RESUMEN
Cannabinoids are known to exert immunosuppressive activities. However, the mechanisms which contribute to these effects are unknown. Using lipopolysaccharide (LPS) to activate BV-2 microglial cells, we examined how Δ(9)-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, and cannabidiol (CBD) the non-psychoactive component, modulate the inflammatory response. Microarray analysis of genome-wide mRNA levels was performed using Illumina platform and the resulting expression patterns analyzed using the Ingenuity Pathway Analysis to identify functional subsets of genes, and the Ingenuity System Database to denote the gene networks regulated by CBD and THC. From the 5338 transcripts that were differentially expressed across treatments, 400 transcripts were found to be upregulated by LPS, 502 by CBD+LPS and 424 by THC+LPS, while 145 were downregulated by LPS, 297 by CBD+LPS and 149 by THC+LPS, by 2-fold or more (p≤0.005). Results clearly link the effects of CBD and THC to inflammatory signaling pathways and identify new cannabinoid targets in the MAPK pathway (Dusp1, Dusp8, Dusp2), cell cycle related (Cdkn2b, Gadd45a) as well as JAK/STAT regulatory molecules (Socs3, Cish, Stat1). The impact of CBD on LPS-stimulated gene expression was greater than that of THC. We attribute this difference to the fact that CBD highly upregulated several genes encoding negative regulators of both NFκB and AP-1 transcriptional activities, such as Trib3 and Dusp1 known to be modulated through Nrf2 activation. The CBD-specific expression profile reflected changes associated with oxidative stress and glutathione depletion via Trib3 and expression of ATF4 target genes. Furthermore, the CBD affected genes were shown to be controlled by nuclear factors usually involved in regulation of stress response and inflammation, mainly via Nrf2/Hmox1 axis and the Nrf2/ATF4-Trib3 pathway. These observations indicate that CBD, and less so THC, induce a cellular stress response and that this response underlies their high immunosuppressant activities.
Asunto(s)
Cannabinoides/farmacología , Perfilación de la Expresión Génica , Lipopolisacáridos/farmacología , Microglía/efectos de los fármacos , Microglía/metabolismo , Transducción de Señal , Animales , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los ResultadosRESUMEN
In the vibrissal system, touch information is conveyed by a receptorless whisker hair to follicle mechanoreceptors, which then provide input to the brain. We examined whether any processing, that is, meaningful transformation, occurs in the whisker itself. Using high-speed videography and tracking the movements of whiskers in anesthetized and behaving rats, we found that whisker-related morphological phase planes, based on angular and curvature variables, can represent the coordinates of object position after contact in a reliable manner, consistent with theoretical predictions. By tracking exposed follicles, we found that the follicle-whisker junction is rigid, which enables direct readout of whisker morphological coding by mechanoreceptors. Finally, we found that our behaving rats pushed their whiskers against objects during localization in a way that induced meaningful morphological coding and, in parallel, improved their localization performance, which suggests a role for pre-neuronal morphological computation in active vibrissal touch.
Asunto(s)
Vías Aferentes/fisiología , Mapeo Encefálico , Folículo Piloso/fisiología , Mecanorreceptores/fisiología , Movimiento/fisiología , Vibrisas/inervación , Análisis de Varianza , Anestésicos/farmacología , Animales , Fenómenos Biomecánicos , Masculino , Estimulación Física , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Factores de Tiempo , Grabación en Video , VigiliaRESUMEN
Whisking mediated touch is an active sense whereby whisker movements are modulated by sensory input and behavioral context. Here we studied the effects of touching an object on whisking in head-fixed rats. Simultaneous movements of whiskers C1, C2, and D1 were tracked bilaterally and their movements compared. During free-air whisking, whisker protractions were typically characterized by a single acceleration-deceleration event, whisking amplitude and velocity were correlated, and whisk duration correlated with neither amplitude nor velocity. Upon contact with an object, a second acceleration-deceleration event occurred in about 25% of whisk cycles, involving both contacting (C2) and non-contacting (C1, D1) whiskers ipsilateral to the object. In these cases, the rostral whisker (C2) remained in contact with the object throughout the double-peak phase, which effectively prolonged the duration of C2 contact. These "touch-induced pumps" (TIPs) were detected, on average, 17.9 ms after contact. On a slower time scale, starting at the cycle following first touch, contralateral amplitude increased while ipsilateral amplitude decreased. Our results demonstrate that sensory-induced motor modulations occur at various timescales, and directly affect object palpation.
Asunto(s)
Retroalimentación Sensorial/fisiología , Tacto , Vibrisas , Animales , Movimientos de la Cabeza , Masculino , Ratas , Ratas Wistar , Percepción del Tacto/fisiologíaRESUMEN
BACKGROUND AND PURPOSE: Apart from their effects on mood and reward, cannabinoids exert beneficial actions such as neuroprotection and attenuation of inflammation. The immunosuppressive activity of cannabinoids has been well established. However, the underlying mechanisms are largely unknown. We previously showed that the psychoactive cannabinoid Δ(9) -tetrahydrocannabinol (THC) and the non-psychoactive cannabidiol (CBD) differ in their anti-inflammatory signalling pathways. EXPERIMENTAL APPROACH: To characterize the transcriptional effects of CBD and THC, we treated BV-2 microglial cells with these compounds and performed comparative microarray analysis using the Illumina MouseRef-8 BeadChip platform. Ingenuity Pathway Analysis was performed to identify functional subsets of genes and networks regulated by CBD and/or THC. KEY RESULTS: Overall, CBD altered the expression of many more genes; from the 1298 transcripts found to be differentially regulated by the treatments, 680 gene probe sets were up-regulated by CBD and 58 by THC, and 524 gene products were down-regulated by CBD and only 36 by THC. CBD-specific gene expression profile showed changes associated with oxidative stress and glutathione depletion, normally occurring under nutrient limiting conditions or proteasome inhibition and involving the GCN2/eIF2α/p8/ATF4/CHOP-TRIB3 pathway. Furthermore, CBD-stimulated genes were shown to be controlled by nuclear factors known to be involved in the regulation of stress response and inflammation, mainly via the (EpRE/ARE)-Nrf2/ATF4 system and the Nrf2/Hmox1 axis. CONCLUSIONS AND IMPLICATIONS: These observations indicated that CBD, but much less than THC, induced a cellular stress response in microglial cells and suggested that this effect could underlie its anti-inflammatory activity. LINKED ARTICLES: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.
Asunto(s)
Cannabidiol/farmacología , Dronabinol/farmacología , Microglía/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Animales , Línea Celular , Perfilación de la Expresión Génica , Glutatión/genética , Ratones , Microglía/metabolismo , Estrés OxidativoRESUMEN
In order to identify basic aspects in the process of tactile perception, we trained rats and humans in similar object localization tasks and compared the strategies used by the two species. We found that rats integrated temporally related sensory inputs ('temporal inputs') from early whisk cycles with spatially related inputs ('spatial inputs') to align their whiskers with the objects; their perceptual reports appeared to be based primarily on this spatial alignment. In a similar manner, human subjects also integrated temporal and spatial inputs, but relied mainly on temporal inputs for object localization. These results suggest that during tactile object localization, an iterative motor-sensory process gradually converges on a stable percept of object location in both species.
Asunto(s)
Propiocepción/fisiología , Sensación/fisiología , Vibrisas/fisiología , Animales , Conducta Animal/fisiología , Conducta Exploratoria/fisiología , Femenino , Humanos , Masculino , Ratas , Percepción Espacial/fisiología , Estadísticas no Paramétricas , Grabación en VideoRESUMEN
Whisking is controlled by multiple, possibly functionally segregated, motor sensory-motor loops. While testing for effects of endocannabinoids on whisking, we uncovered the first known functional segregation of channels controlling whisking amplitude and timing. Channels controlling amplitude, but not timing, were modulated by cannabinoid receptor type 1 (CB1R). Systemic administration of CB1R agonist Δ(9)-tetrahydrocannabinol (Δ(9)-THC) reduced whisking spectral power across all tested doses (1.25-5 mg/kg), whereas whisking frequency was affected at only very high doses (5 mg/kg). Concomitantly, whisking amplitude and velocity were significantly reduced in a dose-dependent manner (25-43 and 26-50%, respectively), whereas cycle duration and bilateral synchrony were hardly affected (3-16 and 3-9%, respectively). Preadministration of CB1R antagonist SR141716A blocked Δ(9)-THC-induced kinematic alterations of whisking, and when administered alone, increased whisking amplitude and velocity but affected neither cycle duration nor synchrony. These findings indicate that whisking amplitude and timing are controlled by separate channels and that endocannabinoids modulate amplitude control channels.
Asunto(s)
Antagonistas de Receptores de Cannabinoides , Moduladores de Receptores de Cannabinoides/fisiología , Dronabinol/farmacología , Vibrisas/fisiología , Animales , Relación Dosis-Respuesta a Droga , Femenino , Piperidinas/farmacología , Pirazoles/farmacología , Ratas , Ratas Wistar , Rimonabant , Vibrisas/efectos de los fármacosRESUMEN
Cannabinoids have been shown to exert anti-inflammatory activities in various in vivo and in vitro experimental models as well as ameliorate various inflammatory degenerative diseases. However, the mechanisms of these effects are not completely understood. Using the BV-2 mouse microglial cell line and lipopolysaccharide (LPS) to induce an inflammatory response, we studied the signaling pathways engaged in the anti-inflammatory effects of cannabinoids as well as their influence on the expression of several genes known to be involved in inflammation. We found that the two major cannabinoids present in marijuana, Delta(9)-tetrahydrocannabinol (THC) and cannabidiol (CBD), decrease the production and release of proinflammatory cytokines, including interleukin-1beta, interleukin-6, and interferon (IFN)beta, from LPS-activated microglial cells. The cannabinoid anti-inflammatory action does not seem to involve the CB1 and CB2 cannabinoid receptors or the abn-CBD-sensitive receptors. In addition, we found that THC and CBD act through different, although partially overlapping, mechanisms. CBD, but not THC, reduces the activity of the NF-kappaB pathway, a primary pathway regulating the expression of proinflammatory genes. Moreover, CBD, but not THC, up-regulates the activation of the STAT3 transcription factor, an element of homeostatic mechanism(s) inducing anti-inflammatory events. Following CBD treatment, but less so with THC, we observed a decreased level of mRNA for the Socs3 gene, a main negative regulator of STATs and particularly of STAT3. However, both CBD and THC decreased the activation of the LPS-induced STAT1 transcription factor, a key player in IFNbeta-dependent proinflammatory processes. In summary, our observations show that CBD and THC vary in their effects on the anti-inflammatory pathways, including the NF-kappaB and IFNbeta-dependent pathways.
Asunto(s)
Cannabidiol/farmacología , Dronabinol/farmacología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lipopolisacáridos/farmacología , Microglía/efectos de los fármacos , FN-kappa B/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Inflamación/metabolismo , Interferón beta/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Ratones , Microglía/citología , Microglía/metabolismo , Factores de Transcripción STAT/metabolismoRESUMEN
We examined how lipopolysaccharide (LPS) and interferon gamma (IFN-gamma), known to differentially activate microglia, affect the expression of G protein-coupled receptor 55 (GPR55), a novel cannabinoid receptor. We found that GPR55 mRNA is significantly expressed in both primary mouse microglia and the BV-2 mouse microglial cell line, and that LPS down-regulates this message. Conversely, IFN-gamma slightly decreases GPR55 mRNA in primary microglia, while it upregulates this message in BV-2 cells. Moreover, the GPR55 agonist, lysophosphatidylinositol, increases ERK phosphorylation in BV-2 stimulated with IFN-gamma, in correlation with the increased amount of GPR55 mRNA. Remarkably, these stimuli-induced changes in GPR55 expression are similar to those observed with CB(2)-R, suggesting that both receptors might be involved in neuroinflammation and that their expression is concomitantly controlled by the state of microglial activation.
Asunto(s)
Microglía/metabolismo , Receptores de Cannabinoides/genética , Receptores de Cannabinoides/metabolismo , Animales , Secuencia de Bases , Agonistas de Receptores de Cannabinoides , Línea Celular , Células Cultivadas , Cartilla de ADN/genética , Expresión Génica/efectos de los fármacos , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Lisofosfolípidos/farmacología , Ratones , Microglía/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas RecombinantesRESUMEN
Anandamide (AEA) is a lipid molecule belonging to the family of endocannabinoids. Various studies report neuroprotective activity of AEA against toxic insults, such as ischemic conditions and excitotoxicity, whereas some show that AEA has pro-apoptotic effects. Here we have shown that AEA confers a protective activity in N18TG2 murine neuroblastoma cells subjected to low serum-induced apoptosis. We have demonstrated that the protection from apoptosis by AEA is not mediated via the CB1 receptor, the CB2 receptor, or the vanilloid receptor 1. Interestingly, breakdown of AEA by fatty acid amide hydrolase is required for the protective effect of AEA. Furthermore, the ethanolamine (EA) generated in this reaction is the metabolite responsible for the protective response. The elevation in the levels of reactive oxygen species during low serum-induced apoptosis is not affected by AEA or EA. On the other hand, AEA and EA reduce caspase 3/7 activity, and AEA attenuates the cleavage of PARP-1. Taken together, our results demonstrate a role for AEA and EA in the protection against low serum-induced apoptosis.
Asunto(s)
Apoptosis , Ácidos Araquidónicos/farmacología , Etanolamina/metabolismo , Alcamidas Poliinsaturadas/farmacología , Amidohidrolasas/metabolismo , Animales , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Endocannabinoides , Citometría de Flujo , Isquemia , Ratones , Neuroblastoma/metabolismo , Especies Reactivas de Oxígeno , Receptores de Cannabinoides/metabolismo , Canales Catiónicos TRPV/metabolismoRESUMEN
Using their large mystacial vibrissas, rats perform a variety of tasks, including localization and identification of objects. We report on the discriminatory thresholds and behavior of rats trained in a horizontal object localization task. Using an adaptive training procedure, rats learned to discriminate offsets in horizontal (anteroposterior) location with all, one row, or one arc of whiskers intact, but not when only a single whisker (C2) was intact on each cheek. However, rats initially trained with multiple whiskers typically improved when retested later with a single whisker intact. Individual rats reached localization thresholds as low as 0.24 mm (approximately 1 degree). Among the tested groups, localization acuity was finest (<1.5 mm) with rats that were initially trained with all whiskers and then trimmed to one arc of whiskers intact. Horizontal acuity was finer than the typical inter-vibrissal spacing (approximately 4.8 mm at contact points). Performance correlated with the net whisking spectral power in the range of 5-25 Hz but not in nonwhisking range of 30-50 Hz. Lesioning the facial motor nerves reduced performance to chance level. We conclude that horizontal object localization in the rat vibrissal system can reach hyperacuity level and is an active sensing process: whisker movements are both required and beneficiary, in a graded manner, for making accurate positional judgments.
Asunto(s)
Discriminación en Psicología/fisiología , Movimiento/fisiología , Ratas/fisiología , Ratas/psicología , Sensación/fisiología , Vibrisas/fisiología , Animales , Aprendizaje Discriminativo/fisiología , Movimientos de la Cabeza/fisiología , Masculino , Ratas Wistar , Factores de TiempoRESUMEN
Mammals acquire much of their sensory information by actively moving their sensory organs. Rats, in particular, scan their surrounding environment with their whiskers. This form of active sensing induces specific patterns of temporal encoding of sensory information, which are based on a conversion of space into time via sensor movement. We investigate the ways in which object location is encoded by the whiskers and decoded by the brain. We recorded from first-order neurons located in the trigeminal ganglion (TG) of anaesthetized rats during epochs of artificial whisking induced by electrical stimulation of the facial motor nerve. We found that TG neurons encode the three positional coordinates with different codes. The horizontal coordinate (along the backward-forward axis) is encoded by two encoding schemes, both relying on the firing times of one type of TG neuron, the 'contact cell'. The radial coordinate (from face outward) is encoded primarily by the firing magnitude of another type of TG neurons, the 'pressure cell'. The vertical coordinate (from ground up) is encoded by the identity of activated neurons. The decoding schemes of at least some of these sensory cues, our data suggest, are also active: cortical representations are generated by a thalamic comparison of cortical expectations with incoming sensory data.