Antimicrobial Activity the Essential Oil from Croton pluriglandulosus Carn. Leaves against Microorganisms of Clinical Interest.

Multiresistant pathogens pose a serious threat to human health. The genus Candida is one class of human pathogenic yeasts responsible for infections affecting healthy and immunocompromised patients. In this context, plant essential oils emerged as a future natural alternative to control the diseases caused by these pathogens. Based on that, the present study aimed to evaluate the antimicrobial potential of essential oil from C. pluriglandulosus and understand the mechanism of action. Here, it highlighted antimicrobial activity and the mechanisms of action of the essential oil extracted from C. pluriglandulosus Carn.-Torres & Riina (CpEO) leaves on human pathogenic microorganisms in planktonic and biofilm lifestyles. In addition, for the first time, the oil composition was revealed by GC-MS analysis and the toxicity to human red blood cells (HRBC). Twenty-six chemical compounds were identified in CpEO, elemicin, bicyclogermacrene, caryophyllene, brevifolin, and 2,4,6-trimethoxy-styrene. Through hemolytic assay, it was shown that CpEO has no toxicity to human RBCs. At the concentration of 50 µg mL-1, CpEO did not show great antibacterial potential. However, promising data were found for C. krusei and C. parapsilosis inhibiting by 89.3% and 80.7% of planktonic cell growth and 83.5% and 77.9% the biofilm formation, respectively. Furthermore, the mechanisms of action CpEO were elucidated by fluorescence. Scanning electron microscopy revealed damage to the cell membrane and pore formation, ROS overproduction, and induction of apoptosis in candida cells. Our results reinforce the potential of CpEO as an effective alternative molecule of pharmaceutical interest.

Essential Oil from Croton blanchetianus Leaves: Anticandidal Potential and Mechanisms of Action.

Antimicrobial drugs are becoming ineffective given the resistance acquired by microorganisms. As such, it is imperative to seek new antimicrobial molecules that could provide a basis for the development of new drugs. Therefore, this work aimed to evaluate the antimicrobial potential and the mechanisms of action of the essential oil extracted from leaves of Croton blanchetianus (named CbEO) on different fungi and bacteria of clinical importance in both planktonic and biofilm lifestyles. GC-MS/MS analysis revealed the presence of twenty-two different compounds in the CbEO, which were identified using the Kovats retention index. Among these, the most abundant were amorphene (20.03%), spathulenol (5%), bicyclogermacrene (1.49%), caryophyllene oxide (4.55%), and eucalyptol (5.62%). CbOE (50 µg mL-1) barely inhibited the growth of Bacillus subtilis (23%), Pseudomonas aeruginosa (27%), and Salmonella enterica (28%), and no inhibition was obtained against Enterobacter aerogenes and Klebsiella pneumoniae. Additionally, no activity against bacterial biofilm was detected. In contrast, CbEO was active against Candida species. C. albicans and C. parapsilosis were inhibited by 78 and 75%, respectively. The antibiofilm potential also was favorable against C. albicans and C. parapsilosis, inhibiting 44 and 74% of biofilm formation and reducing around 41 and 27% of the preformed biofilm, respectively. CbOE caused membrane damage and pore formation, overproduction of ROS, and apoptosis on C. albicans and C. parapsilosis cells, as well as not inducing hemolysis in human red cells. The results obtained in this work raise the possibility of using the essential oil of C. blanchetianus leaves as an alternative to fight infections caused by C. albicans and C. parapsilosis.

Top-down projections of the prefrontal cortex to the ventral tegmental area, laterodorsal tegmental nucleus, and median raphe nucleus.

Anatomical and functional evidence suggests that the PFC is fairly unique among all cortical regions, as it not only receives input from, but also robustly projects back to mesopontine monoaminergic and cholinergic cell groups. Thus, the PFC is in position to exert a powerful top-down control over several state-setting modulatory transmitter systems that are critically involved in the domains of arousal, motivation, reward/aversion, working memory, mood regulation, and stress processing. Regarding this scenario, the origin of cortical afferents to the ventral tegmental area (VTA), laterodorsal tegmental nucleus (LDTg), and median raphe nucleus (MnR) was here compared in rats, using the retrograde tracer cholera toxin subunit b (CTb). CTb injections into VTA, LDTg, or MnR produced retrograde labeling in the cortical mantle, which was mostly confined to frontal polar, medial, orbital, and lateral PFC subdivisions, along with anterior- and mid-cingulate areas. Remarkably, in all of the three groups, retrograde labeling was densest in layer V pyramidal neurons of the infralimbic, prelimbic, medial/ventral orbital and frontal polar cortex. Moreover, a lambda-shaped region around the apex of the rostral pole of the nucleus accumbens stood out as heavily labeled, mainly after injections into the lateral VTA and LDTg. In general, retrograde PFC labeling was strongest following injections into MnR and weakest following injections into VTA. Altogether, our findings reveal a fairly similar set of prefrontal afferents to VTA, LDTg, and MnR, further supporting an eminent functional role of the PFC as a controller of major state-setting mesopontine modulatory transmitter systems.

Habenular connections with the dopaminergic and serotonergic system and their role in stress-related psychiatric disorders.

The habenula (Hb) is a phylogenetically old epithalamic structure differentiated into two nuclear complexes, the medial (MHb) and lateral habenula (LHb). After decades of search for a great unifying function, interest in the Hb resurged when it was demonstrated that LHb plays a major role in the encoding of aversive stimuli ranging from noxious stimuli to the loss of predicted rewards. Consistent with a role as an anti-reward center, aberrant LHb activity has now been identified as a key factor in the pathogenesis of major depressive disorder. Moreover, both MHb and LHb emerged as new players in the reward circuitry by primarily mediating the aversive properties of distinct drugs of abuse. Anatomically, the Hb serves as a bridge that links basal forebrain structures with monoaminergic nuclei in the mid- and hindbrain. So far, research on Hb has focused on the role of the LHb in regulating midbrain dopamine release. However, LHb/MHb are also interconnected with the dorsal (DR) and median (MnR) raphe nucleus. Hence, it is conceivable that some of the habenular functions are at least partly mediated by the complex network that links MHb/LHb with pontomesencephalic monoaminergic nuclei. Here, we summarize research about the topography and transmitter phenotype of the reciprocal connections between the LHb and ventral tegmental area-nigra complex, as well as those between the LHb and DR/MnR. Indirect MHb outputs via interpeduncular nucleus to state-setting neuromodulatory networks will also be commented. Finally, we discuss the role of specific LHb-VTA and LHb/MHb-raphe circuits in anxiety and depression.

Regulation and neurochemical identity of melanin-concentrating hormone neurones in the preoptic area of lactating mice.

Neurones expressing the melanin-concentrating hormone (MCH) can be found in the medial preoptic area (mPOA) and ventral aspects of the periventricular preoptic nucleus of rats by mid-to-late lactation and this expression disappears after weaning. The transitory expression of MCH in the preoptic area suggests a role for these neurones in the control of the end of lactation. However, the neurochemical identity of mPOA MCH neurones and the regulatory factors that control the transient MCH expression remain largely unknown, especially in the mouse. In the present study, we showed that mice also present the transitory expression of MCH in the mPOA at late lactation. mPOA MCH cells did not colocalise significantly with markers of GABAergic (VGAT), glutamatergic (VGLUT2 and VGLUT3) or dopaminergic (tyrosine hydroxylase) neurones. mPOA MCH cells also did not express Kiss1 or oxytocin. By contrast, approximately 70% and 90% of mPOA MCH neurones colocalised with oestrogen receptor α and prolactin-induced phosphorylated signal transducer and activator of transcription 5 (STAT5), respectively. Finally, we demonstrated that the number of MCH neurones in the mPOA is significantly higher in females during the first lactation, compared to mice on the second lactation or pregnant mice during the first lactation or brain-specific STAT5 knockout mice during the first lactation. In summary, our findings indicate that MCH neurones in the mPOA of lactating mice are sensitive to oestrogens and prolactin. Thus, mPOA MCH expression is possibly influenced by hormonal variations. Furthermore, the STAT5 signalling pathway is likely involved in the regulation of MCH expression in the mPOA of lactating mice.

Connections of the laterodorsal tegmental nucleus with the habenular-interpeduncular-raphe system.

The laterodorsal tegmental nucleus (LDTg) is a hindbrain cholinergic cell group thought to be involved in mechanisms of arousal and the control of midbrain dopamine cells. Nowadays, there is increasing evidence that LDTg is also engaged in mechanisms of anxiety/fear and promotion of emotional arousal under adverse conditions. Interestingly, LDTg appears to be connected with other regulators of aversive motivational states, including the lateral habenula (LHb), medial habenula (MHb), interpeduncular nucleus (IP), and median raphe nucleus (MnR). However, the circuitry between these structures has hitherto not been systematically investigated. Here, we placed injections of retrograde or anterograde tracers into LDTg, LHb, IP, and MnR. We also examined the transmitter phenotype of LDTg afferents to IP by combining retrograde tracing with immunofluorescence and in situ hybridization techniques. We found LHb inputs to LDTg mainly emerging from the medial division of the LHb (LHbM), which also receives axonal input from LDTg. The bidirectional connections between IP and LDTg displayed a lateralized organization, with LDTg inputs to IP being predominantly GABAergic or cholinergic and mainly directed to the contralateral IP. Moreover, we disclosed reciprocal LDTg connections with structures involved in the modulation of hippocampal theta rhythm including MnR, nucleus incertus, and supramammillary nucleus. Our findings indicate that the habenula is linked with LDTg either by direct reciprocal projections from/to LHbM or indirectly via the MHb-IP axis, supporting a functional role of LDTg in the regulation of aversive behaviors, and further characterizing LHb as a master controller of ascending brainstem state-setting modulatory projection systems.

Relationship of α-MSH and AgRP axons to the perikarya of melanocortin-4 receptor neurons.

The central melanocortin system is composed of neurons that express either the proopiomelanocortin (POMC) or the agouti-related protein (AgRP). POMC is cleaved in bioactive peptides, including the α-melanocyte-stimulating hormone (α-MSH). α-MSH activates the melanocortin-4 receptor (MC4R) inducing satiety, whereas AgRP acts as an inverse agonist of MC4R. However, only limited information is available regarding possible area-specific differences in the interaction between α-MSH and AgRP terminals on MC4R-expressing cells. Therefore, the objective of the present study was to compare the distribution pattern of α-MSH and AgRP terminals on the perikarya of MC4R-expressing neurons. We performed a triple-label immunofluorescence reaction in brain series of MC4R-reporter mice to visualize MC4R-expressing neurons together with AgRP and α-MSH terminals. POMC and AgRP neurons project to areas that contain MC4R-expressing cells, although several brain nuclei exhibit AgRP and α-MSH terminals, but they do no express MC4R, while other brain areas contain MC4R-expressing cells and receive no apparent innervation of AgRP and POMC neurons. AgRP terminals make more presumptive appositions than α-MSH on the soma of MC4R-expressing neurons of the medial preoptic area and paraventricular nucleus of the hypothalamus (Pa). Additionally, a higher percentage of MC4R cells receive at least one presumptive apposition from AgRP terminals in the median preoptic nucleus and Pa, compared to α-MSH appositions. Thus, our study revealed area-specific differences in the interaction between α-MSH and AgRP terminals and the soma of MC4R-expressing neurons. These findings provide new insights about the relationship between first- and second-order neurons of the central melanocortin system.

Annonalide and derivatives: Semisynthesis, cytotoxic activities and studies on interaction of annonalide with DNA.

The cytotoxic activity of the pimarane diterpene annonalide (1) and nine of its semisynthetic derivatives (2-10) was investigated against the human tumor cell lines HL-60 (leukemia), PC-3 (prostate adenocarcinoma), HepG2 (hepatocellular carcinoma), SF-295 (glioblastoma) and HCT-116 (colon cancer), and normal mouse fibroblast (L929) cells. The preparation of 2-10 involved derivatization of the side chain of 1 at C-13. Except for 2, all derivatives are being reported for the first time. Most of the tested compounds presented IC50s below 4.0 µM, being considered potential antitumor agents. The structures of all new compounds were elucidated by spectroscopic analyses including 2D NMR and HRMS. Additionally, the interaction of annonalide (1) with ctDNA was evaluated using spectroscopic techniques, and the formation of a supramolecular complex with the macromolecule was confirmed. Competition assays with fluorescent probes (Hoechst and ethidium bromide) and theoretical studies confirmed that 1 interacts preferentially via DNA intercalation with stoichiometric ratio of 1:1 (1:ctDNA). The ΔG value was calculated as -28.24 kJ mol-1, and indicated that the interaction process occurs spontaneously. Docking studies revealed that van der Walls is the most important interaction in 1-DNA and EB-DNA complexes, and that both ligands (1 and EB) interact with the same DNA residues (DA6, DA17 and DT19).

SOCS3 expression in SF1 cells regulates adrenal differentiation and exercise performance.

Many hormones/cytokines are secreted in response to exercise and cytokine signaling may play a pivotal role in the training adaptations. To investigate the importance of cytokine signaling during vertical ladder climbing, a resistance exercise model, we produced mice lacking SOCS3 protein exclusively in steroidogenic factor-1 (SF1) cells (SF1 Socs3 KO mice). SF1 expression is found in steroidogenic cells of the adrenal cortex and gonads, as well as in neurons of the ventromedial nucleus of the hypothalamus. Histological markers of the fetal adrenal zone (or X-zone in rodents) were still present in adult males and postpartum SF1 Socs3 KO females, suggesting a previously unrecognized effect of SOCS3 on the terminal differentiation of the adrenal gland. This change led to a distinct distribution of lipid droplets along the adrenal cortex. Under basal conditions, adult SF1 Socs3 KO mice exhibited similar adrenal weight, and plasma ACTH and corticosterone concentrations. Nonetheless, SF1 Socs3 KO mice exhibited a blunted ACTH-induced corticosterone secretion. The overall metabolic responses induced by resistance training remained unaffected in SF1 Socs3 KO mice, including changes in body adiposity, glucose tolerance and energy expenditure. However, training performance and glucose control during intense resistance exercise were impaired in SF1 Socs3 KO mice. Furthermore, a reduced counter-regulatory response to 2-deoxy-d-glucose was observed in mutant mice. These findings revealed a novel participation of SOCS3 regulating several endocrine and metabolic aspects. Therefore, cytokine signaling in SF1 cells exerts an important role to sustain training performance possibly by promoting the necessary metabolic adjustments during exercise.

The lateral habenula and the serotonergic system.

The habenula (Hb) is an epithalamic structure differentiated into two nuclear complexes, medial (MHb) and lateral habenula (LHb). After decades of relative neglect, interest in the Hb resurged when it was demonstrated that LHb neurons play a key role in encoding disappointments and expectation of punishments. Consistent with such a role, the LHb has been implicated in a broad array of functions and pathologic conditions, notably in mechanisms of stress and pain, as well as in the pathophysiology of mood disorders. So far, the vast majority of research involving the LHb has focused on its role in regulating midbrain dopamine release. However, the LHb is also robustly interconnected in a reciprocal manner with a set of rostral serotonin (5-HT) nuclei. Thus, there is increasing evidence that the LHb is amply linked to the dorsal (DR) and median raphe nucleus (MnR) by a complex network of parallel topographically organized direct and indirect pathways. Here, we summarize research about the interconnections of the LHb with different subregions of the DR and MnR, as well as findings about 5-HT-dependent modulation of LHb neurons. Finally, we discuss the contribution of distinct LHb-raphe loops to stress and stress-related psychiatric disorders including anxiety and depression.

Afferent and efferent connections of the interpeduncular nucleus with special reference to circuits involving the habenula and raphe nuclei.

The habenula is an epithalamic structure differentiated into two nuclear complexes, medial (MHb) and lateral habenula (LHb). Recently, MHb together with its primary target, the interpeduncular nucleus (IP), have been identified as major players in mediating the aversive effects of nicotine. However, structures downstream of the MHb-IP axis, including the median (MnR) and caudal dorsal raphe nucleus (DRC), may contribute to the behavioral effects of nicotine. The afferent and efferent connections of the IP have hitherto not been systematically investigated with sensitive tracers. Thus, we placed injections of retrograde or anterograde tracers into different IP subdivisions or the MnR and additionally examined the transmitter phenotype of major IP and MnR afferents by combining retrograde tract tracing with immunofluorescence and in situ hybridization techniques. Besides receiving inputs from MHb and also LHb, we found that IP is reciprocally interconnected mainly with midline structures, including the MnR/DRC, nucleus incertus, supramammillary nucleus, septum, and laterodorsal tegmental nucleus. The bidirectional connections between IP and MnR proved to be primarily GABAergic. Regarding a possible topography of IP outputs, all IP subnuclei gave rise to descending projections, whereas major ascending projections, including focal projections to ventral hippocampus, ventrolateral septum, and LHb originated from the dorsocaudal IP. Our findings indicate that IP is closely associated to a distributed network of midline structures that modulate hippocampal theta activity and forms a node linking MHb and LHb with this network, and the hippocampus. Moreover, they support a cardinal role of GABAergic IP/MnR interconnections in the behavioral response to nicotine.

Changes in Leptin Signaling by SOCS3 Modulate Fasting-Induced Hyperphagia and Weight Regain in Mice.

Weight regain frequently follows interventions that reduce body weight, leading to a failure in long-term obesity treatment. Inhibitory proteins of the leptin signaling pathway, such as the suppressor of cytokine signaling 3 (SOCS3), have been studied in conditions that predispose animals to obesity. However, whether SOCS3 modulates postrestriction hyperphagia and weight regain remains unknown. Mice lacking SOCS3 protein specifically in leptin receptor (LepR)-expressing cells (LepR SOCS3 knockout [KO]) were generated and studied in fasting and refeeding conditions. LepR SOCS3 KO mice exhibited increased leptin sensitivity in the hypothalamus. Notably, LepR SOCS3 KO males and females showed attenuated food intake and weight regain after 48 hours of fasting. Postrestriction hyperleptinemia was also prevented in LepR SOCS3 KO mice. Next, we studied possible mechanisms and neural circuits involved in the SOCS3 effects. SOCS3 deletion did not prevent fasting- or refeeding-induced c-Fos expression in the arcuate nucleus of the hypothalamus (ARH) nor fasting-induced increased excitability of ARH LepR-expressing cells. On the other hand, SOCS3 ablation reduced the mRNA levels of hypothalamic orexigenic neuropeptides during fasting (neuropeptide Y, agouti-related protein, orexin, and melanin-concentrating hormone). In summary, our findings suggest that increased leptin sensitivity contributes to the maintenance of a reduced body weight after food deprivation. In addition, the attenuated postrestriction food intake observed in mutant mice was not explained by fasting-induced changes in the activity of ARH neurons but exclusively by a lower transcription of orexigenic neuropeptides during fasting. These results indicate a partial dissociation between the regulation of neuronal activity and gene expression in ARH LepR-expressing cells.

Leptin receptor-positive and leptin receptor-negative proopiomelanocortin neurons innervate an identical set of brain structures.

Neurons that express the prohormone proopiomelanocortin (POMC) in the arcuate hypothalamic nucleus (Arc) are engaged in the regulation of energy balance and glucose homeostasis. Additionally, POMC neurons are considered key first-order cells regulated by leptin. Interestingly, in the Arc, POMC cells that express the leptin receptor (POMC/LepR+ cells) are found side by side with POMC cells not directly responsive to leptin (POMC/LepR- cells). However, it remains unknown whether these distinct populations innervate different target regions. Therefore, the objective of the present study was to compare the projections of POMC/LepR+ and POMC/LepR- neurons. Using genetically modified LepR-reporter mice to identify leptin receptor-expressing cells and immunohistochemistry to stain POMC-derived peptides (α-MSH or ß-endorphin) we confirmed that approximately 80% of Arc ß-endorphin-positive neurons co-expressed leptin receptors. POMC/LepR+ and POMC/LepR- axons were intermingled in all of their target regions. As revealed by confocal microscopy, we found an elevated degree of co-localization between α-MSH+ axons and the reporter protein (tdTomato) in all brain regions analyzed, with co-localization coefficients ranging from 0.889 to 0.701. Thus, these two populations of POMC neurons seem to project to the same set of brain structures, although one of the two subtypes of POMC axons was sometimes found to be more abundant than the other in distinct subregions of the same nucleus. Therefore, POMC/LepR+ and POMC/LepR- cells may target separate neuronal populations and consequently activate distinct neuronal circuits within some target nuclei. These findings contribute to unravel the neuronal circuits involved in the regulation of energy balance and glucose homeostasis.

The centrally projecting Edinger-Westphal nucleus--I: Efferents in the rat brain.

The oculomotor accessory nucleus, often referred to as the Edinger-Westphal nucleus [EW], was first identified in the 17th century. Although its most well known function is the control of pupil diameter, some controversy has arisen regarding the exact location of these preganglionic neurons. Currently, the EW is thought to consist of two different parts. The first part [termed the preganglionic EW-EWpg], which controls lens accommodation, choroidal blood flow and pupillary constriction, primarily consists of cholinergic cells that project to the ciliary ganglion. The second part [termed the centrally projecting EW-EWcp], which is involved in non-ocular functions such as feeding behavior, stress responses, addiction and pain, consists of peptidergic neurons that project to the brainstem, the spinal cord and prosencephalic regions. However, in the literature, we found few reports related to either ascending or descending projections from the EWcp that are compatible with its currently described functions. Therefore, the objective of the present study was to systematically investigate the ascending and descending projections of the EW in the rat brain. We injected the anterograde tracer biotinylated dextran amine into the EW or the retrograde tracer cholera toxin subunit B into multiple EW targets as controls. Additionally, we investigated the potential EW-mediated innervation of neuronal populations with known neurochemical signatures, such as melanin-concentrating hormone in the lateral hypothalamic area [LHA] and corticotropin-releasing factor in the central nucleus of the amygdala [CeM]. We observed anterogradely labeled fibers in the LHA, the reuniens thalamic nucleus, the oval part of the bed nucleus of the stria terminalis, the medial part of the central nucleus of the amygdala, and the zona incerta. We confirmed our EW-LHA and EW-CeM connections using retrograde tracers. We also observed moderate EW-mediated innervation of the paraventricular nucleus of the hypothalamus and the posterior hypothalamus. Our findings provide anatomical bases for previously unrecognized roles of the EW in the modulation of several physiologic systems.