Adeno-associated virus (AAV) vectors can deliver transgenes to diverse cell types and are therefore useful for basic research and gene therapy. Although AAV has many advantages over other viral vectors, its relatively small packaging capacity limits its use for delivering large genes. The available transgene size is further limited by the existence of additional elements in the expression cassette without which the gene expression level becomes much lower. By using alternative combinations of shorter elements, we generated a series of AAV expression cassettes and systematically evaluated their expression efficiency in neurons to maximize the transgene size available within the AAV packaging capacity while not compromising the transgene expression. We found that the newly developed smaller expression cassette shows comparable expression efficiency with an efficient vector generally used for strong gene expression. This new expression cassette will allow us to package larger transgenes without compromising expression efficiency.

In this review, we focus on the role of the Shank family of proteins in autism. In recent years, autism research has been flourishing. With genetic, molecular, imaging and electrophysiological studies being supported by behavioural studies using animal models, there is real hope that we may soon understand the fundamental pathology of autism. There is also genuine potential to develop a molecular-level pharmacological treatment that may be able to deal with the most severe symptoms of autism, and clinical trials are already underway. The Shank family of proteins has been strongly implicated as a contributing factor in autism in certain individuals and sits at the core of the alleged autistic pathway. Here, we analyse studies that relate Shank to autism and discuss what light this sheds on the possible causes of autism.

The dorsal striatum, with its functional microcircuits galore, serves as the primary gateway of the basal ganglia and is known to play a key role in implicit learning. Initially, excitatory inputs from the cortex and thalamus arrive on the direct and indirect pathways, where the precise flow of information is then regulated by local GABAergic interneurons. The balance of excitatory and inhibitory transmission in the dorsal striatum is modulated by neuromodulators such as dopamine and acetylcholine. Under pathophysiological states in the dorsal striatum, an alteration in excitatory and inhibitory transmission may underlie dysfunctional motor control. Here, we review the cellular connections and modulation of striatal microcircuits and propose that modulating the excitatory and inhibitory balance in synaptic transmission of the dorsal striatum is important for regulating locomotion.The dorsal striatum is best known for its role in decision-making, especially in action selection and initiation through the convergence of sensorimotor, cognitive, and motivational information (DeLong 1990;Smith et al. 1998;Balleine et al. 2007). As the primary input of the basal ganglia, the striatum receives glutamatergic inputs from the cortex and thalamus and in turn projects GABAergic outputs to the globus pallidus and substantia nigra pars reticulata (SNr). Inputs from the cortex and thalamus both form excitatory synaptic connections on medium spiny neurons (MSN) in which cortical afferents are from the sensory, motor, and associational cortex (Bolam et al. 2000), and thalamic afferents are from the intralaminar thalamic nuclei (Doig et al. 2010). These glutamatergic inputs are then processed in the dorsal striatum where numerous connections between various types of neurons exist. Thus, the complexity of neuronal circuits has made it difficult to elucidate the functional roles of the striatum. Recently, studies focused on interneurons that reside in the dorsal striatum have characterized the physiological features and functional connections. For example, parvalbumin-expressing fastspiking interneurons (PV-FSI) and neuropeptide-Y positive lowthreshold spiking interneurons (NPY-LTS) form synaptic connections with MSNs and regulate the firing activity of the principal neuron MSNs (Koos and Tepper 1999;Gittis et al. 2010;Chuhma et al. 2011). These interneurons were shown to have distinct firing patterns and connections, and thus they may exert different effects on MSNs. Other crucial connections are the cholinergic, dopaminergic, and serotonergic axons that strongly innervate the dorsal striatum. These projections are essential for modulating striatal circuits and disruption of such signaling can result in movement impairments and neurological disorders such as Huntington's disease (Lovinger 2010). This review summarizes recent reports of the microcircuits present in the dorsal striatum, although serotonergic signaling is excluded, and suggests a putative role for striatal microcircuits in motor dysfunction and/or hyperactivity that ...

MicroRNAs are non-coding short (~23 nucleotides) RNAs that mediate post-transcriptional regulation through sequence-specific gene silencing. The role of miRNAs in neuronal development, synapse formation and synaptic plasticity has been highlighted. However, the role of neuronal activity on miRNA regulation has been less focused. Neuronal activity-dependent regulation of miRNA may finetune gene expression in response to synaptic plasticity and memory formation. Here, we provide an overview of miRNA regulation by neuronal activity including highthroughput screening studies. We also discuss the possible molecular mechanisms of activity-dependent induction and turnover of miRNAs.

Memory reconsolidation is ubiquitous across species and various memory tasks. It is a dynamic process in which memory is modified and/or updated. In experimental conditions, memory reconsolidation is usually characterized by the fact that the consolidated memory is disrupted by a combination of memory reactivation and inhibition of protein synthesis. However, under some experimental conditions, the reactivated memory is not disrupted by inhibition of protein synthesis. This so called "boundary condition" of reconsolidation may be related to memory strength. In Pavlovian fear conditioning, the intensity of unconditional stimulus (US) determines the strength of the fear memory. In this study, we examined the effect of the intensity of US on the reconsolidation of contextual fear memory. Strong contextual fear memory, which is conditioned with strong US, is not disrupted by inhibition of protein synthesis after its reactivation; however, a weak fear memory is often disrupted. This suggests that a US of strong intensity can inhibit reconsolidation of contextual fear memory.

Fear conditioning has been used to study pathogenic mechanisms underlying anxiety disorders. Several studies have shown that humans with anxiety disorders exhibit strong fear responses during the acquisition of conditioned fear. However, there have been no studies investigating whether basal anxiety within the normal range is related to conditioned fear in rodents. We hypothesized that individual differences in conditioned fear are correlated to the basal anxiety level of each individual. To test this hypothesis, we measured the basal anxiety of mice by using the elevated-plus maze (EPM) and open field test (OFT) and correlated these data with contextual freezing during contextual fear conditioning (CFC). Strong correlation was found between the basal anxiety level measured in the OFT and contextual freezing in the CFC. Baseline freezing was also strongly correlated with the contextual freezing level during the retrieval phase of CFC. However, the basal anxiety level measured in the EPM was correlated neither with conditioned fear nor with baseline freezing in the CFC. These results suggest that both basal anxiety in the OFT and baseline freezing are related to contextually conditioned fear.

Bake‰ J, J. Illek: Plasma Ceruloplasmin and Fibrinogen during Enzyme Therapy of Mastitis in Dairy Cows. Acta Vet. Brno, 2006, 75: 241-246.The aim of this study was to demonstrate the benefits of topical administration of proteolytic enzymes used in the symptomatic mastitis therapy. Eleven lactating cows with clinical signs of mastitis in one udder quarter at least were divided into two groups. Group A was given an antibiotic alone -cefotaxime (250 mg, intramammary administration, 6 doses in total). Group B was given a lower dose of the same antibiotic (cefotaxime, 100 mg, intramammary administration, 5 doses in total) and a blend of enzymes in one drug form, i.e. both the dose and total number of administrations were lower in this group. Before and after the therapy, milk samples were collected and subjected to microbiological examination, and blood samples were withdrawn to determine fibrinogen and ceruloplasmin as acute phase proteins. Before the beginning of the therapy, common mastitis pathogens were detected in milk of both groups; after the end of the therapy, the test results were negative. Mean plasma concentrations of fibrinogen in Group A increased significantly from 6.0 ± 1.4 to 7.4 ± 1.1 g·l -1 , in Group B a non-significant decrease was observed, from 6.5 ± 0.86 g·l -1 to 6.4 ± 0.9 g·l -1 . However, differences between the groups were significant (p < 0.05). In Group A, mean plasma ceruloplasmin concentration increased significantly from 0.9 ± 0.2 to 1.3 ± 0.18 µmol·l -1 , and in Group B it decreased non-significantly from 0.9 ± 0.36 to 1.1 ± 0.26 µmol·l -1 , and the difference between the groups was non-significant. In the present study, a favourable effect of repeated topical application of a mixture of proteolytic enzymes was demonstrated. The enzyme therapy resulted in a decreased therapeutic dose of the antibiotic, and suppressed an increase in the levels of inflammation markers under investigation. Bromelain, cefotaxime, chymotrypsin, papainParticularly during acute mastitis, the mammary gland shows typical signs of inflammatory process. In order to provide the shortest and most effective therapy of acute mastitis, it is necessary to support causal therapy, focused on the causative agent (antiinfectives) using symptomatic therapy to suppress the inflammation (antiflogistics). In practice, glucocorticoids have been widely used, non-steroid antiflogistics (NAF) and enzymes have been used less frequently. Many studies demonstrated that the effects of enzymes are comparable with those of NAF (Salamberidze et al. 2002). The systemic effect is the most pronounced in glucocorticoids, even when it comes to undesirable effects. Unlike enzymes, glucocorticoides show an anti-inflammatory effect accompanied by immunosuppression. Positively adjuvant effects of enzyme therapy include the potentiation of antibiotic effect at a site of inflammation, an increase in the acute cell response and an anti-oedema effect of trypsin, chymotrypsin and bromelain, and an oedema-protective effect of papain (Kasser...

Phosphoinositide 3-kinases (PI3Ks) play key roles in synaptic plasticity and cognitive functions in the brain. We recently found that genetic deletion of PI3Kγ, the only known member of class IB PI3Ks, results in impaired N-methyl-D-aspartate receptor-dependent long-term depression (NMDAR-LTD) in the hippocampus. The activity of RalA, a small GTP-binding protein, increases following NMDAR-LTD inducing stimuli, and this increase in RalA activity is essential for inducing NMDAR-LTD. We found that RalA activity increased significantly in PI3Kγ knockout mice. Furthermore, NMDAR-LTDinducing stimuli did not increase RalA activity in PI3Kγ knockout mice. These results suggest that constitutively increased RalA activity occludes further increases in RalA activity during induction of LTD, causing impaired NMDARLTD. We propose that PI3Kγ regulates the activity of RalA, which is one of the molecular mechanisms inducing NMDARdependent LTD. [BMB Reports 2013; 46(2): 103-106]