E S Vizi’s research while affiliated with HUN-REN Institute of Experimental Medicine and other places

Background and Purpose Tetrabenazine (TBZ), used for treating hyperkinetic disorders, inhibits vesicular monoamine transporter‐2 (VMAT‐2), which sequesters monoamines into vesicles for exocytosis. However, our knowledge of the effect of TBZ on monoaminergic transmission is limited. Herein, we provide neurochemical evidence regarding the effect of VMAT‐2 inhibition on vesicular neurotransmitter release from the prefrontal cortex (PFC) and striatum (STR) (brain regions involved in characteristic TBZ treatment side effects). The interaction between TBZ and MDMA was also assessed regarding motor behaviour in mice. Experimental Approach Vesicular storage capacity and release of [³H]‐noradrenaline ([³H]‐NA), [³H]‐dopamine ([³H]‐DA), [³H]‐serotonin ([³H]‐5‐HT), and [³H]‐acetylcholine ([³H]‐ACh) was studied in mouse PFC and STR ex vivo slice preparations using electrical field stimulation. Additionally, locomotor activity was assessed in vehicle‐treated mice and compared with that of MDMA, TBZ, and co‐administered animals (n = 6) using the LABORAS system. Key Results TBZ lowered the storage capacity and inhibited the vesicular release of [³H]‐NA and [³H]‐DA from the PFC, and [³H]‐DA and [³H]‐5‐HT from the STR in a concentration‐dependent manner. Unlike vesamicol (vesicular ACh uptake inhibitor), TBZ failed to inhibit the vesicular release of [³H]‐ACh from the PFC. When the vesicular storage of the investigated monoamines was inhibited by TBZ in the PFC and STR, MDMA induced the release of transmitters through transporter reversal; MDMA dose dependently increased locomotor activity in vivo. Conclusion and Implications Our observations provide neurochemical evidence explaining the mechanism of VMAT‐2 inhibitors in the brain and support the involvement of dopaminergic and noradrenergic transmission in hyperkinetic movement disorders.

While our understanding of the nanoscale architecture of anterograde synaptic transmission is rapidly expanding, the qualitative and quantitative molecular principles underlying distinct mechanisms of retrograde synaptic communication remain elusive. We show that a particular form of tonic cannabinoid signaling is essential for setting target cell–dependent synaptic variability. It does not require the activity of the two major endocannabinoid-producing enzymes. Instead, by developing a workflow for physiological, anatomical, and molecular measurements at the same unitary synapse, we demonstrate that the nanoscale stoichiometric ratio of type 1 cannabinoid receptors (CB 1 Rs) to the release machinery is sufficient to predict synapse-specific release probability. Accordingly, selective decrease of extrasynaptic CB 1 Rs does not affect synaptic transmission, whereas in vivo exposure to the phytocannabinoid Δ ⁹ -tetrahydrocannabinol disrupts the intrasynaptic nanoscale stoichiometry and reduces synaptic variability. These findings imply that synapses leverage the nanoscale stoichiometry of presynaptic receptor coupling to the release machinery to establish synaptic strength in a target cell–dependent manner.

ATP-gated P2X7 receptors (P2X7Rs) play a crucial role in brain disorders. However, how they affect normal and pathological synaptic transmission is still largely unclear. Here, by using whole-cell patch-clamp technique to record AMPA- and NMDA receptor-mediated excitatory postsynaptic currents (s/mEPSCs) in dentate gyrus granule cells (DG GCs), we revealed a modulation by P2X7Rs of presynaptic sites, especially originated from entorhinal cortex (EC)-GC path but not the mossy cell (MC)-GC path. The involvement of P2X7Rs was confirmed using a pharmacological approach. Additionally, the acute activation of P2X7Rs directly elevated calcium influx from EC-GC terminals. In postnatal phencyclidine (PCP)-induced mouse model of schizophrenia, we observed that P2X7R deficiency restored the EC-GC synapse alteration and alleviated PCP-induced symptoms. To summarize, P2X7Rs participate in the modulation of GC excitatory neurotransmission in the DG via EC-GC pathway, contributing to pathological alterations of neuronal functions leading to neurodevelopmental disorders.

Phenylephrine (PE) is a canonical α 1 -adrenoceptor-selective agonist. However, unexpected effects of PE have been observed in preclinical and clinical studies, that cannot be easily explained by its actions on α 1 -adrenoceptors. The probability of the involvement of α 2 - and β-adrenoceptors in the effect of PE has been raised. In addition, our earlier study observed that PE released noradrenaline (NA) in a [Ca ²⁺ ] o -independent manner. To elucidate this issue, we have investigated the effects of PE on [ ³ H]NA release and α 1 -mediated smooth muscle contractions in the mouse vas deferens (MVD) as ex vivo preparation. The release experiments were designed to assess the effects of PE at the presynaptic terminal, whereas smooth muscle isometric contractions in response to electrical field stimulation were used to measure PE effect postsynaptically. Our results show that PE at concentrations between 0.3 and 30 µM significantly enhanced the resting release of [ ³ H]NA in a [Ca ²⁺ ] o -independent manner. In addition, prazosin did not affect the release of NA evoked by PE. On the contrary, PE-evoked smooth muscle contractions were inhibited by prazosin administration indicating the α 1 -adrenoceptor-mediated effect. When the function of the NA transporter (NAT) was attenuated with nisoxetine, PE failed to release NA and the contractions were reduced by approximately 88%. The remaining part proved to be prazosin-sensitive. The present work supports the substantial indirect effect of PE which relays on the cytoplasmic release of NA, which might explain the reported side effects for PE.

The histamine H3 receptor is a favourable target for the treatment of cognitive deficits. Here we report the in vitro and in vivo profile of RGH-235, a new potent, selective, and orally active H3 receptor antagonist/inverse agonist developed by Gedeon Richter Plc. Radioligand binding and functional assays were used for in vitro profiling. Procognitive efficacy was investigated in rodent cognitive tests, in models of attention deficit hyperactive disorder (ADHD) and in cognitive tests of high translational value (rat touch screen visual discrimination test, primate fixed-foreperiod visual reaction time task). Results were supported by pharmacokinetic studies, neurotransmitter release, sleep EEG and dipsogenia. RGH-235 displayed high affinity to H3 receptors (Ki = 3.0–9.2 nM, depending on species), without affinity to H1, H2 or H4 receptors and >100 other targets. RGH-235 was an inverse agonist ([³⁵S] GTPγS binding) and antagonist (pERK1/2 ELISA), showing favourable kinetics, inhibition of the imetit-induced dipsogenia and moderate effects on sleep-wake EEG. RGH-235 stimulated neurotransmitter release both in vitro and in vivo. RGH-235 was active in spontaneously hypertensive rats (SHR), generally considered as a model of ADHD, and revealed a robust pro-cognitive profile both in rodent and primate tests (in 0.3–1 mg/kg) and in models of high translational value (e.g. in a rodent touch screen test and in non-human primates). The multiple and convergent procognitive effects of RGH-235 support the view that beneficial cognitive effects can be linked to antagonism/inverse agonism of H3 receptors.

Abstract Neuronal networks cause changes in behaviorally important information processing through the vesicular release of neurotransmitters governed by the rate and timing of action potentials (APs). Herein, we provide evidence that dopamine (DA), nonquantally released from the cytoplasm, may exert similar effects in vivo. In mouse slice preparations, (+/−)‐3,4‐methylenedioxy‐methamphetamine (MDMA, or ecstasy) and β‐phenylethylamine (β‐PEA)‐induced DA release in the striatum and nucleus accumbens (NAc), two regions of the brain involved in reward‐driven and social behavior and inhibited the axonal stimulation‐induced release of tritiated acetylcholine ([3H]ACh) in the striatum. The DA transporter (DAT) inhibitor (GBR‐12909) prevented MDMA and β‐PEA from causing DA release. GBR‐12909 could also restore some of the stimulated acetylcholine release reduced by MDMA or β‐PEA in the striatum confirming the fundamental role of DAT. In addition, hypothermia could prevent the β‐PEA‐induced release in the striatum and in the NAc. Sulpiride, a D2 receptor antagonist, also prevented the inhibitory effects of MDMA or β‐PEA on stimulated ACh release, suggesting they act indirectly via binding of DA. Reflecting the neurochemical interactions in brain slices at higher system level, MDMA altered the social behavior of rats by preferentially enhancing passive social behavior. Similar to the in vitro effects, GBR‐12909 treatment reversed specific elements of the MDMA‐induced changes in behavior, such as passive social behavior, while left others including social play unchanged. The changes in behavior by the high level of extracellular DA–– a significant amount originating from cytoplasmic release––suggest that in addition to digital computation through synapses, the brain also uses analog communication, such as DA signaling, to mediate some elements of complex behaviors, but in a much longer time scale.

Age-related hearing loss (ARHL), a sensorineural hearing loss of multifactorial origin, increases its prevalence in aging societies. Besides hearing aids and cochlear implants, there is no FDA approved efficient pharmacotherapy to either cure or prevent ARHL. We hypothesized that selegiline, an antiparkinsonian drug, could be a promising candidate for the treatment due to its complex neuroprotective, antioxidant, antiapoptotic, and dopaminergic neurotransmission enhancing effects. We monitored by repeated Auditory Brainstem Response (ABR) measurements the effect of chronic per os selegiline administration on the hearing function in BALB/c and DBA/2J mice, which strains exhibit moderate and rapid progressive high frequency hearing loss, respectively. The treatments were started at 1 month of age and lasted until almost a year and 5 months of age, respectively. In BALB/c mice, 4 mg/kg selegiline significantly mitigated the progression of ARHL at higher frequencies. Used in a wide dose range (0.15–45 mg/kg), selegiline had no effect in DBA/2J mice. Our results suggest that selegiline can partially preserve the hearing in certain forms of ARHL by alleviating its development. It might also be otoprotective in other mammals or humans.

Background: It has been consistently reported that the deficiency of the adenosine triphosphate (ATP) sensitive purinergic receptor P2X7 (P2X7R) ameliorates symptoms in animal models of brain diseases. Objective: This study aimed to investigate the role of P2X7R in rodent models of acute and subchronic schizophrenia based on phencyclidine (PCP) delivery in animals lacking or overexpressing P2X7R, and to identify the underlying mechanisms involved. Methods: The psychotomimetic effects of acute i.p. PCP administration in C57Bl/6J wild-type, P2X7R knockout (P2rx7−/−) and overexpressing (P2X7-EGFP) young adult mice were quantified. The medial prefrontal cortex (mPFC) of P2rx7−/− and heterozygous P2X7-EGFP acutely treated animals was characterized through immunohistochemical staining. The prefrontal cortices of young adult P2rx7−/− and P2rx7tg/+ mice were examined with tritiated dopamine release experiments and the functional properties of the mPFC pyramidal neurons in layer V from P2rx7−/− mice were assessed by patch-clamp recordings. P2rx7−/− animals were subjected to a 7 days subchronic systemic PCP treatment. The animals working memory performance and PFC cytokine levels were assessed. Results: Our data strengthen the hypothesis that P2X7R modulates schizophrenia-like positive and cognitive symptoms in NMDA receptor antagonist models in a receptor expression level-dependent manner. P2X7R expression leads to higher medial PFC susceptibility to PCP-induced circuit hyperactivity. The mPFC of P2X7R knockout animals displayed distinct alterations in the neuronal activation pattern, microglial organization, specifically around hyperactive neurons, and were associated with lower intrinsic excitability of mPFC neurons. Conclusions: P2X7R expression exacerbated PCP-related effects in C57Bl/6J mice. Our findings suggest a pleiotropic role of P2X7R in the mPFC, consistent with the observed behavioral phenotype, regulating basal dopamine concentration, layer-specific neuronal activation, intrinsic excitability of neurons in the mPFC, and the interaction of microglia with hyperactive neurons. Direct measurements of P2X7R activity concerning microglial ramifications and dynamics could help to further elucidate the molecular mechanisms involved. © Copyright © 2020 Calovi, Mut-Arbona, Tod, Iring, Nicke, Mato, Vizi, Tønnesen and Sperlagh.

The release of [³H]dopamine ([³H]DA) and [³H]noradrenaline ([³H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca²⁺ further increased the release rates of [³H]DA and [³H]NA induced by ischemic conditions. This finding indicated that the Na⁺/Ca²⁺ exchanger (NCX), working in reverse in the absence of extracellular Ca²⁺, fails to trigger the influx of Ca²⁺ in exchange for Na⁺ and fails to counteract ischemia by further increasing the intracellular Na⁺ concentration ([Na⁺]i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca²⁺ was removed. Hypothermia inhibited the excessive release of [³H]DA in response to ischemia, even in the absence of Ca²⁺. These findings further indicate that the NCX plays an important role in maintaining a high [Na⁺]i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [³H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H2O2), a mediator of ischemic brain injury enhanced the striatal resting release of [³H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca²⁺]o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na⁺/Cl⁻-dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects.

Neuropathic pain is a chronic pain caused by central and peripheral nerve injury, long-term diabetes or treatment with chemotherapy drugs, and it is dissimilar to other chronic pain conditions. Chronic pain usually seriously affects the quality of life, and its drug treatment may result in increased costs of social and medical care. As in the USA and Canada, in Europe, the demand for pain-relieving medicines used in chronic pain has also significantly increased, but most European countries are not experiencing an opioid crisis. In this review, the role of various endogenous transmitters (noradrenaline, dopamine, serotonin, met- and leu-enkephalins, β-endorphin, dynorphins, cannabinoids, ATP) and various receptors (α2, μ, etc.) in the innate pain-relieving system will be discussed. Furthermore, the modulation of pain processing pathways by transmitters, focusing on neuropathic pain and the role of the sympathetic nervous system in the side effects of excessive opioid treatment, will be explained.