Sónia Puga

A role of supraspinal galanin in behavioural hyperalgesia in the rat

Introduction In chronic pain disorders, galanin (GAL) is able to either facilitate or inhibit nociception in the spinal cord but the contribution of supraspinal galanin to pain signalling is mostly unknown. The dorsomedial nucleus of the hypothalamus (DMH) is rich in galanin receptors (GALR) and is involved in behavioural hyperalgesia. In this study, we evaluated the contribution of supraspinal GAL to behavioural hyperalgesia in experimental monoarthritis. Methods In Wistar-Han males with a four week kaolin/carrageenan-induced monoarthritis (ARTH), paw-withdrawal latency (PWL) was assessed before and after DMH administration of exogenous GAL, a non-specific GALR antagonist (M40), a specific GALR1 agonist (M617) and a specific GALR2 antagonist (M871). Additionally, the analysis of c-Fos expression after GAL injection in the DMH was used to investigate the potential involvement of brainstem pain control centres. Finally, electrophysiological recordings were performed to evaluate whether pronociceptive On- or antinociceptive Off-like cells in the rostral ventromedial medulla (RVM) relay the effect of GAL. Results Exogenous GAL in the DMH decreased PWL in ARTH and SHAM animals, an effect that was mimicked by a GALR1 agonist (M617). In SHAM animals, an unselective GALR antagonist (M40) increased PWL, while a GALR2 antagonist (M871) decreased PWL. M40 or M871 failed to influence PWL in ARTH animals. Exogenous GAL increased c-Fos expression in the RVM and dorsal raphe nucleus (DRN), with effects being more prominent in SHAM than ARTH animals. Exogenous GAL failed to influence activity of RVM On- or Off-like cells of SHAM and ARTH animals. Conclusions Overall, exogenous GAL in the DMH had a pronociceptive effect that is mediated by GALR1 in healthy and arthritic animals and is associated with alterations of c-Fos expression in RVM and DRN that are serotonergic brainstem nuclei known to be involved in the regulation of pain.

Inorganic mercury accumulation in brain following waterborne exposure elicits a deficit on the number of brain cells and impairs swimming behavior in fish (white seabream—Diplodus sargus)

The current study contributes to fill the knowledge gap on the neurotoxicity of inorganic mercury (iHg) in fish through the implementation of a combined evaluation of brain morphometric alterations (volume and total number of neurons plus glial cells in specific regions of the brain) and swimming behavior (endpoints related with the motor activity and mood/anxiety-like status). White seabream (Diplodus sargus) was exposed to realistic levels of iHg in water (2μgL(-1)) during 7 (E7) and 14 days (E14). After that, fish were allowed to recover for 28 days (PE28) in order to evaluate brain regeneration and reversibility of behavioral syndromes. A significant reduction in the number of cells in hypothalamus, optic tectum and cerebellum was found at E7, accompanied by relevant changes on swimming behavior. Moreover, the decrease in the number of neurons and glia in the molecular layer of the cerebellum was followed by a contraction of its volume. This is the first time that a deficit on the number of cells is reported in fish brain after iHg exposure. Interestingly, a recovery of hypothalamus and cerebellum occurred at E14, as evidenced by the identical number of cells found in exposed and control fish, and volume of cerebellum, which might be associated with an adaptive phenomenon. After 28 days post-exposure, the optic tectum continued to show a decrease in the number of cells, pointing out a higher vulnerability of this region. These morphometric alterations coincided with numerous changes on swimming behavior, related both with fish motor function and mood/anxiety-like status. Overall, current data pointed out the iHg potential to induce brain morphometric alterations, emphasizing a long-lasting neurobehavioral hazard.

Metabotropic glutamate 5 receptor in the infralimbic cortex contributes to descending pain facilitation in healthy and arthritic animals.

The involvement of the prefrontal cortex in pain processing has been recently addressed. We studied the role of the infralimbic cortex (IL) and group I metabotropic glutamate receptors (mGluRs) in descending modulation of nociception in control and monoarthritic (ARTH) conditions. Nociception was assessed using heat-induced paw withdrawal while drugs were microinjected in the IL of rats. Local anesthesia of the IL or the adjacent prelimbic cortex (PL) facilitated nociception, indicating that IL and PL are tonically promoting spinal antinociception. Phasic activation with glutamate (GLU) revealed opposing roles of the PL and IL; GLU in the PL had a fast antinociceptive action, while in the IL it had a slow onset pronociceptive action. IL administration of a local anesthetic or GLU produced identical results in ARTH and control animals. An mGluR5 agonist in the IL induced a pronociceptive effect in both groups, while mGluR5 antagonists had no effect in controls but induced antinociception in ARTH rats. Activation of the IL mGluR1 (through co-administration of mGluR1/5 agonist and mGluR5 antagonist) did not alter nociception in controls but induced antinociception in ARTH animals. IL administration of an mGluR1 antagonist failed to alter nociception in either experimental group. Finally, mGluR5 but not mGluR1 antagonists blocked the pronociceptive action of GLU in both groups. The results indicate that IL contributes to descending modulation of nociception. mGluR5 in the IL enhance nociception in healthy control and monoarthritic animals, an effect that is tonic in ARTH. Moreover, activation of IL mGluR1s attenuates nociception following the development of monoarthritis.

Unveiling the neurotoxicity of methylmercury in fish (Diplodus sargus) through a regional morphometric analysis of brain and swimming behavior assessment

The current study aims to shed light on the neurotoxicity of MeHg in fish (white seabream - Diplodus sargus) by the combined assessment of: (i) MeHg toxicokinetics in the brain, (ii) brain morphometry (volume and number of neurons plus glial cells in specific brain regions) and (iii) fish swimming behavior (endpoints associated with the motor performance and the fear/anxiety-like status). Fish were surveyed for all the components after 7 (E7) and 14 (E14) days of dietary exposure to MeHg (8.7μgg-1), as well as after a post-exposure period of 28days (PE28). MeHg was accumulated in the brain of D. sargus after a short time (E7) and reached a maximum at the end of the exposure period (E14), suggesting an efficient transport of this toxicant into fish brain. Divalent inorganic Hg was also detected in fish brain along the experiment (indicating demethylation reactions), although levels were 100-200 times lower than MeHg, which pinpoints the organic counterpart as the great liable for the recorded effects. In this regard, a decreased number of cells in medial pallium and optic tectum, as well as an increased hypothalamic volume, occurred at E7. Such morphometric alterations were followed by an impairment of fish motor condition as evidenced by a decrease in the total swimming time, while the fear/anxiety-like status was not altered. Moreover, at E14 fish swam a greater distance, although no morphometric alterations were found in any of the brain areas, probably due to compensatory mechanisms. Additionally, although MeHg decreased almost two-fold in the brain during post-exposure, the levels were still high and led to a loss of cells in the optic tectum at PE28. This is an interesting result that highlights the optic tectum as particularly vulnerable to MeHg exposure in fish. Despite the morphometric alterations reported in the optic tectum at PE28, no significant changes were found in fish behavior. Globally, the effects of MeHg followed a multiphasic profile, where homeostatic mechanisms prevented circumstantially morphometric alterations in the brain and behavioral shifts. Although it has become clear the complexity of matching brain morphometric changes and behavioral shifts, motor-related alterations induced by MeHg seem to depend on a combination of disruptions in different brain regions.

Oxidative stress profiles in brain point out a higher susceptibility of fish to waterborne divalent mercury compared to dietary organic mercury

This study examines, for the first time, the neurotoxicity of Hg(II) and MeHg in fish (Diplodus sargus) in a time-course comparative perspective and considering realistic exposure levels and routes. Both forms followed an identical time-variation pattern of accumulation in the brain, but dietary MeHg was more efficiently transported to the brain. MeHg was substantially eliminated from the brain in 28days of depuration, which did not occur for Hg(II). Moreover, Hg(II) displayed a high neurotoxicity potential, as unveiled by the poor activation of brain antioxidant defenses and recurrent oxidative damage (as protein oxidation), while the opposite was recorded upon MeHg exposure. These results highlight the need to include Hg(II) in future environmental health assessment plans, preventing an underestimation of the risk for wild fish populations, which has probably been occurring due to the long-standing idea of the higher toxicity of MeHg in comparison with inorganic Hg forms.

Minocycline reduces mechanical allodynia and depressive-like behaviour in type-1 diabetes mellitus in the rat

HighlightsExperimental rat model of type‐1 diabetes was induced by streptozotocin.Diabetes induced in 4 weeks hypersensitivity to cold and mechanical stimulation.Hypersensitivity was accompanied by depression‐like behaviour.Minocycline treatment attenuated mechanical allodynia and depression.Minocycline failed to attenuate cold allodynia or mechanical hyperalgesia. Abstract A common and devastating complication of diabetes mellitus is painful diabetic neuropathy (PDN) that can be accompanied by emotional disorders such as depression. A few studies have suggested that minocycline that inhibits microglia may attenuate pain hypersensitivity in PDN. Moreover, a recent study reported that minocycline has an acute antidepressive‐like effect in diabetic animals. Here we studied whether (i) prolonged minocycline treatment suppresses pain behaviour in PDN, (ii) the minocycline effect varies with submodality of pain, and (iii) the suppression of pain behaviour by prolonged minocycline treatment is associated with antidepressive‐like effect. The experiments were performed in streptozotocin‐induced rat model of type‐1 diabetes. Pain behaviour was evoked by innocuous (monofilaments) and noxious (paw pressure) mechanical stimulation, innocuous cold (acetone drops) and noxious heat (radiant heat). Depression‐like behaviour was assessed using forced swimming test. Minocycline treatment (daily 80 mg/kg per os) of three‐week duration started four weeks after induction of diabetes. Diabetes induced mechanical allodynia and hyperalgesia, cold allodynia, heat hypoalgesia, and depression‐like behaviour. Minocycline treatment significantly attenuated mechanical allodynia and depression‐like behaviour, while it failed to produce significant changes in mechanical hyperalgesia, cold allodynia or heat hypoalgesia. The results indicate that prolonged per oral treatment with minocycline has a sustained mechanical antiallodynic and antidepressive‐like effect in PDN. These results support the proposal that minocycline might provide a treatment option for attenuating sensory and comorbid emotional symptoms in chronic PDN.

Brain morphometric profiles and their seasonal modulation in fish (Liza aurata) inhabiting a mercury contaminated estuary

Mercury (Hg) is a potent neurotoxicant known to induce important adverse effects on fish, but a deeper understanding is lacking regarding how environmental exposure affects the brain morphology and neural plasticity of specific brain regions in wild specimens. In this work, it was evaluated the relative volume and cell density of the lateral pallium, hypothalamus, optic tectum and molecular layer of the cerebellum on wild Liza aurata captured in Hg-contaminated (LAR) and non-contaminated (SJ) sites of a coastal system (Ria de Aveiro, Portugal). Given the season-related variations in the environment that fish are naturally exposed, this assessment was performed in the winter and summer. Hg triggered a deficit in cell density of hypothalamus during the winter that could lead to hormonal dysfunctions, while in the summer Hg promoted larger volumes of the optic tectum and cerebellum, indicating the warm period as the most critical for the manifestation of putative changes in visual acuity and motor-dependent tasks. Moreover, in fish from the SJ site, the lateral pallium relative volume and the cell density of the hypothalamus and optic tectum were higher in the winter than in summer. Thus, season-related stimuli strongly influence the size and/or cell density of specific brain regions in the non-contaminated area, pointing out the ability of fish to adapt to environmental and physiological demands. Conversely, fish from the Hg-contaminated site showed a distinct seasonal profile of brain morphology, presenting a larger optic tectum in the summer, as well as a larger molecular layer of the cerebellum with higher cell density. Moreover, Hg exposure impaired the winter-summer variation of the lateral pallium relative size (as observed at SJ). Altogether, seasonal variations in fish neural morphology and physiology should be considered when performing ecotoxicological studies in order to better discriminate the Hg neurotoxicity.

Metals(loids) targeting fish eyes and brain in a contaminated estuary - Uncovering neurosensory (un)susceptibility through bioaccumulation, antioxidant and morphometric profiles

This study examined the susceptibility of fish (Liza aurata) eyes and brain to metals(loids) contamination under realistic exposure conditions. A multidimensional approach was applied to fish caught at a chronically contaminated site (BAR) and at a reference site of the Tagus estuary (Portugal), which comprised metals(loids) accumulation in eyes and brain together with a battery of enzymatic and non-enzymatic antioxidants, as well as brain morphometry (i.e. cell density). Trace element levels in the blood, gills, liver and kidney allowed interpretations on their preferential pathway(s) to the eyes and brain. Metals(loids) accumulation pointed out the elevated vulnerability of the fish eyes at BAR, probably related with the direct waterborne uptake. Pb uptake in L. aurata eyes could be associated both with water and indirect pathways. At the most contaminated site, metals(loids) were on the basis of pro-oxidant conditions in the ocular tissues, while no indication of toxicity was recorded in the brain. Overall, the results disclosed a differential bioaccumulation among fish organs, suggesting that, in the L. aurata population studied, metal organotropism underlie the lower susceptibility of the brain comparing to the eyes. However, mechanisms remain little understood and further work is needed.