The effectiveness of evidence summaries on health policymakers and health system managers use of evidence from systematic reviews: a systematic review

Systematic reviews are important for decision makers. They offer many potential benefits but are often written in technical language, are too long, and do not contain contextual details which make them hard to use for decision-making. There are many organizations that develop and disseminate derivative products, such as evidence summaries, from systematic reviews for different populations or subsets of decision makers. This systematic review aimed to (1) assess the effectiveness of evidence summaries on policymakers' use of the evidence and (2) identify the most effective summary components for increasing policymakers' use of the evidence. We present an overview of the available evidence on systematic review derivative products.

Adenomatous polyposis coli protein deletion leads to cognitive and autism-like disabilities.

Intellectual disabilities (IDs) and autism spectrum disorders link to human APC inactivating gene mutations. However, little is known about adenomatous polyposis coli's (APC's) role in the mammalian brain. This study is the first direct test of the impact of APC loss on central synapses, cognition and behavior. Using our newly generated APC conditional knock-out (cKO) mouse, we show that deletion of this single gene in forebrain neurons leads to a multisyndromic neurodevelopmental disorder. APC cKO mice, compared with wild-type littermates, exhibit learning and memory impairments, and autistic-like behaviors (increased repetitive behaviors, reduced social interest). To begin to elucidate neuronal changes caused by APC loss, we focused on the hippocampus, a key brain region for cognitive function. APC cKO mice display increased synaptic spine density, and altered synaptic function (increased frequency of miniature excitatory synaptic currents, modestly enhanced long-term potentiation). In addition, we found excessive ß-catenin levels and associated changes in canonical Wnt target gene expression and N-cadherin synaptic adhesion complexes, including reduced levels of presenilin1. Our findings identify some novel functional and molecular changes not observed previously in other genetic mutant mouse models of co-morbid cognitive and autistic-like disabilities. This work thereby has important implications for potential therapeutic targets and the impact of their modulation. We provide new insights into molecular perturbations and cell types that are relevant to human ID and autism. In addition, our data elucidate a novel role for APC in the mammalian brain as a hub that links to and regulates synaptic adhesion and signal transduction pathways critical for normal cognition and behavior.

Homocysteine thiolactone induces cardiac dysfunction: role of oxidative stress.

This study investigates the cardiac functioning in male Wistar rats after treatments with methionine and homocysteine thiolactone (HcyT). The rats were distributed into 3 groups and treated for 8 weeks. Group I was the control (CO) group, given water, group II was treated with methionine, and group III with HcyT (100 mg/kg). Morphometric and functional cardiac parameters were evaluated by echocardiography. Superoxide dismutase (SOD), catalase, and glutathione S-transferase activities, chemiluminescence, thiobarbituric acid reactive substances, and immunocontent were measured in the myocardium. Hyperhomocysteinemiawas observed in rats submitted to the both treatments. The results showed diastolic function was compromised in HcyT group, seen by the increase of E/A (peak velocity of early (E) and late (A) diastolic filling) ratio, decrease in deceleration time of E wave and left ventricular isovolumic relaxation time. Myocardial performance index was increased in HcyT group and was found associated with increased SOD immunocontent. HcyT group demonstrated an increase in SOD, catalase, and glutatione S-transferase activity, and chemiluminescence and thiobarbituric acid reactive substances. Overall, these results indicated that HcyT induces a cardiac dysfunction and could be associated with oxidative stress increase in the myocardium.

Synaptic interactions regulate gephyrin expression in avian cholinergic neurons in vivo.

Our recent studies of chick parasympathetic ciliary ganglion (CG) neurons demonstrate a unique postsynaptic receptor microheterogeneity - under one presynaptic terminal, excitatory nicotinic acetylcholine receptor (nAChR) clusters and separate inhibitory glycine receptor (GlyR) clusters coexist in distinct membrane microregions. Gephyrin, a peripheral membrane protein that is required for GlyR clustering at synapses in the rodent central nervous system, is also expressed in chick CG neurons where it codistributes with GlyRs, but not nAChRs. We now extend these findings by characterizing the regulation of gephyrin expression in chick CG neurons in vivo. We show that developmental increases in gephyrin transcript levels occur during pre- and postganglionic synapse formation. The increases are induced by both innervation and target tissue interactions, with the target tissues having the greater regulatory influence. The time course of the developmental rise in gephyrin mRNA levels most closely resembles that reported for functional GlyR expression, but not that of functional nAChRs nor GABA(A) receptors. We also demonstrate that gephyrin is concentrated in the postsynaptic density of a subset of synapses on both the ciliary and choroid neurons in the CG and is stably expressed from embryonic to adult stages. Altogether, our results suggest that gephyrin is a synapse organizing molecule that functions to localize GlyRs, but not nAChRs, to discrete postsynaptic membrane microregions in chick CG neurons in vivo.

Receptor targeting and heterogeneity at interneuronal nicotinic cholinergic synapses in vivo.

Within a single neuron the correct targeting of the diverse neurotransmitter receptor types to discrete synaptic regions is crucial for proper function. However, the molecular mechanisms that underlie neuronal receptor clustering and targeting are still largely undefined. Here we report advances in defining the mechanisms that mediate nicotinic acetylcholine receptor (nAChR) targeting to interneuronal synapses. Recent in vivo studies have demonstrated that one subunit plays a critical role in the differentiation of nicotinic cholinergic synapses on vertebrate autonomic neurons. The major cytoplasmic loop of the alpha3 subunit targets specific nAChR subtypes to the synapse. In contrast, nAChR complexes that lack the alpha3 targeting domain are excluded and are perisynaptic. Additional studies have demonstrated a greater complexity to alpha3-nAChR targeting due to a unique postsynaptic receptor microheterogeneity - under one presynaptic terminal, alpha3-nAChR clusters are separate, but proximal to, glycine receptor (GlyR) clusters in discrete postsynaptic membrane microregions. The surprising coexistence under one nerve ending of separate clusters of receptors that respond to different fast-acting transmitters with opposing functions may represent a novel mechanism for modulating synaptic activity. Overall, the receptor targeting and clustering studies reviewed in this issue suggest that a common mechanism underlies the formation of the diverse types of interneuronal synapses but differs from that responsible for neuromuscular junction assembly in vertebrates.

Receptors with opposing functions are in postsynaptic microdomains under one presynaptic terminal.

Fast excitatory synaptic transmission through vertebrate autonomic ganglia is mediated by postsynaptic nicotinic acetylcholine receptors (nAChRs). We demonstrate a unique postsynaptic receptor microheterogeneity on chick parasympathetic ciliary ganglion neurons-under one presynaptic terminal, nAChRs and glycine receptors formed separate but proximal clusters. Terminals were loaded with [3H]glycine via the glycine transporter-1 (GlyT-1), which localized to the cholinergic presynaptic terminal membrane; depolarization evoked [3H]glycine release that was calcium independent and blocked by the GlyT-1 inhibitor sarcosine. Ganglionic synaptic transmission mediated by nAChRs was attenuated by glycine. Coexistence of separate clusters of receptors with opposing functions under one terminal contradicts Dale's principle and provides a new mechanism for modulating synaptic activity in vivo.

Innervation and target tissue interactions induce Rab-GDP dissociation inhibitor (GDI) expression during peripheral synapse formation in developing chick ciliary ganglion neurons in situ.

Regulated exocytosis of neurotransmitter from synaptic vesicles involves the function of a small GTP-binding protein, Rab3A. Rab-GDP dissociation inhibitor (GDI) is an important modulator of Rab function and subcellular distribution. We have characterized the respective roles of innervation and target tissue interactions in regulating GDI expression during synapse formation in chick ciliary ganglion (CG) neurons developing in situ. Here we report the first full-length chick GDI cDNA sequence. It is highly homologous to mammalian GDI isoforms and includes all of the sequence-conserved regions critical for Rab3A binding. This chick GDI mRNA is predominantly expressed in neurons as judged by Northern blot analysis of tissue distribution and by in situ hybridization of CG sections. Developmental increases in CG GDI mRNA levels occur in two phases as determined by reverse transcription (RT)-PCR and by Northern analysis of both normal-developing and input- or target tissue-deprived ganglia. The initial phase appears to be independent of cell-cell interactions. In contrast, the second, larger increase is induced by both presynaptic inputs and postganglionic target tissues but does not occur until target tissue innervation. Synaptic interaction with the target seems necessary for the regulatory response to both inputs and target tissues. GDI protein levels show similar changes. The developmentally delayed ability of inputs and targets to influence GDI levels differs from the regulation of neurotransmitter receptor expression in CG neurons. These results suggest that distinct extrinsic regulatory signals influence the expression of synapse-related components at the presynaptic axon terminal versus postsynaptic membrane in an individual neuron.

The long internal loop of the alpha 3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo.

Different types of neurotransmitter receptors coexist within single neurons and must be targeted to discrete synaptic regions for proper function. In chick ciliary ganglion neurons, nicotinic acetylcholine receptors (nAChRs) containing alpha 3 and alpha 5 subunits are concentrated in the postsynaptic membrane, whereas alpha-bungarotoxin receptors composed of alpha 7 subunits are localized perisynaptically and excluded from the synapse. Using retroviral vector-mediated gene transfer in vivo, we show that the long cytoplasmic loop of alpha 3 targets chimeric alpha 7 subunits to the synapse and reduces endogenous nAChR surface levels, whereas the alpha 5 loop does neither. These results show that a particular domain of one subunit targets specific receptor subtypes to the interneuronal synapse in vivo. Moreover, our findings suggest a difference in the mechanisms that govern assembly of interneuronal synapses as compared to the neuromuscular junction in vertebrates.

Changes in the regulatory effects of cell-cell interactions on neuronal AChR subunit transcript levels after synapse formation.

Nicotinic acetylcholine receptors (AChRs) mediate excitatory synaptic transmission in the chick ciliary ganglion. AChR protein and mRNA levels are increased by both innervation and retrograde signals from target tissues during synapse formation. We now show that AChR alpha3, beta4, and alpha5 subunit transcript levels stop increasing after synaptogenesis. Moreover, maintenance of these mRNA levels requires the continued presence of regulatory signals from both pre- and postganglionic tissues. Unilateral preganglionic denervation or postganglionic axotomy causes declines in alpha3, beta4, and alpha5 transcript levels, ranging from twofold to 3. 5-fold, relative to contralateral control neuron values in newly hatched chicks. The reductions are not merely an injury response; cbeta4-tubulin mRNA levels are not affected by either axotomy or denervation. Further, similar decreases in AChR mRNA levels are observed after local application of colchicine to the postganglionic nerves, which blocks fast transport without disturbing axonal integrity. These results also demonstrate a developmental change in the regulatory effects of target tissues. Reductions in alpha5 mRNA levels caused by axotomy or colchicine treatment after peripheral synapse formation contrast with the lack of an effect on alpha5 when synapse formation with the target tissue is prevented. The ability of the target tissue to regulate alpha5 mRNA levels after synaptogenesis is interesting, because this subunit may be necessary for the formation of high-conductance AChRs. The specific regulatory effects of target tissues and inputs at different developmental stages demonstrate that neurons continually depend on signals from their pre- and postsynaptic tissues to accomplish mature levels of AChR subunit expression and optimal functioning of that neuronal circuit.

Differential display protocol with selected primers that preferentially isolates mRNAs of moderate- to low-abundance in a microscopic system.

A modified reverse transcription polymerase chain reaction (RT-PCR)-based differential display procedure with selected primers (SPR) was developed to increase the bias toward isolating moderate- to low-abundance transcripts that are differentially expressed during synapse formation in a microscopic neuronal system, the embryonic chicken ciliary ganglion. Major modifications, in comparison with available arbitrarily primed RT-PCR protocols, include the use of (i) experimentally selected primer pairs (50% GC-rich 15-21-mers) that avoid the amplification of highly abundant ribosomal and mitochondrial transcripts; (ii) a higher PCR annealing temperature (50 degrees C instead of 40 degrees C); (iii) selection of sequencing gel bands that are dependent on the two primers for amplification; (iv) tests for reproducibility by SPR amplification of independent sets of RNA extractions and Southern blot analysis of the products with an isolated radiolabeled clone; and (v) quantitative RT-PCR, instead of Northern blot analysis, to confirm the differential expression of individual cDNAs. Thirty-six cDNAs were isolated and sequenced using SPR. None showed significant homology to highly abundant transcripts. In contrast, when no criterion for primer or band selection was applied, 22% of 55 cDNAs were identical to ribosomal and mitochondrial transcripts. Reproducible amplification of 9 out of 10 SPR-isolated cDNAs was established by Southern blot analysis. Differential expression was then confirmed for 4 selected sequences by quantitative RT-PCR. Thus, SPR is a reproducible and efficient procedure for identifying differentially regulated transcripts of moderate- to low-abundance in microscopic biological systems.

Agrin gene expression in ciliary ganglion neurons following preganglionic denervation and postganglionic axotomy.

Agrin is an extracellular matrix protein that has been implicated as a synaptogenic agent in the peripheral and central nervous systems. Both the level of expression and pattern of alternative splicing of agrin mRNA are developmentally regulated. As a step toward identifying signals important in regulating agrin gene expression in neurons, we examined the effects of postganglionic axotomy or preganglionic denervation on agrin mRNA levels and alternative splicing in ciliary ganglia of posthatch chicks. In comparison to unoperated age-matched controls, in situ hybridization with a pan-specific agrin cRNA probe demonstrated a significant decrease in neuronal agrin mRNA expression as a result of axotomy. Reverse transcription-polymerase chain reaction analysis demonstrated that axotomy also resulted in changes in the pattern of alternative splicing of agrin mRNA. Underlying these changes are decreases in the molar amounts of transcripts encoding the neuron-specific isoforms agrin8 and agrin19, homologous to rat agrin proteins that have high AChR aggregating activity. Similar, but less dramatic changes in agrin expression following axotomy were also observed in unoperated neurons on the contralateral side. In contrast, the only significant change in agrin gene expression following ganglionic denervation was a small decline in the relative abundance of agrin 8 mRNA in operated versus unoperated age-matched control ganglia. Major changes in agrin gene expression following axotomy but not denervation are consistant with the notion that agrin synthesized by ganglionic neurons exerts its effects in the periphery rather than at synapses formed between ciliary ganglion neurons and their preganglionic input. These data suggest that the pattern of alternative splicing and the absolute amount of agrin mRNA in ciliary ganglion neurons may be regulated by target tissue interactions.

Innervation and target tissue interactions differentially regulate acetylcholine receptor subunit mRNA levels in developing neurons in situ.

Neurons engage in two distinct types of cell-cell interactions: they receive innervation and establish synapses on target tissues. Regulatory events that influence synapse formation and function on developing neurons are largely undefined. We show here that nicotinic acetylcholine receptor (AChR) subunit transcript levels are differentially regulated by innervation and target tissue interactions in developing chick ciliary ganglion neurons in situ. Using ganglia that have developed in the absence of pre- or postganglionic tissues and quantitative RT-PCR, we demonstrate that alpha 3 and beta 4 transcript levels are increased by innervation and target tissue interactions. In contrast, alpha 5 transcript levels are increased by innervation, but target tissues have little effect. Whole-cell ACh-induced currents, used to estimate the number of functional AChRs, change in correlation with alpha 3 and beta 4, but not alpha 5, transcript levels. A model is proposed in which the changes in AChR subunit expression regulate levels of synaptic activity, which is a critical determinant of synapse stabilization and elimination, and neuronal cell death.

Target tissues and innervation regulate the characteristics of K+ currents in chick ciliary ganglion neurons developing in situ.

The expression of appropriate ensembles of ionic channels is necessary for the differentiation and normal function of vertebrate neurons. Cell-cell interactions may regulate the expression and properties of ionic channels in embryonic neurons. Previous studies have shown that the expression of A-type K+ channels (IA) and Ca2+-activated K+ channels (lK[Ca]) is abnormal in chick ciliary ganglion neurons developing in vitro in the absence of normal cell-cell interactions. Other voltage-activated currents develop normally under these conditions. The present studies were designed to establish the role of the target tissues and the preganglionic innervation in regulating the expression of these currents in embryonic chick ciliary ganglion neurons developing in situ. Surgical manipulations were used to remove the developing optic vesicle, which contains the target tissues, the mid-dorsal region of the midbrain primordium, which contains the preganglionic nucleus, or both, all prior to the formation of the ciliary ganglion. IA and IK[Ca] were then examined in acutely isolated neurons that developed in ovo in the presence (OV+) or absence (OV-) of the normal target tissues, in the presence (MB+) or absence (MB-) of preganglionic innervation, and in the absence of both preganglionic innervation and target tissues (OV-/MB-). The amplitude of IA was unaffected by the operations. However, the activation and inactivation kinetics of IA were two- to threefold faster in OV- or OV-/MB- cells compared to neurons isolated from control OV+ ganglia at embryonic days 11-14 (E11-E14). There were no changes in the voltage dependence of activation or steady-state inactivation, or in the time course of recovery from inactivation. By contrast, neurons isolated from MB- ganglia expressed an IA with amplitude, voltage dependence, and kinetics that were indistinguishable from those of control MB+ and OV+ ganglia. Therefore, interactions with target tissues in the eye play a role in determining the characteristics of IA in developing ciliary ganglion neurons, whereas preganglionic innervation does not. Furthermore, the amplitude of IK[Ca] was reduced by 90-100% in OV-, MB-, and OV-/MB- neurons isolated at E12-E14 as compared to MB+ and OV+ controls. Voltage-activated Ca2+ currents were present at normal amplitudes in all of these neurons. Thus, the expression of IK[Ca] in chick ciliary ganglion neurons is regulated by both target tissue interactions and preganglionic innervation. Therefore, cell-cell interactions are necessary for the expression of a normal ensemble of ionic channels in chick ciliary ganglion neurons developing in situ.

Reduced levels of acetylcholine receptor expression in chick ciliary ganglion neurons developing in the absence of innervation.

Chick ciliary ganglion neurons receive innervation from a single source, the accessory oculomotor nucleus (AON), and nicotinic ACh receptors (AChRs) mediate chemical synaptic transmission through the ganglion. Previous experiments examining the developmental expression of AChRs in embryonic chick ciliary ganglion neurons in situ have shown that AChR levels increase substantially in the neurons at the time of innervation. Prior to synapse formation, few AChRs are detected in the neurons. In the present experiments, the role of presynaptic inputs in inducing an increase in AChRs was established by examining AChR levels in ciliary ganglion neurons that have been deprived of innervation by surgical ablation of the AON prior to synapse formation. AChR levels were dramatically reduced in neurons of input-deprived ganglia as compared to control innervated neurons at all developmental stages examined from embryonic day (ED) 5 to ED 12 as determined by indirect immunocytochemical labeling of frozen ganglion sections with the anti-AChR monoclonal antibody mAb 35, and light microscopy. In contrast, neuronal somata of input-deprived and control ganglia had equivalent levels of immunolabeling for three other components, a transmembrane glycoprotein of synaptic vesicles, SV2, and two microtubule-associated proteins, MAP 1B and MAP 2, from ED 5 up to ED 10. The results demonstrate that presynaptic inputs specifically increase the levels of AChR expression in developing neurons. In addition, changes in the levels of immunolabeling for AChRs, SV2, MAP 1B, and MAP 2 in neuronal somata after ED 10 demonstrate that other major developmental events also influence the levels of these components in neurons. Declines in the intensity of AChR, SV2, MAP 1B, and MAP 2 immunolabeling within a subset of neuronal somata in both operated and control ganglia at ED 10 and 12 coincide with the period of neuronal cell death. Increases in AChR labeling in the rest of the neuronal population of input-deprived ganglia at ED 12 suggest that, in addition to innervation, synapse formation with the peripheral target tissue influences AChR levels in developing neurons in situ.

Acetylcholine receptor expression in developing chick ciliary ganglion neurons.

Little is known about the levels of nicotinic ACh receptors (AChRs) in neurons prior to innervation and whether the distribution and number of receptors change in response to innervation. In the present study, AChR levels were examined in developing chick ciliary ganglion neurons in situ at stages preceding and during early and late phases of synaptogenesis. AChRs were localized in surface and intracellular pools of intact and saponin-permeabilized ganglionic neurons, respectively, by using a highly sensitive immunocytochemical approach that included the binding of an anti-AChR monoclonal antibody (mAb) followed by a biotinylated secondary antibody and an avidin-biotinylated HRP complex. At older stages of development, embryonic day (ED) 7-7.5 and ED 11, when all of the neurons are known to be receiving synaptic contacts, AChRs were present in both internal and surface pools. Within the neurons, AChRs were associated with organelles that function in the biosynthesis, processing, and transport of integral plasma membrane proteins. On the surface of the neurons, AChRs were predominantly localized in the specialized postsynaptic membrane, with low levels of AChRs being present in extrasynaptic regions. The earliest stage at which synapses could be detected in the ganglion was ED 4.5. Synapses were detected by light microscopic immunocytochemical labeling with anti-SV2, an mAb to a synaptic vesicle protein, and by ultrastructural analysis. At this stage, most of the neurons were not labeled by the anti-AChR mAb, while a few neurons had dense deposits of reaction product on the rough endoplasmic reticulum and portions of the nuclear envelope. Low levels of reaction product were also found on the surface of a small number of neurons, being localized predominantly on the specialized postsynaptic membrane of the few immature synapses present. Occasionally, small patches of labeling were observed in extrasynaptic regions. In contrast, little internal and no surface anti-AChR immunolabeling was detected in ciliary ganglion neurons prior to innervation, at ED 3.5-4. The finding of a large increase in both internal and surface AChR levels in the neurons at the time of innervation suggests that signals from the presynaptic input play an important role in the induction of AChR expression in neurons.