Daniela Braida

PHARMACOLOGIC RESCUE OF IMPAIRED COGNITIVE FLEXIBILITY, SOCIAL DEFICITS, INCREASED AGGRESSION, AND SEIZURE SUSCEPTIBILITY IN OXYTOCIN RECEPTOR NULL MICE: A NEUROBEHAVIORAL MODEL OF AUTISM

BACKGROUND Oxytocin (OT) has been suggested as a treatment to improve social behavior in autistic patients. Accordingly, the OT (Oxt(-/-)) and the OT receptor null mice (Oxtr(-/-)) display autistic-like deficits in social behavior, increased aggression, and reduced ultrasonic vocalization. METHODS Oxtr(-/-) mice were characterized for general health, sociability, social novelty, cognitive flexibility, aggression, and seizure susceptibility. Because vasopressin (AVP) and OT cooperate in controlling social behavior, learning, and aggression, they were tested for possible rescue of the impaired behaviors. Primary hyppocampal cultures from Oxtr(+/+) and Oxtr(-/-) mouse embryos were established to investigate the balance between gamma-aminobutyric acid (GABA) and glutamate synapses and the expression levels of OT and AVP (V1a) receptors were determined by autoradiography. RESULTS Oxtr(-/-) mice display two additional, highly relevant, phenotypic characteristics: 1) a resistance to change in a learned pattern of behavior, comparable to restricted interests and repetitive behavior in autism, and 2) an increased susceptibility to seizures, a frequent and clinically relevant symptom of autism. We also show that intracerebral administration of both OT and AVP lowers aggression and fully reverts social and learning defects by acting on V1a receptors and that seizure susceptibility is antagonized by peripherally administered OT. Finally, we detect a decreased ratio of GABA-ergic versus total presynapses in hippocampal neurons of Oxtr(-/-) mice. CONCLUSIONS Autistic-like symptoms are rescued on administration of AVP and OT to young Oxtr(-/-) adult animals. The Oxtr(-/-) mouse is thus instrumental to investigate the neurochemical and synaptic abnormalities underlying autistic-like disturbances and to test new strategies of pharmacologic intervention.

Chronic |[Delta]|9-Tetrahydrocannabinol During Adolescence Provokes Sex-Dependent Changes in the Emotional Profile in Adult Rats: Behavioral and Biochemical Correlates

Few and often contradictory reports exist on the long-term neurobiological consequences of cannabinoid consumption in adolescents. The endocannabinoid system plays an important role during the different stages of brain development as cannabinoids influence the release and action of different neurotransmitters and promote neurogenesis. This study tested whether long-lasting interference by cannabinoids with the developing endogenous cannabinoid system during adolescence caused persistent behavioral alterations in adult rats. Adolescent female and male rats were treated with increasing doses of Δ9-tetrahydrocannabinol (THC) for 11 days (postnatal day (PND) 35–45) and left undisturbed until adulthood (PND 75) when behavioral and biochemical assays were carried out. CB1 receptor level and CB1/G-protein coupling were significantly reduced by THC exposure in the amygdala (Amyg), ventral tegmental area (VTA) and nucleus accumbens (NAc) of female rats, whereas male rats had significant alterations only in the amygdala and hippocampal formation. Neither female nor male rats showed any changes in anxiety responses (elevated plus maze and open-field tests) but female rats presented significant ‘behavioral despair’ (forced swim test) paralleled by anhedonia (sucrose preference). In contrast, male rats showed no behavioral despair but did present anhedonia. This different behavioral picture was supported by biochemical parameters of depression, namely CREB alteration. Only female rats had low CREB activity in the hippocampal formation and prefrontal cortex and high activity in the NAc paralleled by increases in dynorphin expression. These results suggest that heavy cannabis consumption in adolescence may induce subtle alterations in the emotional circuit in female rats, ending in depressive-like behavior, whereas male rats show altered sensitivity to rewarding stimuli.

Changes in hippocampal morphology and neuroplasticity induced by adolescent THC treatment are associated with cognitive impairment in adulthood

Marijuana and hashish are the illicit drugs most frequently used by human adolescents. Given the continued neurodevelopment throughout adolescence, adolescents may be more vulnerable than adults to certain neural consequences of heavy marijuana use. This study aimed to assess whether an experimental model of adolescent chronic exposure to Δ9‐tetrahydrocannabinol (THC), may induce lasting effects on learning and memory. Adolescent rats have been treated with THC or its vehicle from 35 to 45 postnatal days (PND) and left undisturbed until their adulthood (75 PND) when aversive and spatial memory was assessed using the passive avoidance and radial maze tasks. No alteration was found in aversive memory, but in the radial maze THC pretreated animals exhibited a worse performance than vehicles, suggesting a deficit in spatial working memory. To correlate memory impairment to altered neuroplasticity, level of marker proteins was investigated in the hippocampus, the most relevant area mediating spatial memory. A significant decrease in the astroglial marker glial fibrillar acid protein was found as well as in pre‐ and postsynaptic protein expression (VAMP2, PSD95) and NMDA receptor levels in pretreated rats. To parallel these changes to alteration in dendritic morphology, Golgi‐Cox staining was performed in the hippocampal dentate gyrus. Pretreated rats had a significantly lower total dendritic length and number than vehicles, as well as reduced spine density. Our data suggest that THC pretreated rats may establish less synaptic contacts and/or less efficient synaptic connections throughout the hippocampus and this could represent the molecular underpinning of the cognitive deficit induced by adolescent THC treatment.

Conditioned place preference induced by the cannabinoid agonist CP 55,940: interaction with the opioid system

Cannabinoids appear atypical as drugs of abuse since controversial data exist concerning the ability to lower the thresholds for electrical self-stimulation (Stark and Dews, 1980; Gardner et al., 1988; Gardner, 1992) and to support self-administration (Martellotta et al., 1998; Tanda et al., 2000) or conditioned place preference in animals (Lepore et al., 1995; Parker and Gillies, 1995; McGregor et al., 1996; Sañudo-Peña et al., 1997; Chaperon et al., 1998; Hutcheson et al., 1998; Mallet and Beninger, 1998; Cheer et al., 2000; Valjent and Maldonado, 2000). Opioids and cannabinoids share some pharmacological properties (Manzanares et al., 1999). The most interactions were found in antinociception (Welch and Stevens, 1992; Smith et al., 1994) and, to a lesser extent, in drug reinforcement (Chen et al., 1990; Vela et al., 1995; Tanda et al., 1997). In the present study we asked whether: (1) a potent synthetic cannabinoid receptor agonist, [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptil)-phenyl]-trans-4-(3-hydroxy propyl) cyclohexanol] (CP 55,940) (from 10 to 40 microg/kg), which binds to the brain cannabinoid receptors with high affinity (Herkenham et al., 1991), would induce conditioned place preference, in comparison with heroin (from 0.1 to 5 mg/kg); (2) what type of receptor was involved; (3) what kind of interaction there was between the two drugs, when given in combination, on reward. CP 55,940 elicited a conditioned place preference only at a dose of 20 microg/kg similar in intensity to that of heroin (2 mg/kg). The reinforcing properties of the cannabinoid agonist were fully antagonised by pretreatment with the brain cannabinoid receptor-1 (CB(1)) antagonist, [N-piperidino-5-(4-chlorophenyl) 1-(2,4-dichloro-phenyl)-4-methyl pyrazole-3-carboxamide hydrochloride] (SR 141716A) and naloxone. The combination of CP 55,940 and heroin, at the reinforcing doses, led to a reward which did not show any additive effect. Taken together these findings are important for understanding how the cannabinoids produce reward and the interconnection of the opioid and cannabinoid system in the motivation.

Cognitive function in young and adult IL (interleukin)-6 deficient mice

Interleukin-6 (IL-6) is a cytokine shown to affect brain function and to be involved in pathological neurodegenerative disorders such as Alzheimers disease (AD). In the present study we investigated the cognitive function in transgenic mice not expressing IL-6 (IL-6 KO) and in wild type (WT) genotype at 4 and 12 months of age, using a passive avoidance and an eight-arm radial maze tasks. Motor function was quantified using an Animex apparatus. Hippocampal choline acetyltransferase (ChAT) activity was evaluated in both genotypes. No difference was observed in both genotypes for spontaneous motor activity. The mean latency (s) to re-enter the shock box, was similar in both young mutant and WT mice. However, a decreased sensitivity (50%) to scopolamine (1 mg/kg) in mutant compared to WT mice, was obtained. IL-6 KO mice exhibited a facilitation of radial maze learning over 30 days, in terms of a lower number of working memory errors and a higher percentage of animals reaching the criterion as compared with WT genotype tested at both ages. Furthermore, mutant mice, at the age of 12 months, showed a faster acquisition (22 days versus 30 days to reach the criterion). The pattern of arm entry exhibited by IL-6 KO mice showed a robust tendency to enter an adjacent arm at both ages, while WT only at the age of 4 months. ChAT activity was inversely correlated with memory performance. These findings suggest a possible involvement of IL-6 on memory processes, even if the mechanism remains still unclear.

Intracerebral self-administration of the cannabinoid receptor agonist CP 55,940 in the rat : interaction with the opioid system

The effect of CP 55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclo-hesanol], heroin and etonitazene on intracerebroventricular (i.c.v.) self-administration in a free-choice procedure was evaluated in rats. Animals were trained in 1-h daily sessions with a continuous reinforcement schedule to press two active levers to obtain the vehicle of each drug. Then, when a stable baseline was reached, each drug could be self-administered by pressing the lever found to be less preferred during training, while the vehicle came from the other. The number of bar pressings associated with the delivery of increasing unit doses of CP 55,940 (0.1, 0.2, 0.4, 0.8, 1.6 microg/2 microl/infusion), heroin (0.125, 0.25, 0.5, 1, 2 microg/2 microl/infusion) or etonitazene (0.1--0.2--0.5--1 microg/ 2 microl/infusion) and with the delivery of the corresponding vehicle was fitted by symmetrical parabolas. The mean drug intake was linearly related to the log of self-administered drugs. Pretreatment with SR141716A [N-piperidino-5-(4-chlorophenyl)1-(2,4-dichloro-phenyl)-4-methylpyrazole-3-carboxamide] (0.5 mg/kg) or naloxone HCl (2 mg/kg/i.p.) 15 min before each daily session reduced the self-administration of both CP 55,940 and heroin. The combination of CP 55,940 with heroin or etonitazene reduced the number of drug-associated lever pressings compared to that obtained with the maximal reinforcing unit dose of each drug alone. These findings suggest there may be a strong interaction between opioids and the cannabinoid system.

An inverted U-shaped curve for heptylphysostigmine on radial maze performance in rats: comparison with other cholinesterase inhibitors

The potential of heptylphysostigmine tartrate (pyrrolo [2,3b] indol-5-ol, 3,3a,8,8a-hexahydro-1,3a,8-trimethylheptylcarbamate [ester, (3aS-cis)]) (MF201), a new second-generation cholinesterase inhibitor, to antagonize scopolamine-induced amnesia in rats was assessed in an 8-arm radial maze. Upon completing the training session, the rats were orally administered increasing doses of MF201 (2, 3, 4, 6 and 8 mg/kg) 60 min prior to a s.c. injection of scopolamine (0.25 mg/kg). 9-Amino-1,2,3,4-tetrahydroamino-acridine hydrochloride hydrate (tacrine) (0.25, 0.37, 0.5, 1 and 2 mg/kg), 1-benzil-4-[(5,6-dimethoxy-1-indanon)-2-yl]-methyl piperidine (E2020) (0.125, 0.18, 0.25 and 0.5 mg/kg) and physostigmine (0.15, 0.25, 0.5 and 1 mg/kg) were orally administered and rats were tested in the same task. As previously described, scopolamine induced an impairment in radial maze performance, measured in terms of total number of errors, total time taken to complete the task and the percentage of amnesic animals. The reversal of scopolamine-induced impairment was characterized by the presence of an inverted U-shaped dose-response curve. A significant antagonistic effect was achieved with a dose (mg/kg) of 0.25 for E2020, 0.5 for tacrine and physostigmine and 3, 4 and 6 for MF201, the latter manifesting a broader spectrum of activity (3-6 mg/kg). While the maximal active doses restored the scopolamine-induced modified pattern of arm entry, they were ineffective in reducing hypermotility, suggesting the drugs have a specific effect on cognitive function.

Potential anxiolytic- and antidepressant-like effects of salvinorin A, the main active ingredient of Salvia divinorum, in rodents

Background and purpose:  Drugs targeting brain κ‐opioid receptors produce profound alterations in mood. In the present study we investigated the possible anxiolytic‐ and antidepressant‐like effects of the κ‐opioid receptor agonist salvinorin A, the main active ingredient of Salvia divinorum, in rats and mice.

The Depressive Phenotype Induced in Adult Female Rats by Adolescent Exposure to THC is Associated with Cognitive Impairment and Altered Neuroplasticity in the Prefrontal Cortex

We recently demonstrated that Δ9-tetrahydrocannabinol (THC) chronic administration in female adolescent rats induces alterations in the emotional circuit ending in depressive-like behavior in adulthood. Since cognitive dysfunction is a major component of depression, we assessed in these animals at adulthood different forms of memory. Adolescent female rats were treated with THC or its vehicle from 35 to 45 post-natal days (PND) and left undisturbed until their adulthood (75 PND) when aversive and spatial memory was assessed using the passive avoidance and radial maze tasks. No alteration was found in aversive memory, but in the radial maze THC pre-treated animals exhibited a worse performance than vehicles, suggesting a deficit in spatial working memory. To correlate memory impairment to altered neuroplasticity, level of marker proteins was investigated in the hippocampus and prefrontal cortex, the most relevant areas for learning and memory. A significant decrease in synaptophysin and PSD95 proteins was found in the prefrontal cortex of THC pre-treated rats, with no alterations in the hippocampus. Finally, proteomic analysis of the synapses in the prefrontal cortex revealed the presence of less active synapses characterized by reduced ability in maintaining normal synaptic efficiency. This picture demonstrates the presence of cognitive impairment in THC-induced depressive phenotype.

Cellular Mechanisms Underlying the Anxiolytic Effect of Low Doses of Peripheral Δ 9 -Tetrahydrocannabinol in Rats

We investigated the effect of low doses of intraperitoneal Δ9-tetrahydrocannabinol (THC) on anxiety behavior in rats using the elevated plus maze (EPM). An anxiolytic effect was obtained in a range of doses between 0.075 and 1.5 mg/kg, the 0.75 dose being the most effective. Pretreatment with the CB1 receptor antagonist AM251 fully reversed THCs effect, suggesting CB1 receptors were involved. In order to elucidate the neuroanatomical substrates underlying the effect of the maximal effective dose of THC, we investigated cFos expression in anxiety-related brain regions (prefrontal cortex, nucleus accumbens, amygdala, and hippocampus) of rats exposed to the EPM. THC significantly lowered the amount of cFos in prefrontal cortex and amygdala without affecting the other cerebral areas. As there is increasing evidence that CREB function regulates anxiety-like behavior in rats, the second biochemical parameter we measured was phosphorylated CREB in the same brain areas. Rats treated with THC showed a significant increase in CREB activation in the prefrontal cortex and hippocampus. In the prefrontal cortex this increased activation was linked to an increase in ERK activation, whereas in the hippocampus there was a drop in the activity of CAMKII, a kinase with inhibitory effect on CREB activation. All these effects were reversed by AM251 pretreatment, suggesting that stimulation of CB1 receptors is fundamental for triggering the biochemical events. Our results suggest that the stimulation of these receptors in the prefrontal cortex, amygdala, and hippocampus with the subsequent activation of different signaling pathways is the first event underlying the effects of cannabinoids on anxious states.