Christian Chiamulera

Removing Obstacles in Neuroscience Drug Discovery : The Future Path for Animal Models

Despite great advances in basic neuroscience knowledge, the improved understanding of brain functioning has not yet led to the introduction of truly novel pharmacological approaches to the treatment of central nervous system (CNS) disorders. This situation has been partly attributed to the difficulty of predicting efficacy in patients based on results from preclinical studies. To address these issues, this review critically discusses the traditional role of animal models in drug discovery, the difficulties encountered, and the reasons why this approach has led to suboptimal utilization of the information animal models provide. The discussion focuses on how animal models can contribute most effectively to translational medicine and drug discovery and the changes needed to increase the probability of achieving clinical benefit. Emphasis is placed on the need to improve the flow of information from the clinical/human domain to the preclinical domain and the benefits of using truly translational measures in both preclinical and clinical testing. Few would dispute the need to move away from the concept of modeling CNS diseases in their entirety using animals. However, the current emphasis on specific dimensions of psychopathology that can be objectively assessed in both clinical populations and animal models has not yet provided concrete examples of successful preclinical–clinical translation in CNS drug discovery. The purpose of this review is to strongly encourage ever more intensive clinical and preclinical interactions to ensure that basic science knowledge gained from improved animal models with good predictive and construct validity readily becomes available to the pharmaceutical industry and clinical researchers to benefit patients as quickly as possible.

Nicotinic Acetylcholine Receptors in the Mesolimbic Pathway: Primary Role of Ventral Tegmental Area α6β2* Receptors in Mediating Systemic Nicotine Effects on Dopamine Release, Locomotion, and Reinforcement

α6* nicotinic acetylcholine receptors (nAChRs) are highly and selectively expressed by mesostriatal dopamine (DA) neurons. These neurons are thought to mediate several behavioral effects of nicotine, including locomotion, habit learning, and reinforcement. Yet the functional role of α6* nAChRs in midbrain DA neurons is mostly unknown. The aim of this study was to determine the composition and in vivo functional role of α6* nAChR in mesolimbic DA neurons of male rats. Immunoprecipitation and immunopurification techniques coupled with cell-specific lesions showed that the composition of α6* nAChR in the mesostriatal system is heterogeneous, with (non-α4)α6β2* being predominant in the mesolimbic pathway and α4α6β2* in the nigrostriatal pathway. We verified whether α6* receptors mediate the systemic effects of nicotine on the mesolimbic DA pathway by perfusing the selective antagonists α-conotoxin MII (CntxMII) (α3/α6β2* selective) or α-conotoxin PIA (CntxPIA) (α6β2* selective) into ventral tegmental area (VTA). The intra-VTA perfusion of CntxMII or CntxPIA markedly decreased systemic nicotine-elicited DA release in the nucleus accumbens and habituated locomotion; the intra-VTA perfusion of CntxMII also decreased the rate of nicotine infusion in the maintenance phase of nicotine, but not of food, self-administration. Overall, the results of these experiments show that the α6β2* nAChRs expressed in the VTA are necessary for the effects of systemic nicotine on DA neuron activity and DA-dependent behaviors such as locomotion and reinforcement, and suggest that α6β2*-selective compounds capable of crossing the blood–brain barrier may affect the addictive properties of nicotine and therefore be useful in the treatment of tobacco dependence.

Effect of NMDA- and strychnine-insensitive glycine site antagonists on NMDA-mediated convulsions and learning

Intracerebroventricular (ICV) injection of N-methyl-d-aspartate (NMDA) was shown to induce generalized seizures in mice. The competitive NMDA antagonistsdl-2-amino-5-phosphonovaleroate (dl-AP7) and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP), the NMDA “channel blocker” antagonist (+)-5-methyl-10,11-dihydro 5H-dibenzo-[a,d] cycloheptan-5,10-imine maleate (MK-801) and the strychnine-insensitive glycine antagonists kynurenic acid (KYNA) and 7-chloro-kynurenic acid (7-Cl-KYNA), when co-administered (ICV) with NMDA, antagonized NMDA-induced generalized seizures. Administration (ICV) ofdl-AP7, CPP and MK-801 resulted in impared learning performance in a passive avoidance task in mice, with ED50 close to the anticonvulsant dose. The glycine antagonists KYNA and 7-Cl-KYNA at high doses significantly failed to affect performance in the same model of learning. The results indicate that compounds acting at the strychnine-insensitive glycine site may have a larger “therapeutic window” as anticonvulsants than antagonists of the NMDA receptor and channel.

Post-retrieval extinction as reconsolidation interference: methodological issues or boundary conditions?

Memories that are emotionally arousing generally promote the survival of species; however, the systems that modulate emotional learning can go awry, resulting in pathological conditions such as post-traumatic stress disorders, phobias, and addiction. Understanding the conditions under which emotional memories can be targeted is a major research focus as the potential to translate these methods into clinical populations carries important implications. It has been demonstrated that both fear and drug-related memories can be destabilised at their retrieval and require reconsolidation to be maintained. Therefore, memory reconsolidation offers a potential target period during which the aberrant memories underlying psychiatric disorders can be disrupted. Monfils et al. (Science 324:951–955, 2009) have shown for the first time that safe information provided through an extinction session after retrieval (during the reconsolidation window) may update the original memory trace and prevent the return of fear in rats. In recent years, several authors have then tested the effect of post-retrieval extinction on reconsolidation of either fear or drug-related memories in both laboratory animals and humans. In this article, we review the literature on post-reactivation extinction, discuss the differences across studies on the methodological ground, and review the potential boundary conditions that may explain existing discrepancies and limit the potential application of post-reactivation extinction approaches.

Effects of the metabotropic glutamate receptor antagonist MCPG on spatial and context-specific learning

The effects of the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) on performance in a water maze and in context-specific associative learning were examined in rats previously implanted with cannulae. MCPG (20.8 micrograms) injected intraventricularly (i.c.v.) before testing impaired the performance of rats in the spatial version of the Morris water maze, but 1/10 of this dose did not. Memory retention, evaluated 24 hr post-training, was also affected by the high dose of MCPG. However, performance in a cued version of the water maze was not impaired by the high dose, excluding effects of the drug on perceptual faculties. The effects of the MCPG were further characterized on performance in another hippocampus-dependent spatial learning task, the context-dependent fear conditioning task. MCPG (20.8 micrograms, i.c.v.) did not interfere with conditioned freezing to context in this task. For comparison, a group of rats was injected with the NMDA receptor blocker MK801. MK801 at a dose that disrupted the performance in the spatial version of the Morris water maze (0.08 mg/kg), significantly reduced freezing compared to controls. These experiments indicate that MCPG-sensitive metabotropic receptors may be required for only a restricted subset of spatial learning tasks, while NMDA receptors may play an integral role in all spatial learning.

Qualitative and quantitative analysis of the progressive cerebral damage after middle cerebral artery occlusion in mice

The middle cerebral artery occlusion (MCAO) in mice induces a focal cerebral ischaemia at the level of the tempo-parietal cortex. Histological staining and immunohistochemical markers were used to characterize the temporal progression of the cerebral infarct: both qualitative and quantitative analyses were performed at different days after the MCAO. At 3 days after MCAO, an extensive necrosis of the cerebral parenchyma was accompanied by extravasation and by massive oedema. After 7 days, GFAP marker showed a gliotic reaction with alteration of the astrocytes membrane permeability (S100 marker). Positivity for acid phosphatase staining indicated the presence of macrophages. At Day 14 and 21 following MCAO, the histological profile was essentially similar. Interestingly, at Day 7, 14 and 21, a previously unreported gliosis was observed in the subthalamic area. Quantitative analysis showed a significantly large infarct volume at Day 3 (7.88 +/- 1.95 mm3 +/- S.E.M.) compared to Day 7 (4.28 +/- 0.47 mm3 +/- S.E.M.). At Day 14 and Day 21 the infarct volumes were further decreased to 2.00 +/- 0.52 and 1.43 +/- 0.39 mm3 +/- S.E.M., respectively. These results suggest that it is important to consider the time of evaluation of cerebral ischaemia-induced cerebral infarct, especially in studies which aim to evaluate the neuroprotective effect of putative therapeutic agents.

Increases in cholinergic neurotransmission measured by using choline-sensitive microelectrodes: Enhanced detection by hydrolysis of acetylcholine on recording sites?

Previous experiments demonstrated that second-based transient increases in choline concentrations measured by electrodes coated with choline oxidase (ChOx) and the amperometric detection of hydrogen peroxide validly indicate the depolarization-dependent release of acetylcholine (ACh) and its hydrolysis by endogenous acetylcholinesterase (AChE). Therefore, choline-sensitive microelectrodes have become valuable tools in neuropharmacological and behavioral research. The present experiments were designed to test the possibility that co-immobilization of ChOx plus AChE on recording sites increases the level of detection for evoked ACh release in the brain. If newly released ACh is not completely hydrolyzed by endogenous AChE and capable of reaching the extracellular space, currents recorded via sites equipped with both enzymes should be greater when compared with sites coated with ChOx only. Pairs of platinum-recording sites were coated either with AChE plus ChOx or ChOx alone. Potassium or nicotine-evoked currents were recorded throughout the entire dorsal-ventral extent of the medial prefrontal cortex (mPFC). The amplitudes of evoked cholinergic signals did not differ significantly between AChE+ChOx and ChOx-only coated recording sites. Additional experiments controlling for several potential confounds suggested that, in vivo, ACh levels > or =150fmol were detected by recordings sites featuring dual enzyme coating. Collectively, these results indicate that co-coating of microelectrodes with AChE does not enhance the detection of cholinergic activity in the cortex compared with measurements via recording sites coated only with ChOx.

Cigarette smoking knowledge and perceptions among students in four Italian medical schools.

INTRODUCTION Tobacco smoking is the leading cause of premature death in the developed world. Advice and assistance by physicians help smokers quit, but little attention has been paid to the topic of tobacco dependence in the curricula of Italian medical schools. Consequently, few physicians follow the clinical practice guidelines for treating dependence. METHODS This study was conducted on 439 students at 4 Italian medical schools in 2010. Students were asked to complete a 60-item questionnaire. Two scores were computed: Score 1 assessed knowledge of the epidemiology of smoking, risks associated with smoking, and benefits of cessation. Score 2 assessed knowledge of tobacco dependence treatment guidelines and the effectiveness of treatments. A score of less than 60% indicated insufficient knowledge. RESULTS Medical students had limited knowledge of the epidemiology of smoking, attributable morbidity and mortality, and the benefits of cessation. This limited knowledge was reflected by the finding that 70% of students had a total Score 1 less than 60% of available points. Knowledge of clinical guidelines, perceived competence in counseling smokers, and treatment of addiction was also insufficient, as 76% of students achieved a total Score 2 of less than 60%. CONCLUSIONS Our data demonstrate that Italian medical students have limited knowledge about tobacco dependence, how to treat it, and the critical role of the physician in promoting cessation. Taken together with research from other countries, these findings suggest that medical schools do not offer adequate training in tobacco dependence and provide a rationale for modifying the core curriculum to include more information on tobacco dependence treatment.

Musculoskeletal adverse drug reactions: a review of literature and data from ADR spontaneous reporting databases.

The musculoskeletal system can be a target organ for adverse drug reactions (ADRs). Drug-induced muscle, bone or connective tissue injuries may be due to, i), primary direct drug action, or, ii), undirected consequence of generalized drug-induced disease. Musculoskeletal ADRs may be only temporarily disabling, such as muscle cramps, as well as in other cases may be serious and life-threatening, such as rhabdomyolysis. In the last few years there has been an increasing awareness of musculoskeletal ADRs. Some recent drug safety issues dealt with serious or uncommon musculoskeletal reactions like rhabdomyolysis associated to statins and tendon rupture associated to fluoroquinolones. In this review, we firstly selected those drug classes having a significantly high percentage of musculoskeletal disorder reports in the WHO adverse drug reaction database, maintained by the Uppsala Monitoring Centre. Secondly, the different musculoskeletal ADRs were closely analyzed through the data obtained from an Italian interregional ADRs spontaneous reporting database. The findings on drugs associated to different musculoskeletal disorders, have been integrated with a review of the epidemiological data available in the literature. For the most involved drugs (HMG-CoA reductase inhibitors, fluoroquinolones, corticosteroids, bisphosphonates, retinoids) the underlying musculoskeletal ADR mechanisms were also reviewed and discussed.

Sub-chronic nicotine-induced changes in regional cerebral blood volume and transversal relaxation time patterns in the rat : a magnetic resonance study

This preliminary study describes magnetic resonance imaging (MRI) data on the effects of sub-chronic nicotine administration in rats. Nicotine 0.4 mg/kg s.c. free base was given once a day for 4 days to Wistar adult male rats. On day 5, anaesthetized subjects were observed using an MRI tomography system. Regional cerebral blood volume (rCBV) and transversal relaxation time (T2) MRI parameters were measured. Nicotine treatment increased T2 values, with a significant effect in the cingulate cortex. A trend to increase was also observed in the prefrontal cortex and nucleus accumbens. Similarly, the effect of nicotine on rCBV was a significant increase in values compared to saline treatment. Post hoc analysis showed a significant effect of nicotine in the prefrontal cortex, cingulate cortex, mediodorsal thalamus and lateral posterior thalamus. This study showed for the first time that sub-chronic nicotine administration can induce changes in MRI pattern which are (i) similar to human MRI studies, and (ii) common to those described for markers of neuronal metabolic activation in corticolimbic brain regions known to be involved in nicotine dependence.