Acute Functional Adaptations in Isolated Presynaptic Terminals Unveil Synaptosomal Learning and Memory

open1Synaptosomes are used to decipher the mechanisms involved in chemical transmission, since they permit highlighting the mechanisms of transmitter release and confirming whether the activation of presynaptic receptors/enzymes can modulate this event. In the last two decades, important progress in the field came from the observations that synaptosomes retain changes elicited by both "in vivo" and "in vitro" acute chemical stimulation. The novelty of these studies is the finding that these adaptations persist beyond the washout of the triggering drug, emerging subsequently as functional modifications of synaptosomal performances, including release efficiency. These findings support the conclusion that synaptosomes are plastic entities that respond dynamically to ambient stimulation, but also that they "learn and memorize" the functional adaptation triggered by acute exposure to chemical agents. This work aims at reviewing the results so far available concerning this form of synaptosomal learning, also highlighting the role of these acute chemical adaptations in pathological conditions.openPittaluga, AnnaPittaluga, Ann

Presynaptic release-regulating mGlu1 receptors in central nervous system

Group I metabotropic glutamate (mGlu) receptors consists of mGlu1 and mGlu5 receptor subtypes. These receptors are widely distributed in the central nervous system (CNS), where they preferentially mediate facilitatory signaling in neurones and glial cells, mainly by favoring phospholipase (PLC) translocation. Based on the literature so far available, group I Metabotropic glutamate receptors (mGluRs) are preferentially expressed at the postsynaptic side of chemical synapsis, where they participate in the progression of the chemical stimulus. Studies, however, have shown the presence of these receptors also at the presynaptic level, where they exert several functions, including the modulation of transmitter exocytosis. Presynaptic Group I mGluRs can be both autoreceptors regulating release of glutamate and heteroreceptors regulating the release of various transmitters, including GABA, dopamine, noradrenaline, and acetylcholine. While the existence of presynaptic release-regulating mGlu5 receptors is largely recognized, the possibility that mGlu1 receptors also are present at this level has been a matter of discussion for a long time. A large body of evidence published in the last decade, however, supports this notion. This review aims at revisiting the data from in vitro studies concerning the existence and the role of release-regulating mGlu1 receptors presynaptically located in nerve terminals isolated from selected regions of the CNS. The functional interaction linking mGlu5 and mGlu1 receptor subtypes at nerve terminals and their relative contributions as modulators of central transmission will also be discussed. We apologize in advance for omission in our coverage of the existing literature

A new specific neuronal modulatory effect of nicotine: the functional cross talk between nicotinic and glutamate receptors

We here have addressed the topic of the cross-talk between receptors. We provide evidence supporting the co-localization and the functional interaction between nicotinic acetylcholine receptors and some glutamatergic receptors. The recruitment of nicotinic acetylcholine receptors dynamically and negatively modulates the function of both N-methyl-D-aspartic acid (NMDA) and ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors throughout their selective internalization. This dynamic control by cholinergic nicotinic system of NMDA and ?-AMPA receptors is operative even at very low concentrations of nicotine. Nicotinic and glutamatergic receptors have been both implicated in important neurological and psychiatric disorders such as Alzheimer’s and Parkinson’s disease, schizophrenia, and anxiety. Thus, a more extensive and detailed knowledge of this new modulatory role of nicotine may eventually enable us to develop specific therapeutic interventions for these pathologies

Presynaptic mGlu1 Receptors Control GABAB Receptors in an Antagonist-Like Manner in Mouse Cortical GABAergic and Glutamatergic Nerve Endings

Mouse cortical GABAergic synaptosomes possess presynaptic inhibitory GABAB autoreceptors. Accordingly, (\ub1)baclofen (3 \u3bcM) inhibits in a CGP53423-sensitive manner the 12 mM KCl-evoked release of preloaded [3H]GABA. Differently, the existence of presynaptic release-regulating metabotropic glutamate type 1 (mGlu1) heteroreceptors in these terminals is still matter of discussion, although confocal microscopy unveiled the existence of mGlu1\u3b1 with GABAB1 or GABAB2 proteins in cortical VGAT-positive synaptosomes. The group I mGlu agonist 3,5-DHPG failed to modify on its own the 12 mM KCl-evoked [3H]GABA exocytosis from cortical nerve endings, but, when added concomitantly to the GABAB agonist, it significantly reduced the 3 \u3bcM (\ub1)baclofen-induced inhibition of [3H]GABA exocytosis. Conversely, the mGlu1 antagonist LY367385 (0.03\u20131 \u3bcM), inactive on its own on GABA exocytosis, amplified the 3 \u3bcM (\ub1)baclofen-induced inhibition of [3H]GABA overflow. The ( \ub1 )baclofen-induced inhibition of [3H]GABA exocytosis was more pronounced in cortical synaptosomes from Grm1crv4/crv4 mice, which bear a spontaneous mutation of the Grm1 gene leading to the functional inactivation of the mGlu1 receptor. Inasmuch, the expression of GABAB2 receptor protein in cortical synaptosomal lysates from Grm1crv4/crv4 mice was increased when compared to controls. Altogether, these observations seem best interpreted by assuming that mGlu1 coexist with GABAB receptors in GABAergic cortical synaptosomes, where they control GABA receptors in an antagonist-like manner. We then asked whether the mGlu1-mediated control of GABAB receptors is restricted to GABAergic terminals, or if it occurs also in other subpopulations of nerve endings. Release-regulating GABAB receptors also exist in glutamatergic nerve endings. (\ub1)baclofen (1 \u3bcM) diminished the 12 mM KCl-evoked [3H]D-aspartate overflow. Also in these terminals, the concomitant presence of 1 \u3bcM LY367385, inactive on its own, significantly amplified the inhibitory effect exerted by (\ub1)baclofen on [3H]D-aspartate exocytosis. Confocal microscopy confirmed the colocalization of mGlu1 with GABAB1 and GABAB2 labeling in vesicular glutamate type1 transporter-positive particles. Our results support the conclusion that mGlu1 receptors modulate in an antagonist-like manner presynaptic release-regulating GABAB receptors. This receptor\u2013receptor interaction could be neuroprotective in central disease typified by hyperglutamatergicity

Presynaptic mGlu1 Receptors Control GABAB Receptors in an Antagonist-Like Manner in Mouse Cortical GABAergic and Glutamatergic Nerve Endings

Mouse cortical GABAergic synaptosomes possess presynaptic inhibitory GABAB autoreceptors. Accordingly, (±)baclofen (3 μM) inhibits in a CGP53423-sensitive manner the 12 mM KCl-evoked release of preloaded [3H]GABA. Differently, the existence of presynaptic release-regulating metabotropic glutamate type 1 (mGlu1) heteroreceptors in these terminals is still matter of discussion, although confocal microscopy unveiled the existence of mGlu1α with GABAB1 or GABAB2 proteins in cortical VGAT-positive synaptosomes. The group I mGlu agonist 3,5-DHPG failed to modify on its own the 12 mM KCl-evoked [3H]GABA exocytosis from cortical nerve endings, but, when added concomitantly to the GABAB agonist, it significantly reduced the 3 μM (±)baclofen-induced inhibition of [3H]GABA exocytosis. Conversely, the mGlu1 antagonist LY367385 (0.03–1 μM), inactive on its own on GABA exocytosis, amplified the 3 μM (±)baclofen-induced inhibition of [3H]GABA overflow. The ( ± )baclofen-induced inhibition of [3H]GABA exocytosis was more pronounced in cortical synaptosomes from Grm1crv4/crv4 mice, which bear a spontaneous mutation of the Grm1 gene leading to the functional inactivation of the mGlu1 receptor. Inasmuch, the expression of GABAB2 receptor protein in cortical synaptosomal lysates from Grm1crv4/crv4 mice was increased when compared to controls. Altogether, these observations seem best interpreted by assuming that mGlu1 coexist with GABAB receptors in GABAergic cortical synaptosomes, where they control GABA receptors in an antagonist-like manner. We then asked whether the mGlu1-mediated control of GABAB receptors is restricted to GABAergic terminals, or if it occurs also in other subpopulations of nerve endings. Release-regulating GABAB receptors also exist in glutamatergic nerve endings. (±)baclofen (1 μM) diminished the 12 mM KCl-evoked [3H]D-aspartate overflow. Also in these terminals, the concomitant presence of 1 μM LY367385, inactive on its own, significantly amplified the inhibitory effect exerted by (±)baclofen on [3H]D-aspartate exocytosis. Confocal microscopy confirmed the colocalization of mGlu1 with GABAB1 and GABAB2 labeling in vesicular glutamate type1 transporter-positive particles. Our results support the conclusion that mGlu1 receptors modulate in an antagonist-like manner presynaptic release-regulating GABAB receptors. This receptor–receptor interaction could be neuroprotective in central disease typified by hyperglutamatergicity

Characterization data of water-soluble hydrophilic and amphiphilic dendrimers prodrugs for delivering bioactive chemical entities otherwise non soluble.

More than 40% of bioactive chemical entities (BCEs) developed in pharmaceutical industry are almost water-insoluble, poorly orally bioavailable and/or not via parenteral administrable, and this strongly limits their clinical applications. Drug Delivery (DD) is an engineered technology dealing with the development of delivery systems (DDSs) able to solubilize, transport, target release and maintain therapeutic drugs concentration where needed for long periods. DD frequently makes use of nanosized carriers, often positive charged, including dendrimer such as commercially available and strongly cationic PAMAM and PEI. Nowadays, uncharged dendrimer scaffolds modified with amino acids-modified in their cationic form, are preferred because a more controlled number of nitrogen atoms causes less damage to cells. Then, two hydrophilic (1, 2) [1] (Fig. 1) and three amphiphilic (3-5) [2] (Fig. 2) water-soluble dendrimers were prepared and completely characterized. Once established through proper routine investigations, that these materials could work well as DDSs, they have been used to physically entrap two completely insoluble BCEs i.e. the thiocarbamate (O-TC) 6 [3] and Ellagic Acid (EA) 7 (Fig. 3) with the aim at improving their solubility and in parallel at protecting them from early degradation, at promoting their fast cellular up-take and thus reducing eventual systemic toxicity. Without resorting to toxic excipients and harmful solubilizing agents often used despite the resulting unpleasant side effects, five structurally different nanodispersions (DPXs) loaded with 6 [4] and two with 7 [5] were achieved and completely characterized to confirm their structure and to evaluate their potentiality in biomedical applications

Non-PAMAM amino acids-modified dendrimers nanoparticles for enhancing water-solubility of insoluble bioactive molecules: our state of the art

Non-PAMAM amino acids-modified dendrimers nanoparticles for enhancing water-solubility of insoluble bioactive molecules: our state of the art Silvana Alfei,* Andrea Spallarossa, Silvia Catena, Federica Turrini, Guendalina Zuccari, Anna Pittaluga, Raffaella Boggia Dipartimento di Farmacia, Universit\ue0 di Genova, Viale Cembrano 4, I-16148 Genova, Italy E-mail: [email protected] ABSTRACT Water-solubility is essential for GIT absorbability or parenteral administration of drugs, therefore it is a key parameter to achieve the systemic drug concentration necessary for an effective therapeutic activity. Unfortunately, low aqueous solubility is the major problem with bioactive chemical entities (BCEs), in fact, more than 40% BCEs developed in pharmaceutical industry are practically water-insoluble. As a consequence, great are the research efforts focused on the development of new techniques aiming at enhancing it. Toxic excipients and harmful solubilizing agents were also extensively used for solubilizing and delivering non water-soluble drugs, despite the resulting unpleasant side effects complained of by patients. Nowadays, safer strategies, such as drugs physicochemical modifications or particle size reduction, crystal engineering, salt formation, solid dispersion, use of surfactant and complexation are being exploited. As far as what regards dispersion/complexation techniques, nanoparticles, including dendrimers, are intensely utilized for this purpose, thus in parallel achieving drugs protection from early degradation, more efficient target delivery into cells and tissues and lower systemic toxicity. Synthetic thiocarbamate (O-TC 1) (Fig. 1) is a non-nucleoside HIV-1 reverse transcriptase inhibitor [1] while Ellagic Acid (EA 2) (Fig. 2) is a polyphenol present in some fruits, nuts and seeds endowed with strong antioxidant, anti-inflammatory and other several healthy properties. Unfortunately, both of them are practically insoluble (Table 1), non orally bioavailable, non parenteral administrable, then non usable for therapeutic purposes in their free forms. Fig. 1: Structure of O-TC 1 Fig. 2: Structure of EA 2 Fig. 3: Examples of hydrophilic (left) and amphiphilic (right) dendrimers structure During the last year, these problems have been addressed and successfully resolved by us, and in this communication, the reached promising outcomes have been summarized and the current state of the art provided. Afar from commercially high cytotoxic PAMAM, five non cells-damaging amino acid-modified hydrophilic (3, 4) [2] and amphiphilic (5-7) [3] dendrimers (Fig. 3) have been synthetized and then used as polymer nano-containers to improve 1 and 2 water-solubility. Five (8-12) [4] and two (13, 14) [5] structurally different drugs-loaded nanodispersions (DPXs) were obtained respectively. The structures were confirmed by FT-IR and NMR analysis and all the samples have resulted in being endowed with very good Drug Loading (DL %). Compound 1, totally insoluble except for in highly diluted DMSO when free, once entrapped in dendrimers, shown to be well soluble both in water and in ethanol. In the case of 2, water-solubility was increased even up to 1000 times compared to the free form. For the prerogatives demonstrated in the performed routine analyses, the prepared DPXs could be considered eligible for biomedical and therapeutic applications thus allowing to exploit 1 and 2 pharmacological properties. REFERENCES: 1. A. Spallarossa et al., Eur. J. Med. Chem., 44, 2190 (2009). 2. S. Alfei & S. Catena, Polym. Advan. Technol., 29, 2735 (2018). 3. S. Alfei & S. Catena, Polym. Int., 67, 1572 (2018). 3. S. Alfei et al., Eur. J. Pharm. Sci., 124, 153 (2018). 4. S. Alfei et al., New J. Chem., 2019, DOI: 10.1039/c8nj05657a

Microdispersions of ellagic acid and pomegranate extracts as new potential nutraceutical ingredients

The health properties attributed to several fruits (i.e. pomegranates, raspberries, strawberries, blackberry, chestnuts, walnuts, pecan), herbs (tea) and seeds (berries seeds) are attributed to an important group of natural polyphenols classified as hydrolysable tannins (HT) named Ellagitannins (ETs), that have shown in vitro multi-target biological properties relevant to the treatment of several human diseases. In vivo, ETs are rather not absorbed, and they are hydrolysed providing mainly Ellagic acid (EA). EA is endowed with the same biological properties of ETs and it could be considered as the responsible of their health benefits. Unfortunately, EA cannot be exploited for in vivo applications because of its poor water solubility (9.7 \u3bcg/mL) and accordingly low bioavailability. At first, aiming to increase EA solubility, an EA solid microdispersion (EA-md) was realized by employing only water and low methoxylated pectin, as a food compatible excipient, by applying spray drying technology. EA-md showed a 22% (w/w) Drug Loading (DL), a 30 times improved water solubility maintaining a remarkable radical scavenging activity [1]. It has been analytically characterised and used for in vivo pharmacological treatments in order to evaluate it as potential nutraceutical ingredient. Adult (3-6 months old) and old (20-22 old months) male mice were chronically administered EA-md dissolved in the drinking water (about 150 mg / Kg) for 14 days. During this period, animals were monitored for the spontaneous motor activity and for curiosity before, during and at the end of the EA-md treatment. Adult and old mice were then sacrificed for \u201cex vivo, in vitro\u201d analysis to test the efficiency of noradrenaline release from cortical nerve endings. It is known that noradrenaline exocytosis from cortical nerve endings is significantly impaired during ageing. We found that the chronic administration of EA-md did not alter the noradrenaline exocytosis from cortical nerve endings of adult mice, but significantly recovered the reduced noradrenaline overflow in aged mice. Further investigations are needed to explore the cellular cascade of events accounting for the beneficial effect. In a second step, pomegranate, as a natural source of EA, has been considered to similarly prepare and investigate an analogous formulation. Since pomegranate fruit is recognized as one of the most important sources of ETs, mainly localized in the by-products obtained after industrial juice squeezing, a method to convert the squeezing marcs into a potential nutraceutical ingredient has been explored. In particular, Pulsed Ultrasound-Assisted Extraction (PUAE), using just water as solvent, resulted to be suitable for extracting the water-soluble bioactive molecules (PEx), whose content in hydrolysable tannins, standardized in EA, has been determined. Furthermore, the already mentioned spray drying microdispersion has been employed to formulate and to stabilize it over time. This last formulation (PEx-md) will be subjected to the already mentioned pharmacological experiments in order to study its nutraceutical properties too. [1] S. Alfei, F. Turrini, S. Catena, P. Zunin, B. Parodi, G. Zuccari, A.M. Pittaluga, R. Boggia, New J. Chem, 43, 2438-2448 DOI: 10.1039/C8NJ05657