Boccella et al. Mol Brain (2021) 14:28 https://doi.org/10.1186/s13041-020-00724-z RESEARCH Open Access 2-Pentadecyl-2-oxazoline ameliorates memory impairment and depression-like behaviour in neuropathic mice: possible role of adrenergic alpha2- and H3 histamine autoreceptors Serena Boccella1†, Francesca Guida1†, Monica Iannotta1†, Fabio Arturo Iannotti3,4†, Rosmara Infantino1, Flavia Ricciardi1, Claudia Cristiano2, Rosa Maria Vitale3, Pietro Amodeo3, Ida Marabese1, Carmela Belardo1, Vito de Novellis1, Salvatore Paino1, Enza Palazzo1, Antonio Calignano2, Vincenzo Di Marzo3,4,5, Sabatino Maione1,4,6 and Livio Luongo1,4,6* Abstract Neuropathic pain (NP) remains an untreatable disease due to the complex pathophysiology that involves the whole pain neuraxis including the forebrain. Sensory dysfunctions such as allodynia and hyperalgesia are only part of the symptoms associated with neuropathic pain that extend to memory and afectivity defcits. The development of multi-target molecules might be a promising therapeutic strategy against the symptoms associated with NP. 2-penta- decyl-2-oxazoline (PEA-OXA) is a plant-derived agent, which has shown efectiveness against chronic pain and associ- ated neuropsychiatric disorders. The molecular mechanisms by which PEA-OXA exerts its efects are, however, only partially known. In the current study, we show that PEA-OXA, besides being an alpha2 adrenergic receptor antagonist, also acts as a modulator at histamine H3 receptors, and report data on its efects on sensory, afective and cognitive symptoms associated with the spared nerve injury (SNI) model of neuropathic pain in mice. Treatment for 14 days with PEA-OXA after the onset of the symptoms associated with neuropathic pain resulted in the following efects: (i) allodynia was decreased; (ii) afective/cognitive impairment associated with SNI (depression, spatial, and working memories) was counteracted; (iii) long-term potentiation in vivo in the lateral entorhinal cortex-dentate gyrus (per- forant pathway, LPP) was ameliorated, (iv) hippocampal glutamate, GABA, histamine, norepinephrine and dopamine altered levels after peripheral nerve injury were reversed, (v) expression level of the TH positive neurons in the Locus Coeruleus were normalized. Thus, a 16-day treatment with PEA-OXA alleviates the sensory, emotional, cognitive, elec- trophysiological and neurochemical alterations associated with SNI-induced neuropathic pain. Keywords: Neuropathic pain, Depression, Cognitive impairments, H3 receptors, Locus coeruleus, Mice *Correspondence: [email protected] Introduction † Serena Boccella, Francesca Guida, Monica Iannotta and Fabio Arturo In the last two decades, many investigations have been Iannotti share the frst authorship 1 Department of Experimental Medicine, Pharmacology Division, focused towards plastic changes at supra-spinal corti- University of Campania “L. Vanvitelli”, 80138 Naples, Italy cal [1–3] and non-cortical structures [4, 5] underlying Full list of author information is available at the end of the article cognitive/afective consequences of neuropathic pain. © The Author(s) 2021 corrected publication 2021. 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Mol Brain (2021) 14:28 Page 2 of 24 Although the infuence of neuropathic pain on hip- agonists in neuropathic pain can be partially mediated by pocampal structures began to be studied many dec- α2 adrenoceptor desensitization [23] inspired us to eval- ades ago [6], only recently it has become the subject of uate possible anti-neuropathic properties of PEA-OXA more in-depth studies by several laboratories [7–10] Te and investigate the possible involvement of H3 receptor. involvement of the hippocampus in spatial memory dis- In the present study, we frst checked whether PEA- orders and other comorbidities of neuropathic pain was OXA was also able to modulate the activity of histamine recently shown also in the spared nerve injury (SNI) H3 receptors in vitro. Indeed, this compound belongs to model of neuropathic pain [7]. SNI impairs synaptic plas- a large family of long chain fatty acid-derived biologi- ticity (LTP or STP) at hippocampal dentate gyrus (DG), cally active mediators with multiple molecular targets CA1, and CA3 synapses [7–10], as well as DG neurogen- [31]. Once that its pharmacological activity at the hista- esis [7, 11]. Te entorhinal cortex-hippocampus pathway mine H3 receptor was demonstrated for the frst time, we plays a pivotal role in persistent pain such that cortical evaluated the efect of repeated administrations (16 days projections to the hippocampus can diferently afect starting the treatment 14 days after sciatic nerve surgery) pain perception [12, 13] Te interactions between the of PEA-OXA on: (i) mechanical allodynia, (ii) afective lateral entorhinal cortex (LEC) and the DG are in charge and cognitive (spatial and working memory) behavior, with the integration of important emotional stimuli asso- (iii) neural activity in the LC, (iv) in vivo long term poten- ciated with space–time orientation and place memory tiation in the entorhinal-cortex/dentate gyrus, LPP, and storage [14]. Among the several neurotransmitters and (iv) glutamate, GABA, norepinephrine, dopamine and neuromodulators that orchestrate the complex scenario histamine levels in the hippocampus. All the behavioral, in forebrain chronic pain mechanisms, the glutamater- biochemical and electrophysiological experiments were gic (Glu), GABAergic and the endocannabinoid (eCB) carried out in control and neuropathic mice 30 days after systems have been deeply investigated [2, 15–18]. Fur- the sham or SNI surgery on the sciatic nerve. thermore, a key role of the biogenic amines (i.e. norepi- nephrine, dopamine, serotonin, histamine, etc.) has been Materials and methods highlighted in both chronic pain and some of its mala- Homology modelling of histamine H3 receptor daptive neuroplasticities [19]. Te noradrenergic projec- Te sequences of the histamine H3 receptor was retrieved tions modulating pain originate from cell nuclei in the from the publicly available sequence database www. unipr brainstem, namely the locus coeruleus (LC), A5, and ot. org (UNIPROT: Q9Y5N1). 50 homology models of A7 cell groups [20, 21]. In particular, the noradrenergic receptor were built with MODELLER [32, 33] version system in the LC seems to play a key role in the main- 9.14 using the x-ray structure of the acetylcholine M3 tenance of neuropathic pain-induced allodynia [22]. receptor (PDB id: 4U15) as a template and the structural Like norepinephrine, histamine is gaining great interest alignment. Te best model in term of both Modeller in the central mechanisms of neuroplasticity underlying Objective Function and Dope score was selected for the neuropathic pain [23, 24]. In addition to the known seda- subsequent docking calculations. tive efect of histamine H1 receptor antagonists, interest today has focused on histamine H3 receptors [24–26], Ligand building which, by being expressed at the level of the presynap- To carry out the docking calculations, the starting ligand tic terminals, strongly infuence the release of histamine geometry of histamine and PEA-OXA was built with and various neurotransmitters [27–29]. Although his- UCSF Chimera 1.14 [34] and energy minimized at molec- tamine H3 receptor blockade has been shown to relieve ular mechanics level, using AM1-BCC charges and then neuropathic pain [25, 26], the role of this receptor and optimized using GAMESS program [35] at the Hartree– the mechanisms underlying its supraspinal efects on the Fock level with STO-3G basis set, followed by a single- various components of neuropathic pain are still poorly point HF energy evaluation at the 6-31G* level to derive understood. We recently investigated the behavioral the partial atomic charges for the ligands using the RESP efects of 2-pentadecyl-2-oxazoline (PEA-OXA), a natu- procedure [36]. ral compound found in green and roasted cofee beans, in a murine model of mild traumatic brain injury (mTBI), Molecular docking which develops into a neuro-psychiatric syndrome very Docking studies were performed with AutoDock 4.2 often observed in post-traumatic states. In that study, it [36, 37]. H3 model along with the ligands was pro- was found that the in vitro and in vivo efects of PEA- cessed with AutoDock Tools (ADT) package version OXA were mediated by its action as an antagonist on the 1.5.6rc123 to merge non-polar hydrogens, calculate norepinephrine alpha2 receptor [30]. Te evidence that Gasteiger charges and select rotatable sidechain bonds. the analgesic efects of H3 receptor antagonists/inverse A 60 Å × 60 Å × 70 Å grid, centred in the orthosteric Boccella et al. Mol Brain (2021) 14:28 Page 3 of 24 Fig. 1 Alpha2-mediated efect of PEA-OXA in vitro and in vivo. (a) Scatter plots showing the efect of PEA-OXA in COS cells stably expressing histamine H3 receptors on intracellular cAMP levels. Forskolin 10 μM served as AMPc inducer. Data represent the mean S.E.M. of four separate ± determinations. Data sets were compared using t-Test and ANOVA followed by Tukey’s test. The asterisk indicates a p value 0.05 vs vehicle.