M. Beatrice Passani

Adenosine decreases aspartate and glutamate release from rat hippocampal slices

The effect of adenosine and related compounds on the release of endogenous aspartate and glutamate from isolated, superfused rat hippocampal slices was studied at rest and during electrical stimulation of the stratum radiatum in the CA3/CA2 region, using a sensitive mass-spectrometric technique. Evoked extracellular potentials were recorded from the CA1 region. Adenosine, at 3 X 10(-4) M concentration, inhibited the stimulation-evoked potentials and prevented the stimulation-induced release of aspartate and glutamate. Similarly, 1-phenylisopropyladenosine (10(-6) M) and cyclohexyladenosine (10(-6) M) depressed both electrical and neurochemical responses to stimulation of the stratum radiatum. 8-Phenyltheophylline (5 X 10(-6) M) increased the release of aspartate and glutamate and antagonized the cyclohexyladenosine-induced inhibition of amino acid release. Our results support the hypothesis that adenosine modulates the electrophysiological responses to stimulation of stratum radiatum through a reduction of the release of the excitatory amino acids aspartate and glutamate.

Activation of histaminergic H3 receptors in the rat basolateral amygdala improves expression of fear memory and enhances acetylcholine release

The basolateral amygdala (BLA) is involved in learning that certain environmental cues predict threatening events. Several studies have shown that manipulation of neurotransmission within the BLA affects the expression of memory after fear conditioning. We previously demonstrated that blockade of histaminergic H3 receptors decreased spontaneous release of acetylcholine (ACh) from the BLA of freely moving rats, and impaired retention of fear memory. In the present study, we examined the effect of activating H3 receptors within the BLA on both ACh release and expression of fear memory. Using the microdialysis technique in freely moving rats, we found that the histaminergic H3 agonists R‐α‐methylhistamine (RAMH) and immepip, directly administered into the BLA, augmented spontaneous release of ACh in a similar manner. Levels of ACh returned to baseline on perfusion with control medium. Rats receiving intra‐BLA, bilateral injections of the H3 agonists at doses similar to those enhancing ACh spontaneous release, immediately after contextual fear conditioning, showed stronger memory for the context–footshock association, as demonstrated by longer freezing assessed at retention testing performed 72 h later. Post‐training, bilateral injections of 15 ng oxotremorine also had a similar effect on memory retention, supporting the involvement of the cholinergic system. Thus, our results further support a physiological role for synaptically released histamine, that in addition to affecting cholinergic transmission in the amygdala, modulates consolidation of fear memories

Histamine H3 receptor-mediated impairment of contextual fear conditioning and in-vivo inhibition of cholinergic transmission in the rat basolateral amygdala

We investigated the effects of agents acting at histamine receptors on both, spontaneous release of ACh from the basolateral amygdala (BLA) of freely moving rats, and fear conditioning. Extensive evidence suggests that the effects of histamine on cognition might be explained by the modulation of cholinergic systems. Using the microdialysis technique in freely moving rats, we demonstrated that perfusion of the BLA with histaminergic compounds modulates the spontaneous release of ACh. The addition of 100 mm KCl to the perfusion medium strongly stimulated ACh release, whereas, 0.5 µm tetrodotoxin (TTX) inhibited spontaneous ACh release by more than 50%. Histaminergic H3 antagonists (ciproxifan, clobenpropit and thioperamide), directly administered to the BLA, decreased ACh spontaneous release, an effect fully antagonized by the simultaneous perfusion of the BLA with cimetidine, an H2 antagonist. Local administration of cimetidine alone increased ACh spontaneous release slightly, but significantly. Conversely, the administration of H1 antagonists failed to alter ACh spontaneous release. Rats receiving intra‐BLA, bilateral injections of the H3 antagonists at doses similar to those inhibiting ACh spontaneous release, immediately after contextual fear conditioning, showed memory consolidation impairment of contextual fear conditioning. Post‐training, bilateral injections of 50 µg scopolamine also had an adverse effect on memory retention. These observations provide the first evidence that histamine receptors are involved in the modulation of cholinergic tone in the amygdala and in the consolidation of fear conditioning.

Histamine pharmacology and new CNS drug targets.

During the last decade, the identification of a number of novel drug targets led to the development of promising new compounds which are currently under evaluation for their therapeutic prospective in CNS related disorders. Besides the established pleiotropic regulatory functions in the periphery, the interest in the potential homeostatic role of histamine in the brain was revived following the identification of H3 and H4 receptors some years ago. Complementing classical CNS pharmacology, the development of selective histamine receptor agonists, antagonists, and inverse agonists provides the lead for the potential exploitation of the histaminergic system in the treatment of brain pathologies. Although no CNS disease entity has been associated directly to brain histamine dysfunction until now, the H3 receptor is recognized as a drug target for neuropathic pain, sleep‐wake disorders, including narcolepsy, and cognitive impairment associated with attention deficit hyperactivity disorder, schizophrenia, Alzheimer’s, or Parkinsons disease, while the first H3 receptor ligands have already entered phase I–III clinical trials. Interestingly, the localization of the immunomodulatory H4 receptor in the nervous system exposes attractive perspectives for the therapeutic exploitation of this new drug target in neuroimmunopharmacology. This review focuses on a concise presentation of the current “translational research” approach that exploits the latest advances in histamine pharmacology for the development of beneficial drug targets for the treatment of neuronal disorders, such as neuropathic pain, cognitive, and sleep‐wake pathologies. Furthermore, the role of the brain histaminergic system(s) in neuroprotection and neuroimmunology/inflammation remains a challenging research area that is currently under consideration.

l‐Dopa activates histaminergic neurons

Non‐technical summary  L‐Dopa is an effective medication for Parkinsons disease (PD), but loses efficiency with time. This can be delayed by treatment with histamine antagonists. We show histaminergic neurons in the hypothalamus controlling wakefulness are excited by L‐Dopa, which they can take up and transform to dopamine. They innervate the whole brain, in particular the striatum which is deficient of dopamine in PD. We revealed mechanisms of excitation of histaminergic neurons by L‐Dopa and dopamine opening new avenues for the treatment of PD and sleep disorders.

Effect of the selective 5-HT1A receptor antagonist WAY 100635 on the inhibition of e.p.s.ps produced by 5-HT in the CA1 region of rat hippocampal slices

The actions of N‐(2‐(‐4(2‐methoxyphenyl)‐1‐piperazinyl)ethyl)‐N‐(2‐pyridinyl) cyclohexane carboxamide (WAY 100635), a novel and selective 5‐hydroxytryptamine1A (5‐HT1A) antagonist, on excitatory postsynaptic potentials (e.p.s.ps) were investigated by use of intracellular recordings in pyramidal cells of the CA1 region of rat hippocampal slices. WAY 100635 (10 nM) did not affect any of the investigated parameters of cell excitability such as membrane potential, total input resistance (Rin), firing threshold, action potential amplitude, action potential frequency adaptation, and slow afterhyperpolarization (sAHP) which follows repetitive firing of action potentials. WAY 100635 did not have any effect on either the slope or the amplitude of e.p.s.ps evoked by stimulation of the CA1 stratum radiatum. Bath application of either 5‐hydroxytryptamine (5‐HT, 10–30 μM) or 5‐carboxamidotryptamine (5‐CT, 300 nM) hyperpolarized the membrane potential (ΔVm=−4.1±0.9 and −6.0±0.9 mV, respectively), and reduced Rin (−25±8% and −18±1%, respectively). 5‐HT blocked the action potential frequency adaptation and significantly reduced the amplitude of the sAHP that follows repetitive firing of action potentials. 5‐HT significantly decreased the amplitude of evoked e.p.s.ps (−14±6%). This effect was greater in the presence of the GABAA receptor antagonist bicuculline (10 μM, −45±12%) and was mimicked by 5‐CT (−49±5%). Both AMPA and NMDA components of e.p.s.ps were significantly reduced in amplitude by 5–HT (−38±8%, n=6, and −29±12%, n=3, respectively; P<0.05). WAY 100635 fully antagonized the hyperpolarization, the reduction of Rin, and the decrease in amplitude of e.p.s.ps elicited by 5‐HT, while it did not affect the action of 5‐HT on the action potential frequency adaptation. In the presence of WAY 100635, 5‐HT elicited a depolarization which was blocked by 10–30 μM RS 23597‐190, a selective 5‐HT4 receptor antagonist. Our data demonstrate that WAY 100635 is devoid of direct effects on CA1 pyramidal cell excitability and on evoked e.p.s.ps, while it fully antagonizes the effects of 5‐HT on excitatory synaptic transmission and on hyperpolarization, without affecting the 5‐HT4 receptor‐mediated response. Since WAY 100635 selectively antagonizes 5‐HT1A receptor‐mediated actions of 5‐HT, our data also demonstrate that the inhibitory action of 5‐HT on excitatory synaptic transmission in CA1 is mediated by 5‐HT1A receptors.

The H3 receptor protean agonist proxyfan enhances the expression of fear memory in the rat

Consolidation of fear memory requires neural changes to occur in the basolateral amygdala (BLA), including modulation of histaminergic neurotransmission. We previously demonstrated that local blockade or activation of histamine H3 receptors in the BLA impaired or ameliorated, respectively, retention of fear memory. The histamine H3 receptor is a G-protein-coupled receptor (GPCR) displaying high constitutive activity that regulates histamine neurons in the brain. Proxyfan is a high-affinity histamine H3 receptor protean agonist exhibiting the full spectrum of pharmacological activities, from full agonist to full inverse agonist depending on the competition between constitutively active and quiescent H3 receptors in a given tissue or brain region. Therefore, protean agonists are powerful tools to investigate receptor conformation and may be useful in designing specific compounds selective for the various receptor conformations. In the present study we examined the effect of post-training, systemic or intra-BLA injections of proxyfan on contextual fear memory. Rats receiving intra-BLA, bilateral injections of 1.66 ng proxyfan immediately after fear conditioning showed stronger memory for the context-footshock association, as demonstrated by longer freezing assessed at retention performed 72 hr later compared to controls. Comparable results were obtained when doses as low as 0.04 mg/kg of proxyfan were injected systemically. Hence, our results suggest that proxyfan behaves as an H3 receptor agonist with a low level of constitutive activity of the H3 receptor in the rat BLA.

Differential effect of cannabinoid agonists and endocannabinoids on histamine release from distinct regions of the rat brain

Cannabinoids exert complex actions on neurotransmitter systems involved in cognition, locomotion, appetite, but no information was available so far on the interactions between the endocannabinoid system and histaminergic neurons that command several, similar behavioural states and memory. In this study, we investigated the effect of cannabimimetic compounds on histamine release using the microdialysis technique in the brain of freely moving rats. We found that systemic administration of the cannabinoid receptors 1 (CB1‐r) agonist arachidonyl‐2′chloroethylamide/N‐(2chloroethyl)‐5Z,8Z,11Z,14Z‐eicosatetraenamide (ACEA; 3 mg/kg) increased histamine release from the posterior hypothalamus, where the histaminergic tuberomamillary nuclei (TMN) are located. Local infusions of ACEA (150 nm) or R(+)‐methanandamide (mAEA; 1 µm), another CB1‐r agonist, in the TMN augmented histamine release from the TMN, as well as from two histaminergic projection areas, the nucleus basalis magnocellularis and the dorsal striatum. When the endocannabinoid uptake inhibitor AM404 was infused into the TMN, however, increased histamine release was observed only in the TMN. The cannabinoid‐induced effects on histamine release were blocked by co‐administrations with the CB1‐r antagonist AM251. Using double‐immunofluorescence labelling and confocal laser‐scanning microscopy, CB1‐r immunostaining was found in the hypothalamus, but was not localized onto histaminergic cells. The modulatory effect of cannabimimetic compounds on histamine release apparently did not involve inhibition of γ‐aminobutyric acid (GABA)ergic neurotransmission, which provides the main inhibitory input to the histaminergic neurons in the hypothalamus, as local infusions of ACEA did not modify GABA release from the TMN. These profound effects of cannabinoids on histaminergic neurotransmission may partially underlie some of the behavioural changes observed following exposure to cannabinoid‐based drugs.

8-Phenyltheophylline potentiates the electrical activity evoked in hippocampal slices.

The effects of 8-phenyltheophylline (8PT) were studied on coronal slices of rat hippocampus. 8PT was more potent than theophylline in enhancing the pyramidal cell responses evoked by stimulation of the stratum radiatum. 8PT dose dependently antagonized the depression of the excitatory postsynaptic potentials induced by cyclohexyladenosine and did not change the amplitude of antidromically evoked responses of pyramidal cells. These findings suggest that 8PT is a potentially powerful tool for studying adenosine neuromodulation in the CNS.

Brain histamine depletion enhances the behavioural sequences complexity of mice tested in the open-field: Partial reversal effect of the dopamine D2/D3 antagonist sulpiride

&NA; Markers of histaminergic dysregulation were found in several neuropsychiatric disorders characterized by repetitive behaviours, thoughts and stereotypies. We analysed the effect of acute histamine depletion by means of i. c.v. injections of alpha‐fluoromethylhistidine, a blocker of histidine decarboxylase, on the temporal organization of motor sequences of CD1 mice behaviour in the open‐field test. An ethogram encompassing 9 behavioural components was employed. Durations and frequencies were only slightly affected by treatments. However, as revealed by multivariate t‐pattern analysis, histamine depletion was associated with a striking increase in the number of behavioural patterns. We found 42 patterns of different composition occurring, on average, 520.90 ± 50.23 times per mouse in the histamine depleted (HD) group, whereas controls showed 12 different patterns occurring on average 223.30 ± 20.64 times. Exploratory and grooming behaviours clustered separately, and the increased pattern complexity involved exclusively exploratory patterns. To test the hypothesis of a histamine‐dopamine interplay on behavioural pattern phenotype, non‐sedative doses of the D2/D3 antagonist sulpiride (12.5‐25‐50 mg/kg) were additionally administered to different groups of HD mice. Sulpiride counterbalanced the enhancement of exploratory patterns of different composition, but it did not affect the mean number of patterns at none of the doses used. Our results provide new insights on the role of histamine on repetitive behavioural sequences of freely moving mice. Histamine deficiency is correlated with a general enhancement of pattern complexity. This study supports a putative involvement of histamine in the pathophysiology of tics and related disorders. HighlightsA role of mouse brain histamine on the temporal architecture of behaviour is proposed.Descriptive and t‐pattern analyses in the open field were carried out in the study.Histamine deficient mice had a marked increase in exploratory t‐patterns complexity.D2/D3 antagonism partially reverted the arrangement of exploratory t‐patterns.