Therapeutic induction of energy metabolism reduces neural tissue damage and increases microglia activation in severe spinal cord injury.

Neural tissue has high metabolic requirements. Following spinal cord injury (SCI), the damaged tissue suffers from a severe metabolic impairment, which aggravates axonal degeneration and neuronal loss. Impaired cellular energetic, tricarboxylic acid (TCA) cycle and oxidative phosphorylation metabolism in neuronal cells has been demonstrated to be a major cause of neural tissue death and regeneration failure following SCI. Therefore, rewiring the spinal cord cell metabolism may be an innovative therapeutic strategy for the treatment of SCI. In this study, we evaluated the therapeutic effect of the recovery of oxidative metabolism in a mouse model of severe contusive SCI. Oral administration of TCA cycle intermediates, co-factors, essential amino acids, and branched-chain amino acids was started 3 days post-injury and continued until the end of the experimental procedures. Metabolomic, immunohistological, and biochemical analyses were performed on the injured spinal cord sections. Administration of metabolic precursors enhanced spinal cord oxidative metabolism. In line with this metabolic shift, we observed the activation of the mTORC1 anabolic pathway, the increase in mitochondrial mass, and ROS defense which effectively prevented the injury-induced neural cell apoptosis in treated animals. Consistently, we found more choline acetyltransferase (ChAT)-expressing motor neurons and increased neurofilament-positive corticospinal axons in the spinal cord parenchyma of the treated mice. Interestingly, oral administration of the metabolic precursors increased the number of activated microglia expressing the CD206 marker suggestive of a pro-resolutive, M2-like phenotype. These molecular and histological modifications observed in treated animals ultimately led to a significant, although partial, improvement of the motor functions. Our data demonstrate that rewiring the cellular metabolism can represent an effective strategy to treat SCI.

Environmental Enrichment Induces Meningeal Niche Remodeling through TrkB-Mediated Signaling.

Neural precursors (NPs) present in the hippocampus can be modulated by several neurogenic stimuli, including environmental enrichment (EE) acting through BDNF-TrkB signaling. We have recently identified NPs in meninges; however, the meningeal niche response to pro-neurogenic stimuli has never been investigated. To this aim, we analyzed the effects of EE exposure on NP distribution in mouse brain meninges. Following neurogenic stimuli, although we did not detect modification of the meningeal cell number and proliferation, we observed an increased number of neural precursors in the meninges. A lineage tracing experiment suggested that EE-induced ß3-Tubulin+ immature neuronal cells present in the meninges originated, at least in part, from GLAST+ radial glia cells. To investigate the molecular mechanism responsible for meningeal reaction to EE exposure, we studied the BDNF-TrkB interaction. Treatment with ANA-12, a TrkB non-competitive inhibitor, abolished the EE-induced meningeal niche changes. Overall, these data showed, for the first time, that EE exposure induced meningeal niche remodeling through TrkB-mediated signaling. Fluoxetine treatment further confirmed the meningeal niche response, suggesting it may also respond to other pharmacological neurogenic stimuli. A better understanding of the neurogenic stimuli modulation for meninges may be useful to improve the effectiveness of neurodegenerative and neuropsychiatric treatments.

Complete neural stem cell (NSC) neuronal differentiation requires a branched chain amino acids-induced persistent metabolic shift towards energy metabolism.

Neural stem cell (NSC) neuronal differentiation requires a metabolic shift towards oxidative phosphorylation. We now show that a branched-chain amino acids-driven, persistent metabolic shift toward energy metabolism is required for full neuronal maturation. We increased energy metabolism of differentiating neurons derived both from murine NSCs and human induced pluripotent stem cells (iPSCs) by supplementing the cell culture medium with a mixture composed of branched-chain amino acids, essential amino acids, TCA cycle precursors and co-factors. We found that treated differentiating neuronal cells with enhanced energy metabolism increased: i) total dendritic length; ii) the mean number of branches and iii) the number and maturation of the dendritic spines. Furthermore, neuronal spines in treated neurons appeared more stable with stubby and mushroom phenotype and with increased expression of molecules involved in synapse formation. Treated neurons modified their mitochondrial dynamics increasing the mitochondrial fusion and, consistently with the increase of cellular ATP content, they activated cellular mTORC1 dependent p70S6 K1 anabolism. Global transcriptomic analysis further revealed that treated neurons induce Nrf2 mediated gene expression. This was correlated with a functional increase in the Reactive Oxygen Species (ROS) scavenging mechanisms. In conclusion, persistent branched-chain amino acids-driven metabolic shift toward energy metabolism enhanced neuronal differentiation and antioxidant defences. These findings offer new opportunities to pharmacologically modulate NSC neuronal differentiation and to develop effective strategies for treating neurodegenerative diseases.

Fingolimod Plays Role in Attenuation of Myocardial Injury Related to Experimental Model of Cardiac Arrest and Extracorporeal Life Support Resuscitation.

BACKGROUND: Sudden cardiac arrest is a major global health concern, and survival of patients with ischemia-reperfusion injury is a leading cause of myocardial dysfunction. The mechanism of this phenomenon is not well understood because of the complex pathophysiological nature of the disease. Aim of the study was to investigate the cardioprotective role of fingolimod in an in vivo model of cardiac arrest and resuscitation. METHODS: In this study, an in vivo rat model of cardiac arrest using extracorporeal membrane oxygenation resuscitation monitored by invasive hemodynamic measurement was developed. At the beginning of extracorporeal life support (ECLS), animals were randomly treated with fingolimod (Group A, n = 30) or saline (Group B, n = 30). Half of the animals in each group (Group A1 and B1, n = 15 each) were sacrificed after 1 h, and the remaining animals (Group A2 and B2) after 24 h of reperfusion. Blood and myocardial tissues were collected for analysis of cardiac features, inflammatory biomarkers, and cell signaling pathways. RESULTS: Treatment with fingolimod resulted in activation of survival pathways resulting into reduced inflammation, myocardial oxidative stress and apoptosis of cardiomyocytes. This led to significant improvement in systolic and diastolic functions of the left ventricle and improved contractility index. CONCLUSIONS: Sphingosine1phosphate receptor activation with fingolimod improved cardiac function after cardiac arrest supported with ECLS. Present study findings strongly support a cardioprotective role of fingolimod through sphingosine-1-phosphate receptor activation during reperfusion after circulatory arrest.

The modulation of BDNF expression and signalling dissects the antidepressant from the reinforcing properties of ketamine: Effects of single infusion vs. chronic self-administration in rats.

Ketamine is a drug of abuse with a unique profile, which besides its inherent mechanism of action as a non-competitive antagonist of the NMDA glutamate receptor, displays both antidepressant and reinforcing properties. The major aim of our study was to find a molecular signature of ketamine that may help in discriminating between its reinforcing and antidepressant effects. To this end, we focused our attention on BDNF, a neurotrophin that has been shown to play a role in both antidepressant and reinforcing properties of several drugs. Rats were exposed to self-administer intravenous (IV) ketamine (S/A) for 43 days or to receive a single IV ketamine 0.5mg/kg, or vehicle infusion. Although the dose we employed is lower than that reported by the literature, it however yields Cmax values that correspond to those achieved in humans after antidepressant treatment. Our results show that while the single infusion of ketamine increased the neurotrophin expression in the hippocampus while reducing it in the ventral striatum, a feature shared with other antidepressants, the repeated self-administration reduced mBDNF expression and its downstream signalling in both ventral striatum and hippocampus. Further, we here show that phosphorylation of Akt is oppositely regulated by ketamine, pointing to this pathway as central to the different actions of the drug. Taken together, we here point to BDNF and its downstream signalling pathway as a finely tuned mechanism whose modulation might subserve the different features of ketamine.

Effects of Different Teaching Approaches on Proxy Measures of Physical Fitness of Italian Kindergarten Children.

Developing physical fitness (PF) behaviors early in life enhances physical development and facilitates sustained participation in physical activity and sports across childhood. This study addressed the effect of different teaching approaches on precursors of PF in kindergarten children. A total of 178 children (5.45 ± 0.40 years, female = 92) from 11 classes were organized into three groups. Group 1 (structured activity + free play) and Group 2 (only free play) attended the same playground (PrimoSport0246) for one hour/week for 10 weeks. Group 3 (structured activity + free play in kindergarten) followed their standard physical education curriculum at school. PF tests (standing long jump, medicine ball throw, and 20 m running speed) were administered pre- and post-intervention. Factorial ANOVA was implemented using a percentage change in PF performance (PFC) as the dependent variable, and teaching approaches, gender, and age as factors. Group 1 demonstrated significant improvements in fitness performance compared with Groups 2 and 3. Moderate to large effect sizes (Cohen's d range: 0.68-1.40) were noted in both males and females. Six-year-old demonstrated the greatest improvement in composite PFC compared to Groups 2 and 3. A structured teaching plan, even with a limited dose of once per week, supported the development of PF attributes in kindergarteners.

Children's Physical Activity During the COVID-19 Lockdown: A Cross Cultural Comparison Between Portugal, Brazil and Italy.

The COVID-19 pandemic forced governments to implement measures that disrupted the daily routines of many families worldwide. We studied how the COVID-19 lockdown affected children's routines in Portugal (PT), Brazil (BR), and Italy (IT) to determine if children's age and country impacted their physical activity (PA) and sedentary time. We launched an anonymous online survey to assess how 3-12 years old children adjusted their daily routines to this situation. Parents reported the times each child was engaged in different activities throughout the day, and we used these data to calculate separately overall sedentary and physical activity time. We conducted separate analyses of variance for age and country on the percentage of time spent in the different activities. Results, based on the data from 3045 children in these three countries (PT n = 2044; BR n = 836; IT n = 165), showed that, during lockdown, most children spent most of their awake daily hours in sedentary activities. There was a clear age effect on the way their routines were organized. Percentages of time spent in intellectual activity, playful screen activity, and overall sedentary time were greater in the older age groups, whereas percentages of time spent in play (with and without PA) and in overall PA were greater in the younger groups. We found a main effect of country for all variables except play without PA. The country effect was mainly due to the difference between the routines in BR when compared to PT and IT. Values of playful screen time and overall sedentary activity were higher in BR than in the two European countries. Conversely, values for play with PA, PA, and overall PA (except in the older group) were lower in BR. Patterns of time spent in these activities were similar in IT and PT, but PA and overall PA times were higher in the two younger age groups in IT. In summary, percentage of PA time of confined children was low and decreased with age across all three countries and was particularly low for children in BR relative to those in PT and IT.

Specific proteolytic cleavage of agrin regulates maturation of the neuromuscular junction.

During the initial stage of neuromuscular junction (NMJ) formation, nerve-derived agrin cooperates with muscle-autonomous mechanisms in the organization and stabilization of a plaque-like postsynaptic specialization at the site of nerve-muscle contact. Subsequent NMJ maturation to the characteristic pretzel-like appearance requires extensive structural reorganization. We found that the progress of plaque-to-pretzel maturation is regulated by agrin. Excessive cleavage of agrin via transgenic overexpression of an agrin-cleaving protease, neurotrypsin, in motoneurons resulted in excessive reorganizational activity of the NMJs, leading to rapid dispersal of the synaptic specialization. By contrast, expression of cleavage-resistant agrin in motoneurons slowed down NMJ remodeling and delayed NMJ maturation. Neurotrypsin, which is the sole agrin-cleaving protease in the CNS, was excluded as the physiological agrin-cleaving protease at the NMJ, because NMJ maturation was normal in neurotrypsin-deficient mice. Together, our analyses characterize agrin cleavage at its proteolytic α- and ß-sites by an as-yet-unspecified protease as a regulatory access for relieving the agrin-dependent constraint on endplate reorganization during NMJ maturation.

Nestin- and doublecortin-positive cells reside in adult spinal cord meninges and participate in injury-induced parenchymal reaction.

Adult spinal cord has little regenerative potential, thus limiting patient recovery following injury. In this study, we describe a new population of cells resident in the adult rat spinal cord meninges that express the neural stem/precursor markers nestin and doublecortin. Furthermore, from dissociated meningeal tissue a neural stem cell population was cultured in vitro and subsequently shown to differentiate into functional neurons or mature oligodendrocytes. Proliferation rate and number of nestin- and doublecortin-positive cells increased in vivo in meninges following spinal cord injury. By using a lentivirus-labeling approach, we show that meningeal cells, including nestin- and doublecortin-positive cells, migrate in the spinal cord parenchyma and contribute to the glial scar formation. Our data emphasize the multiple roles of meninges in the reaction of the parenchyma to trauma and indicate for the first time that spinal cord meninges are potential niches harboring stem/precursor cells that can be activated by injury. Meninges may be considered as a new source of adult stem/precursor cells to be further tested for use in regenerative medicine applied to neurological disorders, including repair from spinal cord injury.

Novel role for prepatterned nicotinic acetylcholine receptors during myogenesis.

INTRODUCTION: Before the nerve contacts the skeletal muscle, the nicotinic acetylcholine receptors (nAChRs) form aggregates known as prepatterned clusters. We investigated their role in the occurrence of Ca(2+) spikes and twitching during myogenesis. METHODS: Cultured mouse myotubes were used as cell models. Cells were subjected to a combination of immunostaining, Ca(2+) imaging and electrophysiological analysis. RESULTS: A single prepatterned nAChR cluster per myotube was generally detected. A correlation between formation of the prepatterned clusters and occurrence of Ca(2+) spikes and twitching was observed. Increase in size of the prepatterned clusters raised the frequency of Ca(2+) spikes and twitching. Blockade of the electrical activity triggered by the autocrine activation of prepatterned nAChR induced over-numbered nAChR clusters. CONCLUSIONS: Prepatterned nAChR aggregation is required for Ca(2+) spikes and twitching of developing myotubes. Moreover, prepatterned nAChR-driven electrical activity preserves the distribution of nAChRs, mimicking the effect of synaptic activity before innervation.

Effects of Free Play and Partly Structured Playground Activity on Motor Competence in Preschool Children: A Pragmatic Comparison Trial.

Both the indoor and the outdoor environments and their organization exert pronounced influence upon physical activity behavior and motor development of preschool children. The aim of this study was to explore whether partly structured activity or free play in a specific playground had different impacts on motor competence development in 4-6-year-old preschoolers. The study had a pretest-post-test design, with two intervention groups and one control. Sixty-two children were included in a partly structured activity group and forty-three children in a free-play group. Both groups participated in playground activities consisting of 10 sessions (once a week), each lasting 1 h, in a specific playground setting. For the partly structured activity group, activities in each session consisted of a combination of both structured and free activity while the free-play group were engaged in unstructured play only. The control group did not attend the playground activities (N = 36). To assess levels of motor skills, each child completed pre- and post-tests using the Movement Assessment Battery for children (MABC-2), the Test of Motor Competence (TMC) and two playground-specific tests. A 3 (study group) and X 2 (gender) ANCOVAs were conducted on post-test scores on each of the test items from TMC, MABC-II and playground tests, with pre-test scores as covariates. Post hoc pairwise multiple comparisons were conducted with the alpha Bonferroni corrected, and the partial eta-squared (η2p) was applied as a measure of effect size. The results indicate no significant differences in motor competence measured by the TMC or the MABC-2 between groups. On the contrary, a significant improvement in performance in the playground-specific tests was observed in the partly structured activity group compared to the free-play and control groups.

Learning to Cycle: A Cross-Cultural and Cross-Generational Comparison.

Background: Learning to cycle is an important milestone for children, but the popularity of cycling and the environmental factors that promote the development and practice of this foundational movement skill vary among cultures and across time. This present study aimed to investigate if country of residence and the generation in which a person was born influence the age at which people learn to cycle. Methods: Data were collected through an online survey between November 2019 and December 2020. For this study, a total of 9,589 responses were obtained for adults (self-report) and children (parental report) living in 10 countries (Portugal, Italy, Brazil, Finland, Spain, Belgium, United Kingdom, Mexico, Croatia, and the Netherlands). Participants were grouped according to their year of birth with 20-year periods approximately corresponding to 3 generations: 1960-79 (generation X; n = 2,214); 1980-99 (generation Y; n = 3,994); 2000-2019 (generation Z; n = 3,381). Results: A two-way ANOVA showed a significant effect of country, F(9,8628) = 90.17, p < 0.001, ηp2 = 0.086, and generation, F(2,8628) = 47.21, p < 0.001, ηp2 = 0.122, on the age at which individuals learn to cycle. Countries with the lowest learning age were the Netherlands, Finland and Belgium and countries with the highest learning age were Brazil and Mexico. Furthermore, the age at which one learns to cycle has decreased across generations. There was also a significant country x generation interaction effect on learning age, F(18,8628) = 2.90, p < 0.001; however, this effect was negligible ( ηp2 = 0.006). Conclusions: These findings support the socio-ecological perspective that learning to cycle is a process affected by both proximal and distal influences, including individual, environment and time.

Meninges: A Widespread Niche of Neural Progenitors for the Brain.

Emerging evidence highlights the several roles that meninges play in relevant brain functions as they are a protective membrane for the brain, produce and release several trophic factors important for neural cell migration and survival, control cerebrospinal fluid dynamics, and embrace numerous immune interactions affecting neural parenchymal functions. Furthermore, different groups have identified subsets of neural progenitors residing in the meninges during development and in the adulthood in different mammalian species, including humans. Interestingly, these immature neural cells are able to migrate from the meninges to the neural parenchyma and differentiate into functional cortical neurons or oligodendrocytes. Immature neural cells residing in the meninges promptly react to brain disease. Injury-induced expansion and migration of meningeal neural progenitors have been observed following experimental demyelination, traumatic spinal cord and brain injury, amygdala lesion, stroke, and progressive ataxia. In this review, we summarize data on the function of meninges as stem cell niche and on the presence of immature neural cells in the meninges, and discuss their roles in brain health and disease. Furthermore, we consider the potential exploitation of meningeal neural progenitors for the regenerative medicine to treat neurological disorders.

Murine cerebral organoids develop network of functional neurons and hippocampal brain region identity.

Brain organoids are in vitro three-dimensional (3D) self-organized neural structures, which can enable disease modeling and drug screening. However, their use for standardized large-scale drug screening studies is limited by their high batch-to-batch variability, long differentiation time (10-20 weeks), and high production costs. This is particularly relevant when brain organoids are obtained from human induced pluripotent stem cells (iPSCs). Here, we developed, for the first time, a highly standardized, reproducible, and fast (5 weeks) murine brain organoid model starting from embryonic neural stem cells. We obtained brain organoids, which progressively differentiated and self-organized into 3D networks of functional neurons with dorsal forebrain phenotype. Furthermore, by adding the morphogen WNT3a, we generated brain organoids with specific hippocampal region identity. Overall, our results showed the establishment of a fast, robust and reproducible murine 3D in vitro brain model that may represent a useful tool for high-throughput drug screening and disease modeling.

Novel stem/progenitor cells with neuronal differentiation potential reside in the leptomeningeal niche.

Stem cells capable of generating neural differentiated cells are recognized by the expression of nestin and reside in specific regions of the brain, namely, hippocampus, subventricular zone and olfactory bulb. For other brain structures, such as leptomeninges, which contribute to the correct cortex development and functions, there is no evidence so far that they may contain stem/precursor cells. In this work, we show for the first time that nestin-positive cells are present in rat leptomeninges during development up to adulthood. The newly identified nestin-positive cells can be extracted and expanded in vitro both as neurospheres, displaying high similarity with subventricular zone-derived neural stem cells, and as homogeneous cell population with stem cell features. In vitro expanded stem cell population can differentiate with high efficiency into excitable cells with neuronal phenotype and morphology. Once injected into the adult brain, these cells survive and differentiate into neurons, thus showing that their neuronal differentiation potential is operational also in vivo. In conclusion, our data provide evidence that a specific population of immature cells endowed of neuronal differentiation potential is resident in the leptomeninges throughout the life. As leptomeninges cover the entire central nervous system, these findings could have relevant implications for studies on cortical development and for regenerative medicine applied to neurological disorders.

Neural agrin changes the electrical properties of developing human skeletal muscle cells.

Recent investigations suggest that the effects of neural agrin might not be limited to neuromuscular junction formation and maintenance and that other aspects of muscle development might be promoted by agrin. Here we tested the hypothesis that agrin induces a change in the excitability properties in primary cultures of non-innervated human myotubes. Electrical membrane properties of human myotubes were recorded using the whole-cell patch-clamp technique. Cell incubation with recombinant chick neural agrin (1 nM) led to a more negative membrane resting potential. Addition of strophanthidin, a blocker of the Na(+)/K(+) ATPase, depolarized agrin-treated myotubes stronger than control, indicating, in the presence of agrin, a higher contribution of the Na(+)/K(+) ATPase in establishing the resting membrane potential. Indeed, larger amounts of both the alpha1 and the alpha2 isoforms of the Na(+)/K(+) ATPase protein were expressed in agrin-treated cells. A slight but significant down-regulation of functional apamin-sensitive K(+) channels was observed after agrin treatment. These results indicate that neural agrin might act as a trophic factor promoting the maturation of membrane electrical properties during differentiation, confirming the role of agrin as a general promoter of muscle development.

Comparing Free Play and Partly Structured Play in 4-5-Years-Old Children in an Outdoor Playground.

The aim of this study was to compare how the organization of a movement session as partly structured play or free play influenced the physical activity engagement in 4-5 years old pre-schoolers. The partly structured playgroup consisted of 46 children and the free playgroup consisted of 33 children. The playground activities consisted of 10 sessions each lasting 1 h, executed once per week in the period Mars to May 2017 at a specific playground setting. The partly structured playgroup conducted a movement activity session that included a combination of both structured- and free play activities. The free playgroup engaged in unstructured play, only. To detect the intensity of the physical activity each child carried an accelerometer 1 h the first week and last week of the intervention. Results indicate a significant difference in physical activity level between the two groups for the 5-year-old in the favor of the partly structured playgroup. There was a significant difference between the four-and 5-year-old in relation to physical activity level. No significant difference between the activity in March and May for the whole group was found.

Expression of small-conductance calcium-activated potassium channels (SK3) in skeletal muscle: regulation by muscle activity.

The type 3 small conductance calcium-activated potassium channel (SK3) is expressed in embryonic and adult denervated skeletal muscles where it contributes to hyperexcitability. This study aimed at determining the role of muscle activity in regulating SK3 channels. Soleus muscles of adult rats were denervated by cutting the sciatic nerve. In reinnervation studies, the soleus nerve was crushed: in one group, muscles were reinnervated with electrically silent axons, by chronic sciatic nerve perfusion with tetrodotoxin. Several groups of denervated muscles were subjected to chronic direct electrical stimulation, using either fast (100 Hz) or slower patterns (20 or 30 Hz). The SK3 mRNA and protein levels in soleus muscle were determined by reverse transcriptional-PCR, Western blot and immunofluorescence. Both denervated and reinnervated-paralysed soleus muscles displayed similar up-regulation of SK3 mRNA and protein. Reinnervation with electrically active axons instead inhibited SK3 up-regulation. Chronic muscle direct stimulation in vivo, irrespective of the pattern used, reversed the denervation-induced up-regulation of SK3 expression or prevented it when initiated at the time of denervation. Chronic electrical stimulation of denervated muscles also completely prevented the development of the after-hyperpolarization (AHP) following the action potential, normally induced in the muscle fibres by denervation. We conclude that action potential activity evoked by motor neurones in muscle fibres is both necessary and sufficient to account for the physiological down-regulation of SK3 channels in the non-junctional membrane of skeletal muscle.

p75 neurotrophin receptor distribution and transport in cultured neurons.

In this work, we define a GFP-tagged version of the p75 neurotrophin receptor (p75GFP) as a useful molecular tool for studying its distribution and cellular dynamics. Expression and subcellular localization of p75GFP have been characterized in non-neuronal (HEK 293) and in neuronal (cortical and hippocampal) cells. By monitoring movements of intracellular p75GFP in living cultured hippocampal neurons, we found that the chimeric protein was transported by tubulo-vesicular structures both anterogradely (0.1-0.5microm/s) and retrogradely (0.1-1.1microm/s), with a faster component in retrogradely moving structures. Movements of the p75GFP-containing structures were inhibited by treatment with the microtubule-disrupting agent nocodazole. Our data indicate that p75GFP is a reliable tool for studying spatial and cellular properties of p75 in CNS neurons and that p75 transport inside neurons is mediated by microtubule-associated motors.

Nicotine-induced phosphorylation of phosphorylated cyclic AMP response element-binding protein (pCREB) in hippocampal neurons is potentiated by agrin.

The scope of this study was to test whether increased levels of the extracellular matrix molecule (ECM) agrin might enhance nicotine effects on those molecular mechanisms that initiate neuroadaptative processes in the hippocampus, a key brain area for learning and memory. We studied the effects of repetitive applications of neuronal agrin to primary hippocampal cell culture on nicotine-induced phosphorylated cyclic AMP response element-binding protein (pCREB) expression, a marker of neuroadaptation, by using immunofluorescence-based assessment of pCREB-positive neurons. We also tested agrin effects on nicotine-induced expression of a marker of metabolic activation, the immediate early gene c-fos. Agrin was shown to significantly enhance nicotine-induced pCREB, but not c-fos, expression. By using Western blotting analysis, cumulative agrin has been shown to increase nicotine-induced pCREB phosphorylation. These analyses, however, showed that inhibition of the CaMKII pathway blocked general pCREB phosphorylation, whereas inhibition of the MAPK pathway potentiated the synergistic effect of cumulative agrin and nicotine. These findings suggest that increasing the concentration of an ECM molecule, i.e. agrin, may enhance nicotine effects on pCREB and that both MAPK and CaMKII signalling may play a regulatory role.