Orexins/hypocretins and aminergic systems.

Orexin/hypocretin neurones in the posterior hypothalamus are mutually connected with noradrenergic, serotonergic, dopaminergic, histaminergic, and cholinergic neurone systems. They activate these targets by direct post-synaptic and indirect pre-synaptic mechanisms and in turn receive inhibitory feedback and excitatory feed forward control. With respect to behavioural state control, orexin/hypocretin neurones are conducting the orchestra of biogenic amines. This review highlights the role of these players in the control of energy administration, sleep-wake architecture, cortical activation, plasticity, and memory functions in health and disease.

Orexins/hypocretins control bistability of hippocampal long-term synaptic plasticity through co-activation of multiple kinases.

AIM: Orexins/hypocretins (OX/Hcrt) are hypothalamic neuropeptides linking sleep-wakefulness, appetite and neuroendocrine control. Their role and mechanisms of action on higher brain functions, such as learning and memory, are not clear. METHODS: We used field recordings of excitatory post-synaptic potentials (fEPSP) in acute mouse brain slice preparations to study the effects of orexins and pharmacological inhibitors of multiple kinases on long-term synaptic plasticity in the hippocampus. RESULTS: Orexin-A (OX-A) but not orexin-B (OX-B) induces a state-dependent long-term potentiation of synaptic transmission (LTP(OX)) at Schaffer collateral-CA1 synapses in hippocampal slices from adult (8- to 12-week-old) mice. In contrast, OX-A applied to slices from juvenile (3- to 4-week-old) animals causes a long-term depression (LTD(OX)) in the same pathway. LTP(OX) is blocked by pharmacological inhibition of orexin receptor-1 (OX1R) and plasticity-related kinases, including serine/threonine- (CaMKII, PKC, PKA, MAPK), lipid- (PI3K), and receptor tyrosine kinases (Trk). Inhibition of OX1R, CaMKII, PKC, PKA and Trk unmasks LTD(OX) in adult animals. CONCLUSION: Orexins control not only the bistability of arousal states and threshold for appetitive behaviours but, in an age- and kinase-dependent manner, also bidirectional long-term synaptic plasticity in the hippocampus, providing a possible link between behavioural state and memory functions.

Dopaminergic-adrenergic interactions in the wake promoting mechanism of modafinil.

Adrenergic signaling regulates the timing of sleep states and sleep state-dependent changes in muscle tone. Recent studies indicate a possible role for noradrenergic transmission in the wake-promoting action of modafinil, a widely used agent for the treatment of excessive sleepiness. We now report that noradrenergic projections from the locus coeruleus to the forebrain are not necessary for the wake-promoting action of modafinil. The efficacy of modafinil was maintained after treatment of C57BL/6 mice with N-(2-chloroethyl)-N-ethyl 2-bromobenzylamine (DSP-4), which eliminates all noradrenaline transporter-bearing forebrain noradrenergic projections. However, the necessity for adrenergic receptors in the wake-promoting action of modafinil was demonstrated by the observation that the adrenergic antagonist terazosin suppressed the response to modafinil in DSP-4 treated mice. The wake-promoting efficacy of modafinil was also blunted by the dopamine autoreceptor agonist quinpirole. These findings implicate non-noradrenergic, dopamine-dependent adrenergic signaling in the wake-promoting mechanism of modafinil. The anatomical specificity of these dopaminergic-adrenergic interactions, which are present in forebrain areas that regulate sleep timing but not in brain stem areas that regulate sleep state-dependent changes in muscle tone, may explain why modafinil effectively treats excessive daytime sleepiness in narcolepsy but fails to prevent the loss of muscle tone that occurs in narcoleptic patients during cataplexy.

Orexins/hypocretins cause sharp wave- and theta-related synaptic plasticity in the hippocampus via glutamatergic, gabaergic, noradrenergic, and cholinergic signaling.

Orexins (OX), also called hypocretins, are bioactive peptides secreted from glucose-sensitive neurons in the lateral hypothalamus linking appetite, arousal and neuroendocrine-autonomic control. Here, OX-A was found to cause a slow-onset long-term potentiation of synaptic transmission (LTPOX) in the hippocampus of young adult mice. LTPOX was induced at Schaffer collateral-CA1 but not mossy fiber-CA3 synapses, and required transient sharp wave-concurrent population field-burst activity generated by the autoassociative CA3 network. Exogenous long theta-frequency stimulation of Schaffer collateral axons erased LTPOX in intact hippocampal slices but not mini slices devoid of the CA3 region. Pharmacological analysis revealed that LTPOX requires co-activation of ionotropic and metabotropic glutamatergic, GABAergic, as well as noradrenergic and cholinergic receptors. Together these data indicate that OX-A induces a state-dependent metaplasticity in the CA1 region associated with sharp-wave and theta rhythm activity as well as glutamatergic, GABAergic, aminergic, and cholinergic transmission. Thus, orexins not only regulate arousal threshold and body weight but also threshold and weight of synaptic connectivity, providing a molecular prerequisite for homeostatic and behavioral state-dependent control of neuronal plasticity and presumably memory functions.

AMPA receptor properties and coexpression with sodium-calcium exchangers in rat hypothalamic neurons.

The histaminergic tuberomamillary (TM) nucleus, a center for the regulation of wakefulness, is excited by glutamatergic, aminergic and peptidergic inputs. AMPA receptor properties in relation to their expression were investigated in acutely isolated TM neurons with the help of whole-cell patch-clamp recordings combined with single-cell RT-PCR. The mRNAs encoding for the AMPA receptor GluR2 (100% of the neurons) and GluR1 (75%) were the most frequently detected, followed by the mRNA for GluR4 (56%), whereas GluR3 cDNA amplification did not yield a PCR product in any neuron. Flip splice variants prevailed over flop, in keeping with a strong glutamate-response potentiation by cyclothiazide. The expression pattern of AMPA subunits in their two splice variants was correlated with the different subtypes of Na+/Ca2+ (NCX) and Na+/Ca2+/K+ (NCKX) exchangers: glutamate receptor subunits GluR1-4 displayed no coordinated pattern with NCX. However, NCKX2 mRNA occurred only in TM cells with a fast desensitizing glutamate response, where it was coexpressed with the GluR4 subunit in the flop splice variant. NCKX3 mRNA was detected in neurons with fast or slow desensitization of glutamate responses. AMPA receptors in TM neurons were Ca2+-impermeable. As reverse Na+/Ca2+ exchange contributes to the immediate rise in intracellular calcium resulting from glutamate receptor activation, we suggest that the coordinated expression of NCKX2 with the fast desensitizing AMPA receptor-type reflects either a receptor-exchanger coupling or separate mechanisms for maintaining calcium homeostasis in neurons with fast or slow glutamate responses.

Co-ordinated expression of 5-HT2C receptors with the NCX1 Na+/Ca2+ exchanger in histaminergic neurones.

The different roles of Na+/Ca2+ (NCX) exchangers and Na+/Ca2+/K+ (NCKX) exchangers in regulation of the ionic homeostasis in neurones are poorly understood. We have previously shown that serotonin excites histaminergic tuberomamillary (TM) neurones by activation of 5-HT2C-receptors and Na+/Ca2+ exchange. With the help of single-cell RT-PCR (sc-RT-PCR) we have now determined the coexpression pattern of different subtypes of NCX and NCKX with serotonin receptors. The majority of TM neurones express NCX1, NCX2 and NCKX3. Serotonin 2C receptor-mRNA was detected in 70% while 5-HT2A mRNA was found in only 10% of TM neurones. In all neurones expressing the 5-HT2C receptor NCX1-mRNA was present. Double immunostaining revealed the presence of the NCX1 protein in histidine decarboxylase-positive neurones. In the majority of TM neurones one or two out of five isoforms, NCX1.4, NCX1.5, NCX1.7, NCX1.14, NCX1.15, were detected by cDNA sequencing and/or by restriction analysis. The alternative splicing region is important for the Ca2+ sensitivity and presumably for the modulation of NCX1 function by second messengers. We conclude that several exchanger-subtypes can be coexpressed in single neurones and that TM cells are heterogeneous with respect to their calcium homeostasis regulation.

Taurine-induced long-lasting enhancement of synaptic transmission in mice: role of transporters.

Taurine, a major osmolyte in the brain evokes a long-lasting enhancement (LLETAU) of synaptic transmission in hippocampal and cortico-striatal slices. Hippocampal LLETAU was abolished by the GABA uptake blocker nipecotic acid (NPA) but not by the taurine-uptake inhibitor guanidinoethyl sulphonate (GES). Striatal LLETAU was sensitive to GES but not to NPA. Semiquantitative PCR analysis and immunohistochemistry revealed that taurine transporter expression is significantly higher in the striatum than in the hippocampus. Taurine transporter-deficient mice displayed very low taurine levels in both structures and a low ability to develop LLETAU in the striatum, but not in the hippocampus. The different mechanisms of taurine-induced synaptic plasticity may reflect the different vulnerabilities of these brain regions under pathological conditions that are accompanied by osmotic changes such as hepatic encephalopathy.

Increased brain histamine levels in Parkinson's disease but not in multiple system atrophy.

We investigated histamine concentration in post-mortem brain samples of patients with Parkinson's disease (PD, n = 24), multiple system atrophy (MSA, n = 8) and age-matched controls (n = 27). Histamine concentrations were significantly increased in the putamen (to 159% of the control mean), substantia nigra pars compacta (to 201%), internal globus pallidus (to 234%) and external globus pallidus (to 200%), i.e. in areas which play a crucial role in the motor behaviour and which show typical functional alterations in PD. In MSA no significant differences were seen. Tele-methylhistamine (histamine metabolite) concentrations were unchanged in PD. These results indicate that histamine concentration, but not its metabolism is increased in PD, but not in MSA. This finding may have implications in developing new drug therapies for PD and in differential diagnosis between PD and MSA.

Orexin/hypocretin excites the histaminergic neurons of the tuberomammillary nucleus.

The hypothalamic orexin (hypocretin) neuropeptides are associated with the regulation of sleep and feeding, and disturbances in orexinergic neurotransmission lead to a narcoleptic phenotype. Histamine has also been shown to play a role in the regulation of sleep and feeding. Therefore, we studied the relationship between the orexin and histamine systems of the CNS using electrophysiology, immunocytochemistry, and the reverse transcriptase (RT)-PCR method. Both orexin-A and orexin-B depolarized the histaminergic tuberomammillary neurons and increased their firing rate via an action on postsynaptic receptors. The depolarization was associated with a small decrease in input resistance and was likely caused by activation of both the electrogenic Na(+)/Ca(2+) exchanger and a Ca(2+) current. In a single-cell RT-PCR study using primers for the two orexin receptors, we found that most tuberomammillary neurons express both receptors and that the expression of the orexin-2 receptor is stronger than that of the orexin-1 receptor. Immunocytochemical studies show that the histamine and orexin neurons are often located very close to each other. The contacts between these two types of neurons seem to be reciprocal, because the orexin neurons are heavily innervated by histaminergic axons. These results suggest a functional connection between the two populations of hypothalamic neurons and that they may cooperate in the regulation of rapid-eye-movement sleep and feeding.

The mechanism of spontaneous firing in histamine neurons.

Histaminergic neurons project to virtually the whole central nervous system and display regular firing related to behavioral state. Electrophysiological studies of histaminergic neurons show that these neurons fire in a beating pacemaker pattern, which is intrinsic to individual neurons. Onset of an action potential occurs as the result of a slow depolarizing potential, which consists of voltage dependent calcium current(s) and non-inactivating sodium current. The calcium component is a voltage-dependent current activated by the return to threshold following the afterhyperpolarization (AHP) while the sodium current appears to be persistent. The action potential is followed by an AHP, which limits firing rate. The AHP is due to two potassium currents, one voltage-, the other calcium-dependent; it determines the amount of voltage-dependent currents available for activation. We show original results indicating that calcium current can be activated during AHP-like ramps and that the amount of calcium current near threshold is strongly dependent on the membrane potential and on the size of the AHP. The amount of calcium entering during the action potential will determine the duration of the AHP and thus, the firing rate.

Glycine receptor mediated responses in rat histaminergic neurons.

Patch-clamp whole-cell recordings were made in the hypothalamic tuberomammillary (TM) nucleus from isolated histaminergic neurons, identified by their expression of histidine decarboxylase. We compared strychnine-sensitive glycine-mediated currents with maximal currents activated by gamma-Aminobutyric acid (GABA, 0.5 mM) which were blocked by gabazine. The maximal glycine response (1 mM) in histaminergic cells with larger somata (25 microm) was about half of the maximal GABA response whereas in the cells with a smaller soma size (19.5 microm) the glycine response was absent or very small. We conclude that histaminergic cells are heterogeneous with respect to their sensitivity to glycine and this correlates with their size.

Serotonin excites tuberomammillary neurons by activation of Na(+)/Ca(2+)-exchange.

We have studied the effects of serotonin on the histaminergic neurons in the hypothalamic tuberomammillary nucleus. Intracellular recordings of the membrane potential were made with sharp electrodes from superfused rat hypothalamic slices. We found that serotonin increased the firing rate of the neurons to 224% of the control rate and depolarized them dose-dependently. Insensitivity to tetrodotoxin indicated a postsynaptic effect, which was unrelated to any conductance change. The involved receptor appeared to be a 5-HT2C receptor. The depolarization was strongly dependent on temperature and replacement of extracellular Na(+) with Li(+) or with N-methyl-D-glucamine suppressed the depolarization. Pretreatment with Ni(2+), 2',4'-dichlorobenzamil or KB-R7943 strongly attenuated the effect. These features indicate that the depolarization is the result of activation of an electrogenic Na(+)/Ca(2+)-exchanger which leads to an net inward current. These results support the view that the Na(+)/Ca(2+)-exchanger can play a role in determining the excitability of neurons. The results also provide a functional connection between two transmitter systems, the histaminergic and serotonergic, which modulate many physiological functions in the brain.

Orexin A excites serotonergic neurons in the dorsal raphe nucleus of the rat.

Orexin A (10-300 nM) strongly excited dorsal raphe serotonergic neurons maintained in vitro. The depolarization persisted in the presence of tetrodotoxin (TTX, 0.5 microM) and was associated with an increase in input resistance. These results have relevance in the context of food intake regulation and the disease, narcolepsy.

Opposite modulation of histaminergic neurons by nociceptin and morphine.

We have studied the effects of nociceptin/orphanin FQ on the histaminergic neurons in the tuberomammillary (TM) nucleus and compared them with the actions of opioid agonists. Intracellular recordings of the membrane potential were made with sharp electrodes from superfused rat hypothalamic slices. Nociceptin strongly inhibited the firing of the TM neurons. In the concentration range 10-300 nM, nociceptin hyperpolarized the neurons in a dose-dependent and reversible manner. Insensitivity to tetrodotoxin indicated a postsynaptic effect which was associated with decreased input resistance. Voltage-current plots suggested the involvement of a potassium conductance which was highly sensitive to Ba(2+) and decreased by Cs(+), in keeping with the activation of an inwardly rectifying potassium channel. Morphine (20-100 microM) depolarized the TM neurons and increased their firing, and this effect was blocked by tetrodotoxin. Dynorphin A(1-13) at 100-300 nM did not affect the TM neurons. Nociceptin and morphine modulate the activity of the TM neurons, and most likely histamine release, in opposite ways. Histamine has an antinociceptive effect in the brain and may be involved in opioid-induced analgesia. Nociceptin might therefore influence pain transmission by inhibiting opioid-induced histamine release from the TM nucleus and also modulate other physiological mechanisms which have been ascribed to the histaminergic system.

Lack of histamine synthesis and down-regulation of H1 and H2 receptor mRNA levels by dexamethasone in cerebral endothelial cells.

The purpose of this work was to determine whether cerebral endothelial cells have the capacity to synthesize histamine or to express mRNA of receptors that specifically respond to available free histamine. The histamine concentrations and the expression of L-histidine decarboxylase (HDC) and histamine H1 and H2 receptor mRNA, both in adult rat brain and in cultured immortalized RBE4 cerebral endothelial cells, were investigated. In this study endothelial cells were devoid of any kind of detectable histamine production, both in vivo and in the immortalized RBE4 cells in culture. Both the immunostainings for histamine and the in situ hybridizations for HDC were negative, as well as histamine determinations by HPLC, indicating that endothelial cells do not possess the capacity to produce histamine. Also, glucocorticoid (dexamethasone) treatment failed to induce histamine production in the cultured cells. Although the cerebral endothelial cells lack histamine production, a nonsaturable uptake in RBE4 cells is demonstrated. The internalized histamine is detected both in the cytoplasm and in the nucleus, which could indicate a role for histamine as an intracellular messenger. Histamine H1 and H2 receptor mRNA was expressed in RBE4 cells, and glucocorticoid treatment down-regulated the mRNA levels of both H1 and H2 receptors. This mechanism may be involved in glucocorticoid-mediated effects on cerebrovascular permeability and brain edema.

Major changes in the brain histamine system of the ground squirrel Citellus lateralis during hibernation.

Hibernation in mammals such as the rodent hibernator Citellus lateralis is a physiological state in which CNS activity is endogenously maintained at a very low, but functionally responsive, level. The neurotransmitter histamine is involved in the regulation of diurnal rhythms and body temperature in nonhibernators and, therefore, could likely play an important role in maintaining the hibernating state. In this study, we show that histamine neuronal systems undergo major changes during hibernation that are consistent with such a role. Immunohistochemical mapping of histaminergic fibers in the brains of hibernating and nonhibernating golden-mantled ground squirrels (C. lateralis) showed a clear increase in fiber density during the hibernating state. The tissue levels of histamine and its first metabolite tele-methylhistamine were also elevated throughout the brain of hibernating animals, suggesting an increase in histamine turnover during hibernation, which occurs without an increase in histidine decarboxylase mRNA expression. This hibernation-related apparent augmentation of histaminergic neurotransmission was particularly evident in the hypothalamus and hippocampus, areas of importance to the control of the hibernating state, in which tele-methylhistamine levels were increased more than threefold. These changes in the histamine neuronal system differ from those reported for the metabolic pattern in other monoaminergic systems during hibernation, which generally indicate a decrease in turnover. Our results suggest that the influence of histamine neuronal systems may be important in controlling CNS activity during hibernation.

Postnatal expression of H1-receptor mRNA in the rat brain: correlation to L-histidine decarboxylase expression and local upregulation in limbic seizures.

Histamine is implicated in the regulation of brain functions through three distinct receptors. Endogenous histamine in the brain is derived from mast cells and neurons, but the importance of these two pools during early postnatal development is still unknown. The expression of histamine H1-receptor in the rat brain was examined using in situ hybridization during postnatal development and in adults. For comparison, the expression of L-histidine decarboxylase (HDC) in the two pools was revealed. H1-receptor was evenly expressed throughout the brain on the first postnatal days, but resembled the adult, uneven pattern already on postnatal day 5 (P5). HDC was expressed in both mast cells and tuberomammillary neurons from birth until P5, after which the mast cell expression was no more detectable. In adult rat brain, high or moderate levels of H1-receptor expression were found in the hippocampus, zona incerta, medial amygdaloid nucleus and reticular thalamic nucleus. In most areas of the adult brain the expression of H1-receptor mRNA correlates well with binding data and histaminergic innervation. A notable exception is the hypothalamus, with high fibre density but moderate or low H1-receptor expression. Systemic kainic acid administration induced increased expression of H1-receptor mRNA in the caudate-putamen and dentate gyrus, whereas no change was seen in the hippocampal subfields CA1-CA3 or in the entorhinal cortex 6 h after kainic acid injections. This significant increase supports the concept that histaminergic transmission, through H1-receptor, is involved in the regulation of seizure activity in the brain.

Neuronal histamine deficit in Alzheimer's disease.

Histamine is known to be a neurotransmitter in the brain, but it has not been clearly implicated in major diseases. All histaminergic neurons reside in the posterior hypothalamus and innervate most brain areas, which is compatible with the concept that histamine is involved in general central regulatory mechanisms. A sensitive high-performance liquid chromatographic fluorimetric method was used to measure histamine contents in post mortem Alzheimer brains and age-matched controls. The cellular storage sites and distribution of histaminergic nerve fibers were examined with a specific immunohistochemical method. The histamine content was significantly reduced in the hypothalamus (42% of control value), hippocampus (43%) and temporal cortex (53%) of Alzheimer brains. Differences in other cortical areas, putamen and substantia nigra were not significant. Histamine-containing nerve fibers were found in the hippocampus, parahippocampal gyrus and subiculum of both Alzheimer brains and controls. No histamine-containing mast cells were seen in these temporal structures. Histamine in the human temporal lobe is stored in nerve fibers originating from the posterior hypothalamus, and not in mast cells. Decrease in brain histamine may contribute to the cognitive decline in Alzheimer's disease directly or through the cholinergic system. Development of drugs that penetrate the blood brain barrier and increase histaminergic activity might be beneficial in Alzheimer's disease.

Development of the histaminergic neurons and expression of histidine decarboxylase mRNA in the zebrafish brain in the absence of all peripheral histaminergic systems.

The histamine-storing neural system in adult and developing zebrafish (Danio rerio) was studied with immunocytochemical and chromatographical methods. Furthermore, the gene for histidine decarboxylase was partially cloned and its expression mapped with in situ hybridization. The histamine-storing neurons were only seen in the caudal hypothalamus, around the posterior recess of the diencephalic ventricle. Almost all parts of the brain, except the cerebellum, contained at least some histamine-immunoreactive fibres. The ascending projections had the rostral part of the dorsal telencephalon as a major target. Descending projections terminated in the torus semicircularis, central grey and inferior olive. A prominent innervation of the optic tectum, which has not been reported in other fish, was seen. The in situ hybridization gave a strong signal in cells with the same anatomical position as the histamine-immunoreactive neurons. The first histamine-immunoreactive neurons appeared in the ventral hypothalamus at about 85 h post-fertilization, and at 90 h, immunoreactive fibres terminated in the dorsal telencephalon. The embryonic histamine production described in mammals was lacking in this species. Both immunocytochemical and chromatographical studies indicated that histamine is absent in all other parts of the zebrafish body, and no specific hybridization was seen in any other part of the fish than the hypothalamus. The zebrafish could therefore be a very useful model for pharmacological in vivo studies of the histaminergic system of the brain, since the powerful peripheral actions of histamine should be lacking in this species.

Nitric oxide synthase in the pharynx of the planarian Dugesia tigrina.

The distribution of the nicotinamide adenine dinucleotide phosphate-(NADPH) diaphorase reaction, an indicator of nitric oxide synthase activity, was studied in the freshwater planarian Dugesia tigrina (Platyhelminthes). The reaction was restricted to the pharynx, where the inner epithelium was intensely stained and the outer epithelium moderately stained. Neurons that innervated the pharynx were also stained. The enzyme activity was studied by high pressure liquid chromatographic quantitation of the formed citrulline. The presumed nitric oxide synthase was dependent on NADPH, whereas no dependency on Ca2+ and calmodulin could be detected. Tetrahydrobiopterin increased the activity about fivefold to 218.2+/-24.9 fmol/mg protein per min. Nomega-nitro-l-arginine depressed the enzyme activity by about 80%. The results indicate that nitric oxide has a role in the feeding behavior of planarians.