All procedures were approved by the University of Georgia Animal Care and Use Committee, and followed the guidelines of the International Association of the Study of Pain and the National Institutes of Health. Rimonabant was obtained from NIDA. URB597 was purchased from Cayman . URB602 was synthesized by reacting diimidazole- 1-ylmethanone with biphenyl-3-yl amine in acetonitrile in the presence of 4-dimethylaminopyradine and subsequently with cyclohexanol as described previously. Animals were anesthetized with a mixture of sodium pentobarbital and ketamine. Stainless steel guide cannulae were unilaterally implanted above either the BLA or CeA using zero points from bregma, the midline suture and the surface of the skull, respectively. Cannulae were fixed to the skull using skull screws and dental acrylic. Five to seven days after surgery, rats were habituated to restraining tubes prior to testing. The latency to remove the tail from a radiant heat source was measured using the tail-flick test. Drug or vehicle was micro-injected using a micro-infusion pump over 60 s into either the BLA, CeA or deliberately off-site. In experiment, rimonabant or vehicle was micro-injected into either the BLA or CeA 5 min prior to foot shock. In experiment, URB597 , URB602 ,rolling tables grow or vehicle was administered to the BLA 32 min prior to foot shock. Doses and delays were selected based upon previous studies demonstrating efficacy of the identical drug treatments following micro-injection into the midbrain PAG.
SIA was induced by exposing rats to continuous foot shock using a Lafayette grid-shock apparatus and quantified behaviorally using the radiant heat tail-flick test. Removal of the tail from the heat source terminated application of thermal stimulation. Tail- flick latencies were monitored over 4 min immediately prior to exposure to the stressor to evaluate changes in basal nociceptive thresholds induced by pharmacological manipulations. Tail withdrawal latencies were measured at 2-min intervals before and after foot shock. A ceiling tail-flick latency of 10 s was employed to prevent tissue damage. In all studies, the experimenter was blinded to the experimental condition. Following testing, rats were euthanized with sodium pentobarbital and perfused with saline followed by formalin. Brains were removed, cryoprotected overnight, cryostat-cut , and mounted onto gelatin-subbed slides. Sections were dried and stained with cresyl violet. Injection sites were confirmed histologically using a light microscope. micro-injection sites were confirmed for thirty-seven animals in the BLA and fifteen animals in the CeA . Eleven animals were used as off-site controls. Only animals with histologically confirmed micro-injection sites were included in data for analysis. Tail flick data were blocked for each subject by averaging every two adjacent tail-flick latencies into a single mean, as described previously. Means of two-trial blocks, calculated for each subject, were subjected to repeated measures analysis of variance and ANOVA, as appropriate. Post hoc comparisons were performed using the Fisher’s protected least squares difference , with P < 0.05 considered significant.In all studies, baseline tail-flick latencies did not differ between groups prior to administration of drug or vehicle. Moreover, latencies recorded just prior to foot shock, following injection of drug or vehicle, were similar between groups, indicating that the injection alone was not sufficient to induce antinociception. In all studies, foot shock increased tail-flick latencies .
Post-shock tail-flick latencies were attenuated in rats receiving intra-BLA micro-injection of rimonabant relative to vehicle 5.764; P < 0.04. Intra-BLA micro-injection of rimonabant also decreased stress antinociception compared with off-site controls receiving the same dose 5.181; P < 0.04. By contrast, intra-CeA micro-injection of rimonabant did not alter tail-flick latencies relative to vehicle . Neither the FAAH inhibitor URB597 nor the MGL inhibitor URB602 altered SIA , at doses that markedly enhanced SIA following micro-injection into the midbrain PAG. The present study demonstrates that pharmacological blockade of CB1 receptors in the BLA attenuates nonopioid SIA induced by continuous foot shock. Micro-injection of rimonabant into the BLA, where CB1 receptors are dense, suppressed stress-induced antinociception relative to control conditions. By contrast, micro-injection of rimonabant into the CeA, where CB1 receptors are largely absent, failed to suppress nonopioid SIA. Micro-injection of rimonabant into regions outside the amygdala also failed to suppress SIA, suggesting that the actions of the cannabinoid antagonist were not due to diffusion to distal sites. Our data are consistent with the observation that CB1 agonists depress monosynaptic evoked inhibitory post-synaptic potentials in the BLA but not in the CeA. Our results, therefore, suggest that CB1 receptors in the BLA modulate local inhibitory networks in the BLA to ultimately regulate expression of SIA. Nonetheless, neither the FAAH inhibitor URB597 nor the MGL inhibitor URB602 enhanced SIA following micro-injection into the BLA, at doses that markedly potentiated SIA following micro-injection into the midbrain dPAG. These differences likely reflect differential modulatory roles of distinct endocannabinoids in the ascending “affective” pain pathway compared to descending pain modulatory systems. Anatomical studies suggest that CB1 is preferentially associated with GABAergic, as opposed to glutamatergic, synapses in the BLA. Nonetheless, in the lateral amygdala, endocannabinoids mediate reductions in both local inhibitory inputs as well as excitatory transmission, whose actions could exert opposing effects. Low frequency stimulation of the lateral amygdala also mobilizes endocannabinoids from BLA neurons to presynaptically induce a long-term depression of inhibitory GABAergic transmission.
Endocannabinoid LTDi in the BLA in turn, enhances excitatory synaptic transmission in the CeA. A specifific role for anandamide, but not 2-AG, in this form of neuronal plasticity is suggested by two complementary observations. First, LTDi is enhanced in FAAH−/− mice, which are impaired in their ability to metabolize anandamide. Second, endocannabinoid mobilization in LTDi apparently requires the activation of the adenylyl-cyclase-protein kinase A pathway in the BLA, but not the phospholipase C-diacylglycerol lipase pathway that is implicated in 2-AG formation.This latter finding is in contrast to our observations that 2-AG appears to be the primary endocannabinoid implicated in nonopioid SIA; a strong temporal correspondence exists between cannabinoid SIA and the accumulation of 2-AG,pipp grow racks but not anandamide, in the PAG. Liquid chromatography mass spectrometric studies are required to determine how foot shock-induced endocannabinoid mobilization in the BLA differs from that observed previously in the PAG. It is also possible that changes in SIA could be more prominent when supraspinally-mediated measures of antinociception are employed that are more sensitive to the affective dimensions of pain . Our data do not preclude the possibility that the doses of FAAH and MGL inhibitors employed here were unable to surmount high levels of FAAH and MGL activity at this site relative to the PAG. It is also possible that enzymes other than FAAH or MGL may participate in endocannabinoid deactivation at these sites. We propose that CB1 receptor activation in the BLA removes inhibitory control over projection neurons innervating the CeA. The CeA thus receives multi-modal sensory information, and in turn, coordinates appropriate behavioral, hormonal, and autonomic responses to stress via efferent projections. The lack of effect of the FAAH inhibitor in our study is consistent with previous work demonstrating that FAAH−/− mice and mice treated with URB597, administered systemically, show similar amygdalar activation in response to restraint stress compared to control mice. In the BLA, restraint stress produced a low level of Fos induction, which was unaffected by cannabinoid treatment, whereas, the combination of restraint stress and CB1 agonist administration produced robust Fos induction within the CeA. These data support a synergistic interaction between environmental stress and CB1 receptor activation in the amygdala that could contribute to the behavioral phenotype observed here. CB1 as well as FAAH and MGL immunore activity are abundant in the BLA, suggesting that incoming stimuli may trigger the on-demand formation of endocannabinoids to activate CB1 receptors prior to undergoing enzymatic hydrolysis. Activation of CB1 receptors in the BLA, in turn, reduce the inhibitory tone exerted on principal neurons, eventually propagating this signal to brain regions implicated in antinociception, most notably the PAG.In the dPAG, foot shock stress stimulates mobilization of the endocannabinoids, 2-AG and anandamide, and micro-injection of CB1 antagonists into this site virtually abolishes nonopioid SIA. Our results suggest that endocannabinoids may be differentially modulated by stress in the amygdala. For example, in the amygdala, anandamide levels are decreased after the first exposure to restraint stress whereas 2-AG levels are unchanged. Our results collectively suggest that the facilitatory effects of endocannabinoids in Specifically enhancing antinociceptive responses to foot shock stress occur downstream of receptor interactions in the BLA.
In a variety of ways, Colorado remains a state in transition. As is the case with many states around the nation, the economy continues to improve from the recession. Demographically, the state is becoming increasingly diverse. According to the census, more than one-fifth of state residents were Hispanic or Latino/a in 2015. Colorado’s population growth of nearly eight percent since 2010 ranks third behind only Texas and North Dakota. Politically, Colorado has been trending blue over the past several election cycles. Some have identified the migration of Californians into Colorado as a contributing factor to the state leaning further in the Democratic direction . The state awarded its Electoral College votes in 2008 and 2012 to the Democratic ticket, and the party appears poised to carry the state in 2016. A streak of two consecutive wins for the Democratic Party’s presidential candidates has not happened since Colorado voters supported Franklin Roosevelt in 1932 and 1936. Democrats emerged victorious in the last three gubernatorial elections and won three consecutive elections to the U.S. Senate beginning in 2004. The state’s march toward blue-state status was interrupted in 2014, however, as Republican Representative Cory Gardner defeated incumbent Democratic Senator Mark Udall in a competitive race. Republican candidates in this election cycle swept three of the remaining four statewide races, and the GOP regained control of one chamber of the state legislature. The single bright spot for Democrats on election night 2014 was the reelection of incumbent Governor John Hickenlooper who narrowly edged out Republican challenger Bob Beauprez. Colorado continues to receive national and international attention over the legalization of recreational marijuana, which has important budgetary implications. Though revenues associated with legalized recreational marijuana have fallen short of early government forecasts, marijuana tax revenue continues to grow steadily. The sale of marijuana to adults over the age of 21 became legal on January 1, 2014. By the end of the year, the state received $44 million in revenue from recreational marijuana. Combined with the preexisting medical marijuana market, the state received $76 million in 2014 .1 Despite an improving economy and a new stream of marijuana tax revenue, legislators remain wary about approving new spending measures. This is partly attributable to uncertainty regarding potential tax rebates mandated by the Taxpayer’s Bill of Rights . When submitting requests to the Office of State Planning and Budget , departments must outline a strategic plan to accompany their request. The governor’s budget request is submitted to the legislature in the fall. After consideration by the Joint Budget Committee , the full legislature typically passes the budget in May in time for the start of the fiscal year on July 1. The constitution mandates a balanced budget. Last November, the priorities reflected in Governor Hickenlooper’s proposed budget for the 2015–2016 fiscal year were similar to those in prior budgets drafted by the governor in collaboration with the OSPB. In his accompanying letter to the six-member JBC, the governor emphasized “enrollment and inflation increases for K-12 education, the return of General Fund support for transportation for the first time since FY 2007‒08 pursuant to S.B. 09‒228, increased caseload in the State’s Medicaid program, an anticipated decrease in the federal Medicaid match rate, the continuation of existing capital construction projects, and essential projects for the state’s information technology infrastructure” . Spending in just two areas—K-12 education and health and human services—constitute two-thirds of all proposed General Fund appropriations. Governor Hickenlooper’s budget letter detailed how economic progress in Colorado has outpaced the nationwide recovery. In that sense, the budget reflects a cautious optimism about the state’s economic well being. The Bureau of Labor Statistics reports that Colorado’s seasonally adjusted unemployment rate in December 2014 was just 4.0 percent. Tied for seventh lowest in the country , it is a product of 35 consecutive months of job growth. The national unemployment rate for the same month was 5.6 percent. After a decrease in gross state product in 2009, the state has logged increases averaging about 2.5 percent a year for the past four years, and per capita income levels have increased. Against this backdrop of economic improvement, the governor’s budget includes $26.8 billion in total spending with a General Fund allocation of $10.3 billion. The sums represent spending increases from the prior fiscal year of 7.0 percent in total funds and 9.6 percent from the General Fund .