Eszter Kirilly

CB1 receptor antagonists: new discoveries leading to new perspectives

CB1 receptor antagonists were among the most promising drug targets in the last decade. They have been explored and found to be effective as therapeutic agents for obesity and related cardiometabolic problems; however, use of rimonabant, the first marketed CB1 receptor antagonist, has been suspended because of its anxiogenic and depressogenic side effects. Because some other antiobesity drugs, like dexfenfluramine or sibutramine, were also suspended, the unmet need for drugs that reduce body weight became enormous. One approach that emerged was the use of CB1 receptor antagonists that poorly cross the blood brain barrier, the second, the development of neutral antagonists instead of inverse agonists, and the third, use of personalized medicine, namely the selection of the patient population without psychiatric side effects. In this review, we dissect the peripheral and central mechanisms involved in the effects of CB1 receptor antagonists and argue that central mechanisms are more or less involved in most cardiometabolic therapeutic effects and thus, among patients with unsatisfactory therapeutic response to compounds with peripheral action, centrally acting antagonists may be needed. An analysis of pharmacogenetic factors may help to identify persons who are at no or low risk for psychiatric adverse effects. Here, we present the models and identify molecular mechanisms and receptors involved in the effects of stress‐, anxiety‐ and depression‐related neurocircuitries sensitive to CB1 receptor antagonists, like the serotonergic, noradrenergic and dopaminergic systems, which are not only regulated by CB1 receptors, but also regulate the synthesis of the endocannabinoid 2‐arachidonoyl‐glycerol.

Partial lesion of the serotonergic system by a single dose of MDMA results in behavioural disinhibition and enhances acute MDMA-induced social behaviour on the social interaction test

The acute effects of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) on anxiety-related behaviours were studied using indices of social interaction in Dark Agouti (DA) both drug naive rats and those pretreated with MDMA (15 mg/kg i.p.) 3 weeks earlier. The functional neuroanatomy of these MDMA effects was visualised using 2-deoxyglucose imaging of local cerebral glucose use (LCMRglu), whilst MDMA-induced serotonergic neurotoxicity was measured by radioligand binding with [3H]paroxetine. Acute MDMA alone markedly decreased most typical elements of social interaction but increased adjacent lying, a behaviour that also contains social elements. In animals pre-exposed to MDMA, decreased [3H]paroxetine binding indicated serotonergic terminal depletion, and in these animals significant increases in locomotor activity, exploratory behaviour and aggressive behaviour were found. Both behavioural effects and also the metabolic activation induced by acute MDMA were potentiated in rats previously exposed to the drug. In conclusion, a single dose of MDMA caused marked changes in social behaviour acutely that might be interpreted either as a decrease or increase in anxiety. Three weeks after MDMA a behavioural disinhibition similar to psychomotor agitation, a symptom connected to depression or mania, and a sensitization to the acute effects of MDMA are apparent in both the behavioural and brain metabolic effects of the drug.

Single dose of MDMA causes extensive decrement of serotoninergic fibre density without blockage of the fast axonal transport in Dark Agouti rat brain and spinal cord.

Prolonged neurotoxicity of the recreational drug, MDMA (3,4‐methylenedioxymethamphetamine) on serotoninergic axon terminals has been suggested. The effect of a single (15 mg/kg) dose of intraperitoneally administered MDMA on serotoninergic fibre density, defined by tryptophan hydroxylase (TpH) and serotonin transporter (5‐HTT) immunoreactivity, has been evaluated in the spinal cord and brain areas in Dark Agouti rats, 7 and 180 days after MDMA applications. Immunostaining for amyloid precursor protein (APP) has been performed to examine possible defects of the fast axonal transport, and 5‐HTT mRNA expressions were quantified in neurones of medullary raphe nuclei. Seven days after MDMA treatment, a substantial decrease in the density of TpH‐immunoreactive fibres was detectable in the frontal cortex, the caudate‐putamen, the CA1 region of the hippocampus, and marked decreases were found in the spinal cord. These changes in TpH density showed a high correlation with 5‐HTT densities. In contrast, APP‐immunoreactive axonal bulbs were not detected in any of the brain regions studied. Seven days after MDMA administrations, significantly elevated 5‐HTT mRNA expressions were found in the raphe pallidus and obscurus. Our results suggest that a single dose of MDMA elicits widespread depletion of TpH and 5‐HTT immunoreactivity in serotoninergic axons without morphological sign of the blockage of the fast anterograde axonal transport. Our results do not support the notion of MDMA‐induced axotomy of serotoninergic neurones. The up‐regulation of 5‐HTT mRNA expressions 1 week after MDMA injections might indicate the potential recovery of the serotonin system.

Decrease in REM latency and changes in sleep quality parallel serotonergic damage and recovery after MDMA: a longitudinal study over 180 days

The recreational drug ecstasy [3,4-methylenedioxymethamphetamine (MDMA)], has been found to selectively damage brain serotonin neurons in experimental animals, and probably in human MDMA users, but detailed morphometric analyses and parallel functional measures during damage and recovery are missing. Since there is evidence that serotonin regulates sleep, we have compared serotonergic markers parallel with detailed analysis of sleep patterns at three time-points within 180 d after a single dose of 15 mg/kg MDMA in male Dark Agouti rats. At 7 d and 21 d after MDMA treatment, significant(30-40%), widespread reductions in serotonin transporter (5-HTT) density were detected in the cerebral cortex, hippocampus, most parts of the hypothalamus, and some of the brainstem nuclei. With the exception of the hippocampus, general recovery was observed in the brain 180 d after treatment. Transient increases followed by decreases were detected in 5-HTT mRNA expression of dorsal and median raphe nuclei at 7 d and 21 d after the treatment. Significant reductions in rapid eye movement (REM) sleep latency, increases in delta power spectra in non-rapid eye movement sleep and increased fragmentation of sleep were also detected, but all these alterations disappeared by the 180th day. The present data provide evidence for long-term, albeit, except for the hippocampus, transient changes in the terminal and cellular regions of the serotonergic system after this drug. Reduced REM latency and increased sleep fragmentation are the most characteristic alterations of sleep consistently described in depression using EEG sleep polygraphy.

Acute and long-term effects of a single dose of MDMA on aggression in Dark Agouti rats.

MDMA causes selective depletion of serotonergic terminals in experimental animals and the consequent decrease in synaptic 5-HT may, inter alia, increase impulsivity. To study the effects of MDMA upon brain function, the behaviour of male Dark Agouti rats exposed to MDMA (15 mg/kg i.p.), two 5-HT1B agonists (CGS-12066A and CP-94,253, both 5 mg/kg i.p.) or saline were investigated in the resident-intruder test. Studies were performed in drug-naive rats and also in rats exposed to MDMA (15 mg/kg i.p.) 21 d earlier. In parallel experiments the functional neuroanatomy of MDMA effects were assessed using 2-deoxyglucose imaging of local cerebral metabolic rate of glucose utilization (LCMRGlu) and neurotoxicity was assessed by measuring [3H]paroxetine binding. There was no significant difference in aggressive behaviour (biting, boxing, wrestling and their latencies) between drug-naive rats and rats previously exposed to MDMA 21 d earlier, despite reduced social behaviour, decreased LCMRGlu in several brain areas involved in aggression, and reductions in paroxetine binding by 30-60% in the forebrain. CGS-12066A, CP-94,253 and acute MDMA produced marked decreases in aggressive behaviours, especially in biting, boxing and kicking found in drug-naive rats. In animals previously exposed to the drug, acute anti-aggressive effects of MDMA were, in general, preserved as were MDMA-induced increases in LCMRGlu. Our studies provide evidence that in the resident-intruder test, where social isolation is a requirement, aggressive behaviour and acute anti-aggressive effects of MDMA and 5-HT1B receptor agonists remain intact 3 wk after a single dose of the drug despite significant damage to the serotonergic system.

Opposing local effects of endocannabinoids on the activity of noradrenergic neurons and release of noradrenaline: relevance for their role in depression and in the actions of CB(1) receptor antagonists.

There is strong evidence that endocannabinoids modulate signaling of serotonin and noradrenaline, which play key roles in the pathophysiology and treatment of anxiety and depression. Most pharmacological and genetic, human and rodent studies suggest that the presence of under-functioning endocannabinoid type-1 (CB1) receptors is associated with increased anxiety and elevated extracellular serotonin concentration. In contrast, noradrenaline is presumably implicated in the mediation of depression-type symptoms of CB1 receptor antagonists. Evidence shows that most CB1 receptors located on axons and terminals of GABA-ergic, serotonergic or glutamatergic neurons stimulate the activity of noradrenergic neurons. In contrast, those located on noradrenergic axons and terminals inhibit noradrenaline release efficiently. In this latter process, excitatory ionotropic or G protein-coupled receptors, such as the NMDA, alpha1 and beta1 adrenergic receptors, activate local endocannabinoid synthesis at postsynaptic sites and stimulate retrograde endocannabinoid neurotransmission acting on CB1 receptors of noradrenergic terminals. The underlying mechanisms include calcium signal generation, which activates enzymes that increase the synthesis of both anandamide and 2-arachidonoylglycerol, while Gq/11 protein activation also increases the formation of 2-arachidonoylglycerol from diacylglycerol during the signaling process. In addition, other non-CB1 receptor endocannabinoid targets such as CB2, transient receptor potential vanilloid subtype, peroxisome proliferator-activated receptor-alpha and possibly GPR55 can also mediate some of the endocannabinoid effects. In conclusion, both neuronal activation and neurotransmitter release depend on the in situ synthesized endocannabinoids and thus, local endocannabinoid concentrations in different brain areas may be crucial in the net effect, namely in the regulation of neurons located postsynaptically to the noradrenergic synapse.

Chronic repeated restraint stress increases prolactin-releasing peptide/tyrosine-hydroxylase ratio with gender-related differences in the rat brain

In this study, we investigated the effect of chronic repeated restraint (RR) on prolactin‐releasing peptide (PrRP) expression. In the brainstem, where PrRP colocalize with norepinephrine in neurons of the A1 and A2 catecholaminergic cell groups, the expression of tyrosine hydroxylase (TH) has also been examined. In the brainstem, but not in the hypothalamus, the basal PrRP expression in female rats was higher than that in the males that was abolished by ovariectomy. RR evoked an elevation of PrRP expression in all areas investigated, with smaller reaction in the brainstems of females. There was no gender‐related difference in the RR‐evoked TH expression. Elevation of PrRP was relatively higher than elevation of TH, causing a shift in PrRP/TH ratio in the brainstem after RR. Estrogen α receptors were found in the PrRP neurons of the A1 and A2 cell groups, but not in the hypothalamus. Bilateral lesions of the hypothalamic paraventricular nucleus did not prevent RR‐evoked changes. Elevated PrRP production parallel with increased PrRP/TH ratio in A1/A2 neurons indicate that: (i) there is a clear difference in the regulation of TH and PrRP expression after RR, and (ii) among other factors this may also contribute to the changed sensitivity of the hypothalamo‐pituitary–adrenal axis during chronic stress.

Transcriptional Evidence for the Role of Chronic Venlafaxine Treatment in Neurotrophic Signaling and Neuroplasticity Including also Glutatmatergic- and Insulin-Mediated Neuronal Processes.

Objectives Venlafaxine (VLX), a serotonine-noradrenaline reuptake inhibitor, is one of the most commonly used antidepressant drugs in clinical practice for the treatment of major depressive disorder (MDD). Despite being more potent than its predecessors, similarly to them, the therapeutical effect of VLX is visible only 3–4 weeks after the beginning of treatment. Furthermore, recent papers show that antidepressants, including also VLX, enhance the motor recovery after stroke even in non depressed persons. In the present, transcriptomic-based study we looked for changes in gene expressions after a long-term VLX administration. Methods Osmotic minipumps were implanted subcutaneously into Dark Agouti rats providing a continuous (40 mg/kg/day) VLX delivery for three weeks. Frontal regions of the cerebral cortex were isolated and analyzed using Illumina bead arrays to detect genes showing significant chances in expression. Gene set enrichment analysis was performed to identify specific regulatory networks significantly affected by long term VLX treatment. Results Chronic VLX administration may have an effect on neurotransmitter release via the regulation of genes involved in vesicular exocytosis and receptor endocytosis (such as Kif proteins, Myo5a, Sv2b, Syn2 or Synj2). Simultaneously, VLX activated the expression of genes involved in neurotrophic signaling (Ntrk2, Ntrk3), glutamatergic transmission (Gria3, Grin2b and Grin2a), neuroplasticity (Camk2g/b, Cd47), synaptogenesis (Epha5a, Gad2) and cognitive processes (Clstn2). Interestingly, VLX increased the expression of genes involved in mitochondrial antioxidant activity (Bcl2 and Prdx1). Additionally, VLX administration also modulated genes related to insulin signaling pathway (Negr1, Ppp3r1, Slc2a4 and Enpp1), a mechanism that has recently been linked to neuroprotection, learning and memory. Conclusions Our results strongly suggest that chronic VLX treatment improves functional reorganization and brain plasticity by influencing gene expression in regulatory networks of motor cortical areas. These results are consonant with the synaptic (network) hypothesis of depression and antidepressant-induced motor recovery after stroke.

Persistent cerebrovascular effects of MDMA and acute responses to the drug

Acutely, 3,4,‐methylenedioxymethamphetamine (MDMA) induces cerebrovascular dysfunction [ Quate et al., (2004)Psychopharmacol., 173, 287–295]. In the longer term the same single dose results in depletion of 5‐hydroxytrptamine (5‐HT) nerve terminals. In this study we examined the cerebrovascular consequences of this persistent neurodegeneration, and the acute effects of subsequent MDMA exposure, upon the relationship that normally exists between local cerebral blood flow (LCBF) and local cerebral glucose utilization (LCMRglu). Dark agouti (DA) rats were pre‐treated with 15 mg/kg i.p. MDMA or saline. Three weeks later, rats from each pre‐treatment group were treated with an acute dose of MDMA (15 mg/kg i.p.) or saline. Quantitative autoradiographic imaging was used to measure LCBF or LCMRglu with [14C]‐iodoantipyrine and [14C]‐2‐deoxyglucose, respectively. Serotonergic terminal depletion was assessed using radioligand binding with [3H]‐paroxetine and immunohistochemistry. Three weeks after MDMA pre‐treatment there were significant reductions in densities of 5‐HT transporter (SERT)‐positive fibres (−46%) and [3H]‐paroxetine binding (−47%). In animals pre‐treated with MDMA there were widespread significant decreases in LCMRglu, but no change in LCBF indicating a persistent loss of cerebrovascular constrictor tone. In both pre‐treatment groups, acute MDMA produced significant increases in LCMRglu, while LCBF was significantly decreased. In 50% of MDMA‐pre‐treated rats, random areas of focal hyperaemia indicated a loss of autoregulatory capacity in response to MDMA‐induced hypertension. These results suggest that cerebrovascular regulatory dysfunction resulting from acute exposure to MDMA is not diminished by previous exposure, despite a significant depletion in 5‐HT terminals. However, there may be a sub‐population, or individual circumstances, in which this dysfunction develops into a condition that might predispose to stroke.

PACAP is transiently expressed in anterior pituitary gland of rats: in situ hybridization and cell immunoblot assay studies.

In this work the expression of PACAP (pituitary adenylate cyclase activating polypeptide) in rat anterior pituitary was demonstrated for the first time using in situ hybridization. The number of cells showing PACAP signal in intact male rats was negligible similarly to that of diestrous rats. In proestrous rats sacrificed at 10h there was a moderate increase in the expression and after a decrease at 16 h and 18 h, there was a transient peak at 20 h and then the number of labeled cells was declined again (22 h). In the cell immunoblot assay study it was observed that the number of PACAP blot forming (PACAP releasing) cells in an anterior pituitary cell culture changed according to a similar pattern as the number of PACAP expressing cells. The number of blots was also the highest when the animals were sacrificed in the evening of proestrus at 20h. The results obtained by in situ hybridization and cell immunoblot assay well correlate with each other. The above-mentioned results support our hypothesis that the enhanced expression and secretion of PACAP in the pituitary gland may be involved in ceasing the LH surge.