Éva Borbély

Neuropeptides in learning and memory

Dementia conditions and memory deficits of different origins (vascular, metabolic and primary neurodegenerative such as Alzheimers and Parkinsons diseases) are getting more common and greater clinical problems recently in the aging population. Since the presently available cognitive enhancers have very limited therapeutical applications, there is an emerging need to elucidate the complex pathophysiological mechanisms, identify key mediators and novel targets for future drug development. Neuropeptides are widely distributed in brain regions responsible for learning and memory processes with special emphasis on the hippocampus, amygdala and the basal forebrain. They form networks with each other, and also have complex interactions with the cholinergic, glutamatergic, dopaminergic and GABA-ergic pathways. This review summarizes the extensive experimental data in the well-established rat and mouse models, as well as the few clinical results regarding the expression and the roles of the tachykinin system, somatostatin and the closely related cortistatin, vasoactive intestinal polypeptide (VIP) and pituitary adenylate-cyclase activating polypeptide (PACAP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), opioid peptides and galanin. Furthermore, the main receptorial targets, mechanisms and interactions are described in order to highlight the possible therapeutical potentials. Agents not only symptomatically improving the functional impairments, but also inhibiting the progression of the neurodegenerative processes would be breakthroughs in this area. The most promising mechanisms determined at the level of exploratory investigations in animal models of cognitive disfunctions are somatostatin sst4, NPY Y2, PACAP-VIP VPAC1, tachykinin NK3 and galanin GALR2 receptor agonisms, as well as delta opioid receptor antagonism. Potent and selective non-peptide ligands with good CNS penetration are needed for further characterization of these molecular pathways to complete the preclinical studies and decide if any of the above described targets could be appropriate for clinical investigations.

A CRPS-IgG-transfer-trauma model reproducing inflammatory and positive sensory signs associated with complex regional pain syndrome.

Summary The transfer of serum IgG from patients with long‐standing complex regional pain syndrome (CRPS) to mice, followed by mild hind paw trauma, causes typical signs of CRPS. ABSTRACT The aetiology of complex regional pain syndrome (CRPS), a highly painful, usually post‐traumatic condition affecting the limbs, is unknown, but recent results have suggested an autoimmune contribution. To confirm a role for pathogenic autoantibodies, we established a passive‐transfer trauma model. Prior to undergoing incision of hind limb plantar skin and muscle, mice were injected either with serum IgG obtained from chronic CRPS patients or matched healthy volunteers, or with saline. Unilateral hind limb plantar skin and muscle incision was performed to induce typical, mild tissue injury. Mechanical hyperalgesia, paw swelling, heat and cold sensitivity, weight‐bearing ability, locomotor activity, motor coordination, paw temperature, and body weight were investigated for 8 days. After sacrifice, proinflammatory sensory neuropeptides and cytokines were measured in paw tissues. CRPS patient IgG treatment significantly increased hind limb mechanical hyperalgesia and oedema in the incised paw compared with IgG from healthy subjects or saline. Plantar incision induced a remarkable elevation of substance P immunoreactivity on day 8, which was significantly increased by CRPS‐IgG. In this IgG‐transfer‐trauma model for CRPS, serum IgG from chronic CRPS patients induced clinical and laboratory features resembling the human disease. These results support the hypothesis that autoantibodies may contribute to the pathophysiology of CRPS, and that autoantibody‐removing therapies may be effective treatments for long‐standing CRPS.

Capsaicin-sensitive sensory nerves exert complex regulatory functions in the serum-transfer mouse model of autoimmune arthritis

Highlights • Capsaicin-sensitive sensory nerves are protective against autoimmune arthritis.• Desensitization of these fibers increase immune cell activation and edema.• Sensory denervation enhances ROS production, MMP activity and arthritic changes.• Late mechanical hyperalgesia is decreased after destroying these sensory nerves.

Transient receptor potential ankyrin 1 (TRPA1) receptor is involved in chronic arthritis: in vivo study using TRPA1-deficient mice

BackgroundThe transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable cation channel that is expressed on capsaicin-sensitive sensory neurons, endothelial and inflammatory cells. It is activated by a variety of inflammatory mediators, such as methylglyoxal, formaldehyde and hydrogen sulphide. Since only few data are available about the role of TRPA1 in arthritis and related pain, we investigated its involvement in inflammation models of different mechanisms.MethodsChronic arthritis was induced by complete Freund’s adjuvant (CFA), knee osteoarthritis by monosodium iodoacetate (MIA) in TRPA1 knockout (KO) mice and C57Bl/6 wildtype mice. For comparison, carrageenan- and CFA-evoked acute paw and knee inflammatory changes were investigated. Thermonociception was determined on a hot plate, cold tolerance in icy water, mechanonociception by aesthesiometry, paw volume by plethysmometry, knee diameter by micrometry, weight distribution with incapacitance tester, neutrophil myeloperoxidase activity and vascular leakage by in vivo optical imaging, and histopathological alterations by semiquantitative scoring.ResultsCFA-induced chronic mechanical hypersensitivity, tibiotarsal joint swelling and histopathological alterations, as well as myeloperoxidase activity in the early phase (day 2), and vascular leakage in the later stage (day 7), were significantly reduced in TRPA1 KO mice. Heat and cold sensitivities did not change in this model. Although in TRPA1 KO animals MIA-evoked knee swelling and histopathological destruction were not altered, hypersensitivity and impaired weight bearing on the osteoarthritic limb were significantly decreased. In contrast, carrageenan- and CFA-induced acute inflammation and pain behaviours were not modified by TRPA1 deletion.ConclusionsTRPA1 has an important role in chronic arthritis/osteoarthritis and related pain behaviours in the mouse. Therefore, it might be a promising target for novel analgesic/anti-inflammatory drugs.

Role of Tachykinin 1 and 4 Gene-Derived Neuropeptides and the Neurokinin 1 Receptor in Adjuvant-Induced Chronic Arthritis of the Mouse

Objective Substance P, encoded by the Tac1 gene, is involved in neurogenic inflammation and hyperalgesia via neurokinin 1 (NK1) receptor activation. Its non-neuronal counterpart, hemokinin-1, which is derived from the Tac4 gene, is also a potent NK1 agonist. Although hemokinin-1 has been described as a tachykinin of distinct origin and function compared to SP, its role in inflammatory and pain processes has not yet been elucidated in such detail. In this study, we analysed the involvement of tachykinins derived from the Tac1 and Tac4 genes, as well as the NK1 receptor in chronic arthritis of the mouse. Methods Complete Freund’s Adjuvant was injected intraplantarly and into the tail of Tac1−/−, Tac4−/−, Tacr1−/− (NK1 receptor deficient) and Tac1−/−/Tac4−/− mice. Paw volume was measured by plethysmometry and mechanosensitivity using dynamic plantar aesthesiometry over a time period of 21 days. Semiquantitative histopathological scoring and ELISA measurement of IL-1β concentrations of the tibiotarsal joints were performed. Results Mechanical hyperalgesia was significantly reduced from day 11 in Tac4−/− and Tacr1−/− animals, while paw swelling was not altered in any strain. Inflammatory histopathological alterations (synovial swelling, leukocyte infiltration, cartilage destruction, bone damage) and IL-1β concentration in the joint homogenates were significantly smaller in Tac4−/− and Tac1−/−/Tac4−/− mice. Conclusions Hemokinin-1, but not substance P increases inflammation and hyperalgesia in the late phase of adjuvant-induced arthritis. While NK1 receptors mediate its antihyperalgesic actions, the involvement of another receptor in histopathological changes and IL-1β production is suggested.

Somatostatin receptor subtype 4 activation is involved in anxiety and depression-like behavior in mouse models.

Somatostatin regulates stress-related behavior and its expression is altered in mood disorders. However, little is known about the underlying mechanisms, especially about the importance of its receptors (sst1-sst5) in anxiety and depression-like behavior. Here we analyzed the potential role of sst4 receptor in these processes, since sst4 is present in stress-related brain regions, but there are no data about its functional relevance. Genetic deletion of sst4 (Sstr4(-/-)) and its pharmacological activation with the newly developed selective non-peptide agonist J-2156 were used. Anxiety was examined in the elevated plus maze (EPM) and depression-like behavior in the forced swim (FST) and tail suspension tests (TST). Neuronal activation during the TST was monitored by Fos immunohistochemistry, receptor expression was identified by sst4(LacZ) immunostaining in several brain regions. Sstr4(-/-) mice showed increased anxiety in the EPM and enhanced depression-like behavior in the FST. J-2156 (100 μg/kg i.p.) exhibited anxiolytic effect in the EPM and decreased immobility in the TST. J-2156 alone did not influence Fos immunoreactivity in intact mice, but significantly increased the stress-induced Fos response in the dorsal raphe nucleus, central projecting Edinger-Westphal nucleus, periaqueductal gray matter, the magnocellular, but not the parvocellular part of the hypothalamic paraventricular nucleus, lateral septum, bed nucleus of the stria terminalis and the amygdala. Notably, sst4(LacZ) immunoreactivity occurred in the central and basolateral amygdala. Together, these studies reveal that sst4 mediates anxiolytic and antidepressant-like effects by enhancing the stress-responsiveness of several brain regions with special emphasis on the amygdala.

Analgesic and Anti-Inflammatory Effects of the Novel Semicarbazide-Sensitive Amine-Oxidase Inhibitor SzV-1287 in Chronic Arthritis Models of the Mouse

Semicarbazide-sensitive amine oxidase (SSAO) catalyses oxidative deamination of primary amines. Since there is no data about its function in pain and arthritis mechanisms, we investigated the effects of our novel SSAO inhibitor SzV-1287 in chronic mouse models of joint inflammation. Effects of SzV-1287 (20 mg/kg i.p./day) were investigated in the K/BxN serum-transfer and complete Freund’s adjuvant (CFA)-evoked active immunization models compared to the reference SSAO inhibitor LJP-1207. Mechanonociception was assessed by aesthesiometry, oedema by plethysmometry, clinical severity by scoring, joint function by grid test, myeloperoxidase activity by luminescence, vascular leakage by fluorescence in vivo imaging, histopathological changes by semiquantitative evaluation, and cytokines by Luminex assay. SzV-1287 significantly inhibited hyperalgesia and oedema in both models. Plasma leakage and keratinocyte chemoattractant production in the tibiotarsal joint, but not myeloperoxidase activity was significantly reduced by SzV-1287 in K/BxN-arthritis. SzV-1287 did not influence vascular and cellular mechanisms in CFA-arthritis, but significantly decreased histopathological alterations. There was no difference in the anti-hyperalgesic and anti-inflammatory actions of SzV-1287 and LJP-1207, but only SzV-1287 decreased CFA-induced tissue damage. Unlike SzV-1287, LJP-1207 induced cartilage destruction, which was confirmed in vitro. SzV-1287 exerts potent analgesic and anti-inflammatory actions in chronic arthritis models of distinct mechanisms, without inducing cartilage damage.

Higher susceptibility of somatostatin 4 receptor gene-deleted mice to chronic stress-induced behavioral and neuroendocrine alterations

The somatostatin 4 receptor (sst4) is widely expressed in stress-related brain areas (e.g. hippocampus, amygdala) and regulates the emotional behavior in acute situations. Since its importance in chronic stress-induced complex pathophysiological alterations is unknown, we investigated the involvement of sst4 in the responsiveness to chronic variable stress (CVS). Sstr4 gene-deficient (Sstr4-/-) mice and their wildtype counterparts (Sstr4+/+) were used to examine the behavioral and neuroendocrine alterations as well as chronic neuronal activity (FosB expression) changes in response to CVS. In Sstr4+/+ mice, there was no behavioral response to the applied CVS paradigm. In contrast, immobility time in the tail suspension test increased after the CVS in the knockouts. In the forced swim test, Sstr4-/- animals showed increased baseline immobility and then it decreased after the CVS. Light-dark box and open field test behaviors and sucrose preference did not respond to the stress in the knockouts. Adrenal weights increased and thymus weights decreased in both Sstr4+/+ and Sstr4-/- mice demonstrating the effect of chronic stress. The relative adrenal weight of stressed knockouts increased to a greater extent, while relative thymus and body weights decreased only in the Sstr4-/- mice. Basal plasma corticosterone concentrations did not change after the CVS in either genotype. FosB immunopositivity in the central and basolateral amygdaloid nuclei was enhanced in stressed knockouts, but not in wild types. This is the first evidence that sst4 activation is involved in the behavioral and neuroendocrine alterations induced by chronic stress with a crucial role of plastic changes in the amygdala.

Hemokinin-1 is an important mediator of endotoxin-induced acute airway inflammation in the mouse

OBJECTIVE Hemokinin-1, the newest tachykinin encoded by the preprotachykinin C (Tac4) gene, is predominatly produced by immune cells. Similarly to substance P, it has the greatest affinity to the tachykinin NK1 receptor, but has different binding site and signaling mechanisms. Furthermore, several recent data indicate the existence of a not yet identified own receptor and divergent non-NK1-mediated actions. Since there is no information on its functions in the airways, we investigated its role in endotoxin-induced pulmonary inflammation. METHODS Acute pneumonitis was induced in Tac4 gene-deleted (Tac4(-/-)) mice compared to C57Bl/6 wildtypes by intranasal E. coli lipopolysaccharide (LPS). Airway responsiveness to inhaled carbachol was measured with unrestrained whole body plethysmography 24h later. Semiquantitative histopathological scoring was performed; reactive oxygen species (ROS) production was measured with luminol bioluminescence, myeloperoxidase activity with spectrophotometry, and inflammatory cytokines with Luminex. RESULTS All inflammatory parameters, such as histopathological alterations (perivascular edema, neutrophil/macrophage accumulation, goblet cell hyperplasia), myeloperoxidase activity, ROS production, as well as interleukin-1beta, interleukin-6, tumor necrosis factor alpha, monocyte chemoattractant protein-1 and keratinocyte chemoattractant concentrations were significantly diminished in the lung of Tac4(-/-) mice. However, bronchial hyperreactivity similarly developed in both groups. Interestingly, in LPS-treated Tac4(-/-) mouse lungs, bronchus-associated, large, follicle-like lymphoid structures developed. CONCLUSIONS We provide the first evidence that hemokinin-1 plays a crucial pro-inflammatory role in the lung by increasing inflammatory cell activities, and might also be a specific regulator of lymphocyte functions.

Noxious heat threshold temperature and pronociceptive effects of allyl isothiocyanate (mustard oil) in TRPV1 or TRPA1 gene-deleted mice

AIMS To investigate the roles of TRPV1 and TRPA1 channels in baseline and allyl isothiocyanate (AITC)-evoked nociceptive responses by comparing wild-type and gene-deficient mice. MAIN METHODS In contrast to conventional methods of thermonociception measuring reflex latencies, we used our novel methods to determine the noxious heat threshold. KEY FINDINGS It was revealed that the heat threshold of the tail measured by an increasing-temperature water bath is significantly higher in TRPV1(-/-), but not TRPA1(-/-), mice compared to respective wild-types. There was no difference between the noxious heat thresholds of the hind paw as measured by an increasing-temperature hot plate in TRPV1(-/-), TRPA1(-/-) and the corresponding wild-type mice. The withdrawal latency of the tail from 0°C water was prolonged in TRPA1(-/-), but not TRPV1(-/-), mice compared to respective wild-types. In wild-type animals, dipping the tail or paw into 1% AITC induced an 8-14°C drop of the noxious heat threshold (heat allodynia) of both the tail and paw, and 40-50% drop of the mechanonociceptive threshold (mechanical allodynia) of the paw measured by dynamic plantar esthesiometry. These AITC-evoked responses were diminished in TRPV1(-/-), but not TRPA1(-/-), mice. Tail withdrawal latency to 1% AITC was significantly prolonged in both gene-deleted strains. SIGNIFICANCE Different heat sensors determine the noxious heat threshold in distinct areas: a pivotal role for TRPV1 on the tail is contrasted with no involvement of either TRPV1 or TRPA1 on the hind paw. Noxious heat threshold measurement appears appropriate for preclinical screening of TRP channel ligands as novel analgesics.