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.

Effects of preganglionic denervation and postganglionic axotomy on acetylcholine receptors in the chick ciliary ganglion.

The regulation of nicotinic acetylcholine receptors (AChRs) in chick ciliary ganglia was examined by using a radiolabeled anti-AChR mAb to quantitate the amount of receptor in ganglion detergent extracts after preganglionic denervation or postganglionic axotomy. Surgical transection of the preganglionic input to the ciliary ganglion in newly hatched chicks caused a threefold reduction in the total number of AChRs within 10 d compared with that present in unoperated contralateral control ganglia. Surgical transection of both the choroid and ciliary nerves emerging from the ciliary ganglion in newly hatched chicks to establish postganglionic axotomy led to a nearly 10-fold reduction in AChRs within 5 d compared with unoperated contralateral ganglia. The declines were specific since they could not be accounted for by changes in ganglionic protein or by decreases in neuronal survival or size. Light microscopy revealed no gross morphological differences between neurons in operated and control ganglia. A second membrane component of cholinergic relevance on chick ciliary ganglion neurons is the alpha-bungarotoxin (alpha-Bgt)-binding component. The alpha-Bgt-binding component also declined in number after either postganglionic axotomy or preganglionic denervation, but appeared to do so with a more rapid time course than did ganglionic AChRs. The results imply that cell-cell interactions in vivo specifically regulate both the number of AChRs and the number of alpha-Bgt-binding components in the ganglion. Regulation of these neuronal cholinergic membrane components clearly differs from that previously described for muscle AChRs.

Surface and intracellular distribution of a putative neuronal nicotinic acetylcholine receptor.

Chick ciliary ganglion neurons have a membrane component that shares an antigenic determinant with the main immunogenic region (MIR) of nicotinic acetylcholine receptors from skeletal muscle and electric organ. Previous studies have shown that the component has many of the properties expected for a ganglionic nicotinic acetylcholine receptor, and that its distribution on the neuron surface in vivo is restricted predominantly to synaptic membrane. Here we report the presence of a large intracellular pool of the putative receptor in embryonic neurons and demonstrate that it is associated with organelles known to comprise the biosynthetic and regulatory pathways of integral plasma membrane proteins. Embryonic chick ciliary ganglia were lightly fixed, saponin-permeabilized, incubated with an anti-MIR monoclonal antibody (mAb) followed by horseradish peroxidase-conjugated secondary antibody, reacted for peroxidase activity, and examined by electron microscopy. Deposits of reaction product were associated with synaptic membrane, small portions of the pseudodendrite surface membrane, most of the rough endoplasmic reticulum, small portions of the nuclear envelope, some Golgi complexes, and a few coated pits, coated vesicles, multivesicular bodies, and smooth-membraned vacuoles. No other labeling was present in the neurons. The labeling was specific in that it was not present when the anti-MIR mAb was replaced with either nonimmune serum or mAbs of different specificity. Chick dorsal root ganglion neurons thought to lack nicotinic acetylcholine receptors were not labeled by the anti-MIR mAb. Substantial intracellular populations have also been reported for the muscle acetylcholine receptor and brain voltage-dependent sodium channel alpha-subunit. This may represent a general pattern for multisubunit membrane proteins during development.

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.

Expression and regulation of neuronal acetylcholine receptor mRNA in chick ciliary ganglia.

A chicken genomic clone encoding a portion of the neuronal acetylcholine receptor (AChR) alpha 3 subunit was used to identify homologous mRNA in embryonic chick ciliary ganglia. In situ hybridization indicated that the mRNA was neuronal. Northern blot analysis revealed a major hybridizing species of 3.5 kb. Protection experiments confirmed that ganglionic RNA contained material indistinguishable by RNAase digestion from the 300 nucleotide probe used. No transcripts were detected by in situ hybridization or Northern blot analysis for chick neuronal AChR alpha 2 or alpha 4 genes. alpha 3 transcripts were present at all times examined (E6 to 1 year posthatch). Both postganglionic axotomy and preganglionic denervation of ciliary ganglia in newly hatched chicks produced declines in alpha 3 mRNA levels, implying regulation of neuronal AChR mRNA by cell-cell interactions.

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.

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.

Regulating the number and function of neuronal acetylcholine receptors.

Nicotinic acetylcholine receptors (AChRs) on vertebrate neurons represent a family of receptors distinct from the well-characterized AChR of skeletal muscle. New probes for neuronal AChRs are now being used to examine the regulation of receptor number and function. The results suggest that neuronal AChRs differ from muscle AChRs in regulation by presynaptic input and by at least one second messenger system, and that neuronal AChRs are additionally regulated by retrograde signals from the synaptic target tissue. The forms of regulation provide potential mechanisms by which cell-cell interactions could stabilize synaptic contacts on neurons and modulate synaptic function.

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.

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.

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.

Neurogenesis in Aplysia californica resembles nervous system formation in vertebrates.

The pattern of neurogenesis of the central nervous system of Aplysia californica was investigated by [3H]thymidine autoradiography. Large numbers of animals at a series of early developmental stages were labeled with [3H]thymidine for 24 or 48 hr and were subsequently sampled at specific intervals throughout the life cycle. I found that proliferative zones, consisting of columnar and placodal ectodermal cells, are established in regions of the body wall adjacent to underlying mesodermal cells. Mitosis in the proliferative zones generates a population of cells which leave the surface and migrate inward to join the nearby forming ganglia. Tracing specific [3H]thymidine-labeled cells from the body wall to a particular ganglion and within the ganglion over time suggests that the final genomic replication of the neuronal precursors occurs before the cells join the ganglion while glial cell precursors and differentiating glial cells continue to divide within the ganglion for some time. Ultrastructural examination of the morphological features of the few mitosing cells observed within the Aplysia central nervous system supports this interpretation. The pattern of neurogenesis in the Aplysia central nervous system resembles the proliferation of cells in the neural tube and the migration of neural crest and ectodermal placode cells in the vertebrate nervous system but differs from the pattern described for other invertebrates.

The distribution of acetylcholine receptors in chick ciliary ganglion neurons following disruption of ganglionic connections.

Chick ciliary ganglion neurons have nicotinic acetylcholine receptors (AChRs) that mediate primary chemical synaptic transmission through the ganglion. Previous studies have shown that preganglionic denervation reduces the total number of AChRs in the ganglion about 3-fold in 10 d, while postganglionic axotomy reduces AChR levels about 10-fold in 5 d. Since the neurons contain large numbers of intracellular AChRs in addition to the surface AChRs, the present studies were undertaken to determine whether either surface or internal AChR pools are changed selectively by the operations. An anti-AChR monoclonal antibody followed by an HRP-conjugated secondary antibody was used to visualize AChR distributions on neurons in ciliary ganglia 5 d after postganglionic axotomy and 10 d after preganglionic denervation. Ganglia were permeabilized by treatment with saponin to obtain access to intracellular receptors. The results show that the operations do not qualitatively change the subcellular localization of AChRs, but they do alter the levels relative to control ganglia. Axotomy produces substantial declines both in the number of synaptic AChRs and in the number of intracellular AChRs. Denervation produces a significant, though less extensive decline in the number of intracellular receptors while having no detectable effect on the number of synaptic AChRs. Small alterations in receptor distribution would have gone undetected by the present techniques. Regulation of neuronal AChRs differs in several respects from that described for muscle AChRs: presynaptic input appears to be less important for controlling neuronal AChRs, while signals from the postsynaptic target tissue may be essential for maintaining synaptic receptors on the neurons.

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.

The ultrastructural localization of alpha-bungarotoxin binding sites in relation to synapses on chick ciliary ganglion neurons.

The distribution of alpha-bungarotoxin binding sites on chick ciliary ganglion neurons was examined at the ultrastructural level by incubating ganglia with horseradish peroxidase-conjugated toxin and examining the peroxidase-stained and thin-sectioned ganglia with the electron microscope. Both in embryonic and in adult ganglia heavy labeling was restricted to the surface membrane of short processes emerging from the ciliary and choroid cell somata in the region of preganglionic innervation. Less dense labeling occasionally was present on the smooth surface membrane of the soma in the same region. In contrast, the pre- and postsynaptic membranes of most synapses were clearly not labeled even in the immediate vicinity of heavily labeled processes. The labeling represented specific binding of the toxin conjugate since it could be prevented by d-tubocurarine and hexamethonium or by unconjugated toxin. The conjugated toxin was not excluded from the synaptic cleft on the basis of size because a substantially larger protein conjugate, a horseradish peroxidase-labeled monoclonal antibody, was able to enter the cleft and heavily label synaptic membranes as well as soma membranes. Even neurons in adult ganglia had very little synaptic labeling after exposure to the conjugated toxin. These results strongly suggest that the high affinity alpha-bungarotoxin binding sites on chick ciliary ganglion neurons are different from the synaptic ACh receptors which would be expected to be concentrated in the postsynaptic membrane. Clustering of the alpha-bungarotoxin binding sites in the vicinity of synapses, however, may reflect a related synaptic function.

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.

Differential effects of nerve transection on the ACh and GABA receptors of chick ciliary ganglion neurons.

Chick ciliary ganglion neurons have nicotinic acetylcholine receptors (AChRs) that mediate chemical transmission through the ganglion, and GABAA receptors of unknown significance. Previous experiments examining the role of cell-cell interactions in regulating neuronal AChRs have shown that postganglionic axotomy of ciliary ganglia in newly hatched chicks causes a 10-fold decline in total AChRs within 5 d compared with unoperated contralateral ganglia and that preganglionic denervation causes a 3-fold decline within 10 d. Many of the AChRs are known to be intracellular; of those present on the cell surface, only a small fraction appears to be functionally available normally. In the present experiments, the effects of the operations on functional AChRs and GABAA receptors in the plasma membrane of the neurons were examined by removing the ganglia 5 d after axotomy or 10 d after denervation, dissociating them into single cells, and immediately measuring their ACh and GABA sensitivities with intracellular recording techniques. The ACh sensitivity of axotomized ciliary ganglion neurons was reduced 10-fold compared with neurons from unoperated contralateral ganglia of the same chicks. The reduction could be largely accounted for by a decrease in the maximum response and did not arise from a change either in the dose-response curve or the acetylcholinesterase activity of the neurons. Autoradiographic studies using a radiolabeled anti-AChR monoclonal antibody also demonstrated a substantial decrease in the total number of surface AChRs associated with axotomized neurons. In contrast, axotomy had no unilateral effect on the GABA response.(ABSTRACT TRUNCATED AT 250 WORDS)