Kerstin Michel

Early age-related cognitive impairment in mice lacking cannabinoid CB1 receptors

The molecular mechanisms contributing to the normal age-related decline of cognitive functions or to pathological learning and memory impairment are largely unknown. We demonstrate here that young mice (6–7 weeks) with a genetic deletion of the cannabinoid CB1 receptor performed as well as WT mice, or often better, in a number of learning and memory paradigms, including animal models of skill-learning, partner recognition, and operant conditioning. In contrast, the performance of mature mice (3–5 months) lacking CB1 receptors was much worse than that of age-matched WT animals. In most tests, these mice performed at the same level as old animals (14–17 months), suggesting that the decline in cognitive functions is accelerated in the absence of CB1 receptors. This rapid decline in CB1-deficient animals is accompanied by a loss of neurons in the CA1 and CA3 regions of the hippocampus.

Control of hormonal stress reactivity by the endogenous opioid system.

Regulations of hormonal stress responses entail the initiation, amplitude and termination of the reaction, as well as its integration with other stress response systems. This study investigates the role of endogenous opioids in the regulation and integration of behavioral, thermal and hormonal stress responses, as these neuromodulators and their receptors are expressed in limbic structures responsible for stress responses. For this purpose, we subjected mice with selective deletion of beta-endorphin, enkephalin or dynorphin to the zero-maze test, a mildly stressful situation, and registered behaviors and stress hormone levels. Behavioral stress reactivity was assessed using zero-maze, light-dark and startle-reactivity paradigms. Animals lacking enkephalin displayed increased anxiety-related behavioral responses in each three, dynorphin knockouts in two models, whereas the responses of beta-endorphin knockouts indicated lower anxiety level in the zero-maze test. All knockout strains showed marked changes in hormonal stress reactivity. Increase in ACTH level after zero-maze test situation, unlike in wild type animals, failed to reach the level of significance in Penk1(-/-) and Pdyn(-/-) mice. Corticosterone plasma levels rapidly increased in all strains, with a lower peak response in knockouts. In wild-type and beta-endorphin-deficient mice, corticosterone levels returned to baseline within 60min after stress exposure. In contrast, mice lacking dynorphin and enkephalin showed longer-lasting elevated corticosterone levels, indicating a delayed termination of the stress reaction. Importantly, the behavioral and hormonal responses correlated in wild-type but not in knockout mice. Hyperthermia elicited by stress was reduced in animals lacking dynorphin and absent in Penk1(-/-) mice, despite of the heightened behavioral anxiety level of these strains. These results demonstrate an important role on the endogenous opioid system in the integration of behavioral and hormonal stress responses.

Behavioral phenotype of pre-proenkephalin-deficient mice on diverse congenic backgrounds

RationaleThe phenotype of genetically modified animals is thought to result from an interaction of gene manipulation with the genetic background and environmental factors.ObjectivesTo test the behavioral and drug responses of Penk1−/− mice on different genetic backgrounds.MethodsCongenic C57BL/6J and DBA/2J mouse strains with a targeted deletion of the Penk1 gene were generated. Behavior and drug effects were tested in models of pain and anxiety.ResultsPenk1−/− mice showed exaggerated responses to painful or threatening environmental stimuli, but the expressivity of the mutant phenotype was strongly dependent on the behavioral paradigm and on the genetic background. For example, elevated levels of anxiety were readily detectable in C57BL/6J-Penk1−/− mice in the light–dark and startle response tests, but not in the social interaction test. In contrast, we found elevated levels of anxiety in DBA/2J-Penk1−/− mice only in the zero-maze and social interaction tests. In some cases, the idiosyncratic behavior masked the appearance of the knockout gene effect. The activity of the anxiogenic drug, m-chlorophenylpiperazine, but not the anxiolytic drug diazepam, was strain and genotype dependent. Mice with the Penk1 mutation on the DBA/2J, but not on other genetic backgrounds, showed an increased opioid-dependent stress-induced analgesia.Conclusions(1) The behavioral effects of the Penk1 gene deletion persists on different genetic backgrounds, but its detection sometimes requires the use of different behavioral paradigms. (2) The behavior of the background strain should be considered in the analysis of knockout mice to avoid floor and ceiling effects, which may mask the phenotype.

Role of CB1 cannabinoid receptors on GABAergic neurons in brain aging

Brain aging is associated with cognitive decline that is accompanied by progressive neuroinflammatory changes. The endocannabinoid system (ECS) is involved in the regulation of glial activity and influences the progression of age-related learning and memory deficits. Mice lacking the Cnr1 gene (Cnr1−/−), which encodes the cannabinoid receptor 1 (CB1), showed an accelerated age-dependent deficit in spatial learning accompanied by a loss of principal neurons in the hippocampus. The age-dependent decrease in neuronal numbers in Cnr1−/− mice was not related to decreased neurogenesis or to epileptic seizures. However, enhanced neuroinflammation characterized by an increased density of astrocytes and activated microglia as well as an enhanced expression of the inflammatory cytokine IL-6 during aging was present in the hippocampus of Cnr1−/− mice. The ongoing process of pyramidal cell degeneration and neuroinflammation can exacerbate each other and both contribute to the cognitive deficits. Deletion of CB1 receptors from the forebrain GABAergic, but not from the glutamatergic neurons, led to a similar neuronal loss and increased neuroinflammation in the hippocampus as observed in animals lacking CB1 receptors in all cells. Our results suggest that CB1 receptor activity on hippocampal GABAergic neurons protects against age-dependent cognitive decline by reducing pyramidal cell degeneration and neuroinflammation.

Dynorphins Regulate Fear Memory: from Mice to Men

Reexposure to trauma reminders is an integral element of trauma-focused cognitive behavioral therapy (Roberts et al., 2009), but little is known about the physiological processes underlying the therapeutic progress. While it is well established that amygdala, prefrontal cortex and hippocampus are key brain structures in fear memory processing (McGaugh, 2004; Herry et al., 2008; Likhtik et al., 2008), it is not well known which neurotransmitters or neuromodulators are involved. Here with a translational approach we investigated the role of dynorphins in the formation and extinction of fear memories in mice and in humans. Mice lacking dynorphin showed an enhanced cue-dependent fear conditioning, as well as delayed extinction in contextual conditioning/extinction paradigms. The pharmacological blockade of κ-opioid receptors before the extinction trials but not before or after the conditioning produced a similar effect. Analysis of neuronal activity, using the immediate early gene c-fos, demonstrated a reduced neuronal activity in key limbic structures during extinction in the absence of dynorphin. Translating these findings into the human domain, fear conditioning and extinction, coupled with functional MRI was then performed in volunteers preselected for a functionally relevant polymorphism in the dynorphin gene. Human volunteers bearing the (T) allele of PDYN (prodynorphin) at rs1997794 showed reduced fear extinction and a significantly diminished functional connectivity between amygdala and ventromedial prefrontal cortex. Our findings establish a role of dynorphin κ-opioid receptor signaling in fear extinction.

A chronic low dose of [Delta]9-tetrahydrocannabinol (THC) restores cognitive function in old mice

The balance between detrimental, pro-aging, often stochastic processes and counteracting homeostatic mechanisms largely determines the progression of aging. There is substantial evidence suggesting that the endocannabinoid system (ECS) is part of the latter system because it modulates the physiological processes underlying aging. The activity of the ECS declines during aging, as CB1 receptor expression and coupling to G proteins are reduced in the brain tissues of older animals and the levels of the major endocannabinoid 2-arachidonoylglycerol (2-AG) are lower. However, a direct link between endocannabinoid tone and aging symptoms has not been demonstrated. Here we show that a low dose of Δ9-tetrahydrocannabinol (THC) reversed the age-related decline in cognitive performance of mice aged 12 and 18 months. This behavioral effect was accompanied by enhanced expression of synaptic marker proteins and increased hippocampal spine density. THC treatment restored hippocampal gene transcription patterns such that the expression profiles of THC-treated mice aged 12 months closely resembled those of THC-free animals aged 2 months. The transcriptional effects of THC were critically dependent on glutamatergic CB1 receptors and histone acetylation, as their inhibition blocked the beneficial effects of THC. Thus, restoration of CB1 signaling in old individuals could be an effective strategy to treat age-related cognitive impairments.

A Common Genetic Predisposition to Stress Sensitivity and Stress-Induced Nicotine Craving

BACKGROUND Clinical studies have shown that stress is one of the main causes for relapse in abstinent smokers. In this article, we have asked whether animals with a genetic predisposition to high or low stress responsivity differ in behaviors relevant to nicotine addiction, in particular stress-induced reinstatement of drug addiction. METHODS First, we selected animals with high, low, and average stress sensitivity from the F2 generation from an intercross of high (C57BL/6J) and low (C3H/J) emotional mouse strains. Next, these animals were trained to self-administer nicotine through a chronic intravenous catheter. After extinction of the operant behavior replacing nicotine with saline, mice were stressed with a foot shock and the reinstatement of drug-seeking behaviors was evaluated. RESULTS Mice with different stress reactivity showed no difference in the acquisition, extinction, or level of nicotine self-administration. We found an immediate reinstatement of drug-seeking behavior in high stress reactive mice, in contrast to low or average stress reactive animals, which showed no significantly increased activity at the active (nicotine-associated) sensor. CONCLUSIONS We conclude that a genetic predisposition to high stress sensitivity contributes to relapse vulnerability but not to the initiation or maintenance of nicotine consumption.

Early onset of aging-like changes is restricted to cognitive abilities and skin structure in Cnr1−/− mice

Genetic deletion of the cannabinoid 1 (CB1) receptor leads to an early onset of learning and memory impairment. In the present study we asked whether the lack of CB1 receptors accelerates aging in general or is selective for cognitive functions. We therefore compared the onset and dynamics of age-dependent changes in social memory, locomotor activity, hearing ability, and in the histopathology of peripheral organs between wild-type and Cnr1 knockout (Cnr1(-/-)) mice. We observed deficits in social memory already in 3-month-old Cnr1(-/-) mice. In contrast, wild-type animals showed such deficits at the age of 6 months. Sensory and motor functions were similar between the genotypes. Thus, hearing loss for higher frequencies and the development of hypomotility showed a similar age-dependent course. In the periphery we detected an early onset of aging-like histological changes in the skin, but not in other organs. We conclude that the lack of CB1 receptor does not induce accelerated aging in general, but induces changes in cognitive function and in skin structure that resemble those associated with aging.

Preproenkephalin knockout mice show no depression-related phenotype

Clinical, preclinical, and pharmacological studies have suggested that decreased enkephalin tone is associated with depression-like symptoms and increase in enkephalin signaling could have a therapeutic value in the treatment of depression. In this study we demonstrate that, surprisingly, animals lacking enkephalin (preproenkephalin, Penk1−/−) showed no depression-related phenotype in the Porsolt forced swimming or tail suspension tests. Moreover, Penk1−/− mice had a lower frequency of depression-related behavior in stress-induced hypoactivity and ultrasonic vocalization models of depression, similar to animals treated with antidepressant drugs, although this effect was specific to the genetic background. In addition, there was no significant difference in the efficacy of antidepressant reference compounds in wild-type and knockout animals. Nialamide and amitriptyline were even slightly more effective in animals with genetic deletion of Penk1, whereas the minimal effective dose of imipramine and fluoxetine was the same in the two genotypes. The dual peptidase inhibitor RB-101 was also effective in Penk1−/− as well as in Penk1−/−/Pdyn−/− animals, although its efficacy was somewhat reduced compared with wild-type animals. This result was also surprising because the antidepressant effects of RB-101 were thought to be due to the elevation of enkephalin levels.

Dynorphins regulate the strength of social memory

Emotionally arousing events like encounter with an unfamiliar con-species produce strong and vivid memories, whereby the hippocampus and amygdala play a crucial role. It is less understood, however, which neurotransmitter systems regulate the strength of social memories, which have a strong emotional component. It was shown previously that dynorphin signalling is involved in the formation and extinction of fear memories, therefore we asked if it influences social memories as well. Mice with a genetic deletion of the prodynorphin gene Pdyn (Pdyn(-/-)) showed a superior partner recognition ability, whereas their performance in the object recognition test was identical as in wild-type mice. Pharmacological blockade of kappa opioid receptors (KORs) led to an enhanced social memory in wild-type animals, whereas activation of KORs reduced the recognition ability of Pdyn(-/-) mice. Partner recognition test situation induced higher elevation in dynorphin A levels in the central and basolateral amygdala as well as in the hippocampus, and also higher dynorphin B levels in the hippocampus than the object recognition test situation. Our result suggests that dynorphin system activity is increased in emotionally arousing situation and it decreases the formation of social memories. Thus, dynorphin signalling is involved in the formation of social memories by diminishing the emotional component of the experience.