Therapeutic potential of inhibitors of endocannabinoid degradation for the treatment of stress-related hyperalgesia in an animal model of chronic pain.

The occurrence of chronic stress, depression, and anxiety can increase nociception in humans and may facilitate the transition from localized to chronic widespread pain. The mechanisms underlying chronic widespread pain are still unknown, hindering the development of effective pharmacological therapies. Here, we exposed C57BL/6J mice to chronic unpredictable stress (CUS) to investigate how persistent stress affects nociception. Next, mice were treated with multiple intramuscular nerve growth factor (NGF) injections, which induced chronic widespread nociception. Thus, combination of CUS and NGF served as a model where psychophysiological impairment coexists with long-lasting hyperalgesia. We found that CUS increased anxiety- and depression-like behavior and enhanced basal nociception in mice. When co-applied with repeated NGF injections, CUS elicited a sustained long-lasting widespread hyperalgesia. In order to evaluate a potential therapeutic strategy for the treatment of chronic pain associated with stress, we hypothesized that the endocannabinoid system (ECS) may represent a target signaling system. We found that URB597, an inhibitor of the anandamide-degrading enzyme fatty acid amide hydrolase (FAAH), and JZL184, an inhibitor of the 2-arachidonoyl glycerol-degrading enzyme monoacylglycerol lipase (MAGL), increased eCB levels in the brain and periphery and were both effective in reducing CUS-induced anxiety measured by the light-dark test and CUS-induced thermal hyperalgesia. Remarkably, the long-lasting widespread hyperalgesia induced by combining CUS and NGF was effectively reduced by URB597, but not by JZL184. Simultaneous inhibition of FAAH and MAGL did not improve the overall therapeutic response. Therefore, our findings indicate that enhancement of anandamide signaling with URB597 is a promising pharmacological approach for the alleviation of chronic widespread nociception in stress-exposed mice, and thus, it could represent a potential treatment strategy for chronic pain associated with neuropsychiatric disorders in humans.

The endocannabinoid system controls food intake via olfactory processes.

Hunger arouses sensory perception, eventually leading to an increase in food intake, but the underlying mechanisms remain poorly understood. We found that cannabinoid type-1 (CB1) receptors promote food intake in fasted mice by increasing odor detection. CB1 receptors were abundantly expressed on axon terminals of centrifugal cortical glutamatergic neurons that project to inhibitory granule cells of the main olfactory bulb (MOB). Local pharmacological and genetic manipulations revealed that endocannabinoids and exogenous cannabinoids increased odor detection and food intake in fasted mice by decreasing excitatory drive from olfactory cortex areas to the MOB. Consistently, cannabinoid agonists dampened in vivo optogenetically stimulated excitatory transmission in the same circuit. Our data indicate that cortical feedback projections to the MOB crucially regulate food intake via CB1 receptor signaling, linking the feeling of hunger to stronger odor processing. Thus, CB1 receptor-dependent control of cortical feedback projections in olfactory circuits couples internal states to perception and behavior.

Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions.

A major goal in current neuroscience is to understand the causal links connecting protein functions, neural activity, and behavior. The cannabinoid CB1 receptor is expressed in different neuronal subpopulations, and is engaged in fine-tuning excitatory and inhibitory neurotransmission. Studies using conditional knock-out mice revealed necessary roles of CB1 receptor expressed in dorsal telencephalic glutamatergic neurons in synaptic plasticity and behavior, but whether this expression is also sufficient for brain functions is still to be determined. We applied a genetic strategy to reconstitute full wild-type CB1 receptor functions exclusively in dorsal telencephalic glutamatergic neurons and investigated endocannabinoid-dependent synaptic processes and behavior. Using this approach, we partly restored the phenotype of global CB1 receptor deletion in anxiety-like behaviors and fully restored hippocampus-dependent neuroprotection from chemically induced epileptiform seizures. These features coincided with a rescued hippocampal depolarization-induced suppression of excitation (DSE), a CB1 receptor-dependent form of synaptic plasticity at glutamatergic neurons. By comparison, the rescue of the CB1 receptor on dorsal telencephalic glutamatergic neurons prolonged the time course of DSE in the amygdala, and impaired fear extinction in auditory fear conditioning. These data reveal that CB1 receptor in dorsal telencephalic glutamatergic neurons plays a sufficient role to control neuronal functions that are in large part hippocampus-dependent, while it is insufficient for proper amygdala functions, suggesting an unexpectedly complex circuit regulation by endocannabinoid signaling in the amygdala. Our data pave the way to a better understanding of neuronal networks in the context of behavior, by fine-tuned interference with synaptic transmission processes.

Developmental programming of energy balance and its hypothalamic regulation.

Developmental programming is an important physiological process that allows different phenotypes to originate from a single genotype. Through plasticity in early life, the developing organism can adopt a phenotype (within the limits of its genetic background) that is best suited to its expected environment. In humans, together with the relative irreversibility of the phenomenon, the low predictive value of the fetal environment for later conditions in affluent countries makes it a potential contributor to the obesity epidemic of recent decades. Here, we review the current evidence for developmental programming of energy balance. For a proper understanding of the subject, knowledge about energy balance is indispensable. Therefore, we first present an overview of the major hypothalamic routes through which energy balance is regulated and their ontogeny. With this background, we then turn to the available evidence for programming of energy balance by the early nutritional environment, in both man and rodent models. A wealth of studies suggest that energy balance can indeed be permanently affected by the early-life environment. However, the direction of the effects of programming appears to vary considerably, both between and within different animal models. Because of these inconsistencies, a comprehensive picture is still elusive. More standardization between studies seems essential to reach veritable conclusions about the role of developmental programming in adult energy balance and obesity.

Neonatal food restriction permanently alters rat body dimensions and energy intake.

Neonatal food restriction (FR) in rats, by means of increased litter size, has been used as a model for developmental programming by several investigators. However, the results reported have been inconsistent and difficult to compare between studies. In the present study, we aim to characterize the effects of this model throughout life in both sexes of one particular strain. On the second day of life, Wistar rat pups were randomly assigned to a litter of 10 (control) or 20 (FR). All litters had an equal number of males and females, and pups were weaned on day 25. Body dimensions and food intake were measured regularly until the age of one year. Serum leptin levels were determined in four subsets of different ages. FR acutely reduced growth in all body dimensions and serum leptin levels. Despite catch-up after weaning, all these parameters remained reduced throughout life. Male and female FR rats had a significantly reduced absolute energy intake throughout life. Male FR rats had significantly higher energy intake adjusted for body weight immediately after weaning. During catch-up growth, both FR males and females showed significantly enhanced feed efficiency. These results suggest that neonatal food restriction programmed both male and female Wistar rats to remain small and lean in adult life, with a lower food intake. Low neonatal leptin levels may play a mechanistic role in this process.

Hypothalamic neuropeptide expression of juvenile and middle-aged rats after early postnatal food restriction.

Rats subjected to early postnatal food restriction (FR) show persistent changes in energy balance. The hypothalamus plays a major role in the regulation of energy balance. Therefore, we hypothesized that early postnatal food restriction induces developmental programming of hypothalamic gene expression of neuropeptides involved in this regulation. In the hypothalamus of juvenile and middle-aged rats that were raised in control (10 pups) or FR litters (20 pups), gene expression was investigated for neuropeptide Y (NPY), agouti-related protein (AgRP), proopiomelanocortin (POMC), and cocaine- and amphetamine-regulated transcript (CART) in the arcuate nucleus (ARC); CRH and TRH in the paraventricular nucleus; and melanin-concentrating hormone (MCH) and orexin in the lateral hypothalamic area. Early postnatal FR acutely and persistently reduced body size. Juvenile FR rats had significantly reduced CART gene expression and increased MCH expression. In middle-aged FR rats, POMC and CART mRNA levels were significantly reduced. The ratio between expression of the ARC orexigenic peptides (NPY and AgRP) and anorexigenic peptides (POMC and CART) was increased in juvenile, but not in middle-aged, FR rats. These results suggest that in neonatal rats, FR already triggers the ARC, and to a lesser extent the lateral hypothalamic area, but not the paraventricular nucleus, to increase expression of orexigenic relative to anorexigenic peptides. In addition, with enduring small body size and normalized hypothalamic gene expression, the adult FR rats appeared to have accepted this smaller body size as normal. This suggests that the body weight set-point was differently programmed in animals with early postnatal FR.

Energy intake and resting energy expenditure in adult male rats after early postnatal food restriction.

Both in man and in animal models, changes in food intake and body composition in later life have been reported after alterations in perinatal nutrition. Therefore, we hypothesised that early postnatal undernutrition in the rat induces permanent changes in energy balance. Food restriction (FR) during lactation was achieved by enlarging litter size to twenty pups, whereas control animals were raised in litters containing ten pups. Energy intake and resting energy expenditure were determined in adult males. Early postnatal FR resulted in acute growth restriction followed by incomplete catch-up in body weight, body length and BMI. At the age of 12 months, middle-aged FR males had significantly lower absolute resting energy expenditure (200 v. 216 kJ/24 h, P = 0.009), absolute energy intake (281 v. 310 kJ/24 h, P = 0.001) and energy intake adjusted for BMI (284 v. 305 kJ/24 h, P = 0.016) than controls, whereas resting energy expenditure adjusted for BMI did not differ significantly between the groups (204 v. 211 kJ/24 h, P = 0.156). The amount of energy remaining for other functions was lower in FR males (80 v. 94 kJ/24 h, P = 0.044). Comparable data were obtained at the age of 6 months. These results indicate that in rats energy balance can be programmed by early nutrition. A low early postnatal food intake appears to programme these animals for a low energy intake and to remain slender in adult life.

Postnatal food restriction in the rat as a model for a low nephron endowment.

A low nephron endowment may be associated with hypertension. Nephrogenesis is the process that leads to the formation of nephrons until week 36 of gestation in humans and may be inhibited by many factors like intrauterine growth restriction and premature birth. To study the consequences of a low glomerular number, animal models have been developed. We describe a model of postnatal food restriction in the rat in which litter size is increased to 20 pups, which leads to growth restriction. In the rat, active nephrogenesis continues until postnatal day 8, which coincides with the growth restriction in our model. Design-based stereological methods were used to estimate glomerular number and volume. Our results show an approximately 25% lower glomerular number in rats after postnatal food restriction (30,800 glomeruli/kidney) compared with control rats (39,600 glomeruli/kidney, P < 0.001). Mean glomerular volume was increased by 35% in the growth-restricted rats (P = 0.006). There was a significant negative correlation between glomerular volume and glomerular number (r = -0.76, P < 0.001). We conclude that postnatal food restriction in the rat leads to a low nephron endowment with compensatory enlargement. It is therefore a suitable model to study the effect of intrauterine growth restriction or prematurity on kidney development and the consequences of a reduced glomerular number in later life.