic days 12 (E12) and 20 (E20) by using a double-immunostaining method. The high-pressure liquid chromatography (HPLC) fluorometric method was used for determination of histamine concentration in different brain regions during the same period of development and synthetic oligonucleotide probes complementary to the rat histidine decarboxylase (HDC) to determine the origin of HA in the brain during the development with in situ hybridization. The immunohistochemical results revealed co-localization of HA and 5-HT within a subgroup of cells in the developing raphe nuclei between E14 and E18. From E18 onwards HA immunoreactivity started to gradually disappear from the rhombencephalon, and was totally abolished by E20, while 5-HT-ir cells continued to establish their adult positions. No significant colocalization of HA and TH immunoreactivities was detected. The biochemical results were in agreement with the immunohistochemical ones and confirmed that histamine detected in the early developing brain is authentic. A positive in situ hybridization signal for HDC was detected in a small area in the ventrolateral pons in the same areas as HA- and HDC-ir cell bodies at E16, suggesting that at least some HA may be synthesized locally. These results confirm that HA is one of the first neurotransmitters to appear in the developing brain. In addition, the transient co-localization of HA and 5-HT immunoreactivities and the transient HDC expression at E16 within the developing pontine raphe nuclei may imply an interesting and a more general role for HA in modification of brain development.

Distribution and characterization of neuropeptide FF-like immunoreactivity in the rat nervous system with a monoclonal antibody.

Monoclonal antibodies against neuropeptide FF were produced and characterized. The antibodies are directed and highly specific to neuropeptide FF, and reactivity requires the C-terminal dipeptide of neuropeptide FF (Arg-Phe-NH2). Tissue extracts from bovine spinal cord, rat spinal cord and hypothalamus were analysed by high-pressure liquid chromatography coupled with radioimmunoassay using the characterized monoclonal antibody. Only one immunoreactive peptide was detected and it coeluted with authentic neuropeptide FF. Using this highly specific monoclonal antibody, the distribution of neuropeptide FF-like immunoreactivity was further studied by indirect immunohistochemistry. Immunoreactivity was seen in two major cell groups in the rat brain. The largest cell group was located in the medial hypothalamus between the dorsomedial and ventromedial nuclei. The other one was found in the nucleus of the solitary tract. Fibres immunoreactive for neuropeptide FF were located in the lateral septal nucleus, amygdala, different hypothalamic areas, nucleus of the solitary tract, ventral medulla, trigeminal complex and the dorsal horn of the spinal cord. Spinal and sympathetic ganglia were non-reactive. No neuropeptide FF immunoreactivity was seen in the gut autonomic nervous system or endocrine cells. The results show that neuropeptide FF-like immunoreactivity has a clearly more limited distribution in the nervous system than typical brain-gut peptides.

Histamine-containing nerve fibers innervate human cerebellum.

Histamine is found in nerve cell bodies of the tuberomammillary nucleus in mammalian brain. This nucleus is prominent in human brain. Samples of human cerebelli obtained from neurosurgical operations were examined for the presence of histamine-containing nerve fibers. In all samples, a moderately dense network of histamine-immunoreactive fibers was seen in the molecular layer. These fibers ran parallel to the Purkinje cell layer after traversing it perpendicularly. Numerous fibers were also seen in the granular cell layer. The results suggest that the human cerebellar cortex receives a direct input from histamine-synthesizing hypothalamic neurons, as no other histamine-containing neurons have been found in human brain.

Histaminergic system in the cat hypothalamus with reference to type B monoamine oxidase.

It is known that histamine (HA) and type B monoamine oxidase (MAO-B), an enzyme involved in its metabolism, are present in the posterior hypothalamus, but the sites where MAO-B intervenes in HA metabolism remain uncertain. The present study examined and compared the detailed distribution and morphology of neurons immunoreactive to HA (HA-ir) and MAO-B (MAO-B-ir) in the cat hypothalamus. HA-ir neurons were localized almost exclusively in the posterior hypothalamus with the largest group in the tuberomammillary nucleus and adjacent areas. MAO-B-ir staining was detected in the vast majority of HA-ir neurons, suggesting that the degradation of tele-methylhistamine (t-MHA), the direct metabolite of HA, may occur within these cells. Nevertheless, a few HA-ir cells showed no detectable or very weak MAO-B-ir labeling; a small group of neurons containing MAO-B alone was detected in the area dorsolateral to the caudal part of the arcuate nucleus. Numerous HA-ir axons and terminal-like structures were distributed unevenly in virtually all hypothalamic regions. One of their principal trajectories ascended through the ventrolateral part of the hypothalamus and rostrally formed an axon column, which ascended into the preoptic area and contributed fibers to the diagonal band of Broca and bed nucleus of the stria terminalis. Other HA-ir axons passed laterally, dorsal to the zona incerta or ventrally through a narrow zone dorsal to the optic tract. Numerous long HA-ir axons coursed dorsomedially from the ventrolateral posterior hypothalamus to the dorsal hypothalamic area. Many are oriented vertically to the thalamus in the midline. MAO-B-ir axons and fibers were detectable throughout the hypothalamus and overlapped the areas distributing HA-ir fibers. They were, however, weaker in staining intensity and apparently fewer than the HA-ir fibers. MAO-B-ir glial cells were numerous in all hypothalamic structures rich in HA-ir fibers. These results suggest that the metabolism of t-MHA may also occur within HA terminals and glial cells.

Origin and distribution of neuropeptide-FF-like immunoreactivity in the spinal cord of rats.

Neuropeptide FF, or F8Famide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2; "morphine-modulating peptide"), isolated from bovine brain, is an FMRF-NH2-like peptide with morphine-modulating effects. Neuropeptide FF (NFF) is highly concentrated in spinal cords of various mammalian species. There is evidence that NFF participates in the modulation of nociceptive mechanisms. The present study was aimed at describing the distribution and origin of the neuropeptide-FF-like immunoreactivity (LI) in the rat spinal cord. For distribution studies spinal cord sections from colchicine-treated animals were processed according to the indirect immunofluorescence method. Retrograde fluorescent tracer was injected in the lumbar dorsal spinal cord to study descending NFF-LI-containing spinal pathways. NFF-immunoreactive (ir) cell bodies were detected in the substantia gelatinosa, marginal zone, laminae III, IV, and X at all levels of the spinal cord, and the dorsolateral funiculus, and dorsal gray commissure of the lumbosacral transition zone. NFF-ir fibers and terminals were identified in laminae I-IV and X, dorsolateral funiculus, intermediolateral cell column, dorsal gray commissure, sacral parasympathetic nucleus, and ventral horn. A spinal NFF-LI-containing descending pathway originating in the lamina X neurons was observed, but a supraspinal origin of descending NFF-ir fibers was not identified. According to this study the NFF-LI of the spinal cord is of intrinsic spinal origin. The anatomical distribution supports the concept that the NFF-like peptides have a role in the nociception. They may also be involved in sensory-visceral reflex arcs.

Neurofibrillary tangles and histamine-containing neurons in Alzheimer hypothalamus.

The location of histamine-immunoreactive (IR) cell bodies in normal aged human brain and in cases of Alzheimer's disease (AD) were compared to the distribution of neurofibrillary tangles (NFT). Cryostat sections were fixed with carbodiimide and processed for histamine-immunohistochemistry using the PAP technique. NFT were visualized in the same sections using thioflavin. Histamine-IR cell bodies in human brain were concentrated in the tuberomammillary (TM) nucleus that embodied a major part of the hypothalamus. Although located in similar large profiles and mainly concentrated in the TM area, the numerous hypothalamic NFT in AD were seldom found within the histamine-IR neurons.

Histamine neurons in human hypothalamus: anatomy in normal and Alzheimer diseased brains.

The anatomy of histamine-immunoreactive cell bodies in normal adult human brain was examined in detail. In addition, the distribution of these cells in three cases of Alzheimer's disease was compared to the distribution of neurofibrillary tangles. Histamine-immunoreactive cell bodies were confined to the tuberal and posterior hypothalamus, forming the tuberomammillary nuclear complex. Most of the about 64,000 histamine neurons were large and multipolar. They comprised four distinct parts: (i) a major ventral part corresponding to the classical tuberomammillary nucleus, (ii) a medial part including the supramammillary nucleus and part of the posterior hypothalamic area, (iii) a caudal paramammillary part, and (iv) a minor lateral part. The parts showed some similarity with the subgroups in rat. In human, as compared to rat, the histamine neurons occupy a larger proportion of the hypothalamus. Numerous neurofibrillary tangles were found in the Alzheimer hypothalami, concentrated in the tuberomammillary area. Most of them were of globular type and extracellular, and only a minority were histamine immunoreactive. They may represent remnants of degenerated tuberomammillary neurons.

Central neuronal pathways containing FLFQPQRFamide-like (morphine-modulating) peptides in the rat brain.

Octapeptide FLFQPQRFamide (FMRFamide-like peptide; morphine-modulating peptide), isolated from bovine brain, has some opiate analgesia modulating effects. Octapeptide FLFQPQRFamide-like immunoreactivity is found in high concentrations in the posterior pituitary, hypothalamus, pons-medulla, and dorsal spinal cord. Octapeptide FLFQPQRFamide-immunoreactive neurons of the brain are localized in the medial hypothalamus and in the nucleus of the solitary tract. High densities of octapeptide FLFQPQRFamide-immunoreactive nerve terminals are found in the median eminence, lateral parabrachial nucleus, and nucleus of the solitary tract. By using the retrograde tract tracing method combined with immunohistochemistry, we studied the central pathways interconnecting the octapeptide FLFQPQRFamide-immunoreactive structures. The octapeptide FLFQPQRFamide-immunoreactive neurons of the hypothalamus sent projections bilaterally to the nucleus of the solitary tract. The octapeptide FLFQPQRFamide-immunoreactive neurons of the nucleus of the solitary tract projected to the contralateral side of the same nucleus, to the lateral parabrachial nuclei bilaterally, and to the ipsilateral periambigual region. The results give neuroanatomical evidence of interacerebral pathways containing recently identified FLFQPQRFamide-like peptides, which may belong to a larger family of peptides. These neuroanatomical findings support the previous pharmacological studies, suggesting that the mammalian FMRFamide-like peptides may, in addition to modulatory effects on nociceptive mechanisms, participate in the regulation of blood pressure, feeding behaviour and endocrine functions.

An analysis of histaminergic efferents of the tuberomammillary nucleus to the medial preoptic area and inferior colliculus of the rat.

The efferent projections of the five histaminergic neuronal subgroups in the tuberomammillary nucleus to the medial preoptic area (MPO) and inferior colliculus (IC) were examined by immunocytochemistry with antihistidine decarboxylase (HDC) antibodies combined with retrograde axonal tracing with Fast Blue (FB). The term "E groups" were used for the histaminergic neuronal subgroups. About 10% of the HDC-immunoreactive (HDCI) neurons were retrogradely labeled after FB injection into the MPO. The labeled neurons were not concentrated in any particular area, but were diffusely distributed bilaterally in all the subgroups. About two-thirds of the labeled neurons were observed on the side ipsilateral to the injection site and one-third on the contralateral side. The percentages of labeled neurons (double-labeled neurons/HDCI neurons) in the five subgroups were not significantly different with each other. The percentages in group E1 and E2 were particularly close, while that in group E4 resembled that in group E5. About 4% of the HDCI neurons were retrogradely labeled after the dye injections into the IC, and about half of the labeled neurons were detected on the ipsilateral side. The percentage of the double-labeled neurons in the five groups were not significantly different. Furthermore, those in E1 and E2, and in E4 and E5 were almost identical, respectively, to the situation following injection of FB into the MPO. These results indicate that each subgroup of histaminergic neurons in the tuberomammillary nucleus has similar efferent projections to the MPO and IC.