A role for the cytoplasmic polyadenylation element in NMDA receptor-regulated mRNA translation in neurons.

The ability of neurons to modify synaptic connections based on activity is essential for information processing and storage in the brain. The induction of long-lasting changes in synaptic strength requires new protein synthesis and is often mediated by NMDA-type glutamate receptors (NMDARs). We used a dark-rearing paradigm to examine mRNA translational regulation in the visual cortex after visual experience-induced synaptic plasticity. In this model system, we demonstrate that visual experience induces the translation of mRNA encoding the alpha-subunit of calcium/calmodulin-dependent kinase II in the visual cortex. Furthermore, this increase in translation is NMDAR dependent. One potential source for newly synthesized proteins is the translational activation of dormant cytoplasmic mRNAs. To examine this possibility, we developed a culture-based assay system to study translational regulation in neurons. Cultured hippocampal neurons were transfected with constructs encoding green fluorescent protein (GFP). At 6 hr after transfection, approximately 35% of the transfected neurons (as determined by in situ hybridization) expressed detectable GFP protein. Glutamate stimulation of the cultures at this time induced an increase in the number of neurons expressing GFP protein that was NMDAR dependent. Importantly, the glutamate-induced increase was only detected when the 3'-untranslated region of the GFP constructs contained intact cytoplasmic polyadenylation elements (CPEs). Together, these findings define a molecular mechanism for activity-dependent synaptic plasticity that is mediated by the NMDA receptor and requires the CPE-dependent translation of an identified mRNA.

Molecular mechanisms for activity-regulated protein synthesis in the synapto-dendritic compartment.

The creation of enduring modifications in synaptic efficacy requires new protein synthesis. Neurons face the formidable challenge of directing these newly made proteins to the appropriate subset of synapses. One attractive solution to this problem is the local translation of mRNAs that are targeted to dendrites and perhaps to synapses themselves. The molecular mechanisms mediating such local protein synthesis, notably CPEB-mediated cytoplasmic polyadenylation, are now being elucidated.

The insulin receptor tyrosine kinase substrate p58/53 and the insulin receptor are components of CNS synapses.

The synapse is the primary locus of cell-cell communication in the nervous system. It is now clear that the synapse incorporates diverse cell signaling modalities in addition to classical neurotransmission. Here we show that two components of the insulin pathway are localized at CNS synapses, where they are components of the postsynaptic density (PSD). An immunochemical screen revealed that polypeptides of 58 and 53 kDa (p58/53) were highly enriched in PSD fractions from rat cerebral cortex, hippocampus, and cerebellum. These polypeptides were purified and microsequenced, revealing that p58/53 is identical to the insulin receptor tyrosine kinase substrate p58/53 (IRSp53). Our analysis of IRSp58/53 mRNA suggests that within rat brain there is one coding region for IRSp58 and IRSp53; we find no evidence of alternative splicing. We demonstrate that IRSp58/53 is expressed in the synapse-rich molecular layer of the cerebellum and is highly concentrated at the synapses of cultured hippocampal neurons, where it co-localizes with the insulin receptor. Together, these data suggest that insulin signaling may play a role at CNS synapses.

Neurotrophins regulate agrin-induced postsynaptic differentiation.

The precise orchestration of synaptic differentiation is critical for efficient information exchange in the nervous system. The nerve-muscle synapse forms in response to agrin, which is secreted from the motor nerve terminal and induces the clustering of acetylcholine receptors (AChRs) and other elements of the postsynaptic apparatus on the subjacent muscle cell surface. In view of the highly restricted spatial localization and the plasticity of neuromuscular junctions, it seems likely that synapse formation and maintenance are regulated by additional, as-yet-unidentified factors. Here, we tested whether neurotrophins modulate the agrin-induced differentiation of postsynaptic specializations. We show that both brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) inhibit agrin-induced AChR clustering on cultured myotubes. Nerve growth factor and NT-3 are without effect. Muscle cells express full-length TrkB, the cognate receptor for BDNF and NT-4. Direct activation of this receptor by anti-TrkB antibodies mimicked the BDNF/NT-4 inhibition of agrin-induced AChR clustering. This BDNF/NT-4 inhibition is likely to be an intrinsic mechanism for regulating AChR clustering, because neutralization of endogenous TrkB ligands resulted in elevated levels of AChR clustering even in the absence of added agrin. Finally, high concentrations of agrin can occlude the BDNF/NT-4 inhibition of AChR clustering. These results indicate that an interplay between agrin and neurotrophins can regulate the formation of postsynaptic specializations. They also suggest a mechanism for the suppression of postsynaptic specializations at nonjunctional regions.

SAP90 binds and clusters kainate receptors causing incomplete desensitization.

The mechanism of kainate receptor targeting and clustering is still unresolved. Here, we demonstrate that members of the SAP90/PSD-95 family colocalize and associate with kainate receptors. SAP90 and SAP102 coimmunoprecipitate with both KA2 and GluR6, but only SAP97 coimmunoprecipitates with GluR6. Similar to NMDA receptors, GluR6 clustering is mediated by the interaction of its C-terminal amino acid sequence, ETMA, with the PDZ1 domain of SAP90. In contrast, the KA2 C-terminal region binds to, and is clustered by, the SH3 and GK domains of SAP90. Finally, we show that SAP90 coexpressed with GluR6 or GluR6/KA2 receptors alters receptor function by reducing desensitization. These studies suggest that the organization and electrophysiological properties of synaptic kainate receptors are modified by association with members of the SAP90/PSD-95 family.

The state of the union. Neuromuscular junction.

Synaptic differentiation is triggered by signals from the ingrowing axon and is shaped by information exchange between the presynaptic and postsynaptic cells. The central role of agrin in this process, and the identity of the signaling component of its receptor, have now been established.

Changes in responsiveness to extracellular ATP in chick skeletal muscle during development and upon denervation.

Skeletal muscles in developing chick embryos were tested for responsiveness to adenosine 5'-triphosphate (ATP), a substance known to depolarize chick skeletal muscle in culture. The sensitivity to extracellular ATP was tested at various stages of development in five different muscles; pectoralis superficia, anterior latissimus dorsi, posterior latissimus dorsi, sartorious, and gastrocnemius. At the earliest time that muscles were tested (Embryonic Day 6, stage 30 of Hamburger and Hamilton, 1951) application of ATP(50-100 microM) elicited vigorous contractions in all five muscles, but within a few days (Embryonic Day 17, stage 43) none of the muscles contracted in response to ATP. Sensitivity declined at approximately the same time in all five of these muscles. Intracellular recordings made from muscle fibers near the time of hatching (Embryonic Days 18-21 or Postnatal Days 1-2) indicated that the loss of the ability to contract in response to ATP was due to the total loss of responsiveness to ATP. Surgical denervation of the anterior latissimus dorsi and posterior latissimus dorsi was performed in a series of chicks 1-2 days after hatching, and the ability of these muscles to contract in response to ATP was tested 3-10 days after the surgery. Contractions in response to ATP were present in many of the muscles. Thus denervation of muscles in newly hatched chicks led to the reappearance of sensitivity to ATP. The disappearance of ATP responsiveness shortly after muscles become innervated and the reappearance of ATP responsiveness following denervation suggest that the expression of ATP responsiveness is regulated by motor neurons.

Immunohistochemical identification of neurons in ganglia of the guinea pig sphincter of Oddi.

The sphincter of Oddi is a smooth muscle sphincter that regulates the flow of bile into the duodenum. To identify potential chemical coding in sphincter of Oddi neurons, immunohistochemistry and histochemistry were employed to assay for putative neurotransmitters and related synthetic enzymes in wholemount preparations, with and without colchicine treatment. Immunoreactivities for enkephalin-endorphin (ENK-END), substance P (SP), nitric oxide synthase, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) were demonstrated within the ganglionated plexus. Roughly half of the neurons in the sphincter of Oddi expressed immunoreactivity for both SP and ENK-END, but not for nitric oxide synthase. About 25% of the neurons expressed nitric oxide synthase immunoreactivity as well as NADPH-diaphorase activity. This contingent of neurons was made up of two subgroups: one that expressed immunoreactivity for VIP, the other for NPY. Neurons that expressed CGRP immunoreactivity were sparse in sphincter of Oddi ganglia; however, many axons immunoreactive for both CGRP and SP were present in the ganglionated plexus. The CGRP/SP fibers are probably visceral afferents that may influence ganglionic output through axon reflex circuits. These results, along with studies of the actions of these neuroactive compounds on sphincter tone, support the view that ganglia of the sphincter of Oddi are largely comprised of excitatory (SP/ENK-END-immunoreactive) and inhibitory (nitric oxide synthase/VIP- or NPY-immunoreactive) neurons, and that sphincter of Oddi tone is controlled by the regulation of the outputs of these two groups of cells.

Actions of cholecystokinin and norepinephrine on vagal inputs to ganglion cells in guinea pig gallbladder.

Previous studies have demonstrated that all guinea pig gallbladder neurons receive nicotinic synaptic input and that cholecystokinin (CCK) and norepinephrine have presynaptic facilitory and inhibitory effects, respectively, on these fast synaptic events. The current study was undertaken to determine the sources of the cholinergic terminals that provide nicotinic input to gallbladder neurons. To stimulate potential extrinsic inputs to gallbladder neurons, a stimulating electrode was placed on the nerve bundles that pass along the cystic duct. Stimulation of these nerves elicited fast excitatory postsynaptic potentials (EPSPs) in gallbladder neurons that were sensitive to hexamethonium, facilitated by CCK, and inhibited by norepinephrine. After vagotomy, most neurons (14 of 18) did not exhibit any nicotinic input. However, some neurons (3 of 18) did exhibit fast EPSPs in response to fiber tract stimulation, but not cystic nerve stimulation, indicating that interganglionic communication does exist amongst gallbladder neurons. These results demonstrate that the vagus nerves provide the major nicotinic input to gallbladder neurons. Furthermore, these data suggest that vagal terminals within gallbladder are a site of neurohormonal modulation of gallbladder ganglionic output by CCK, norepinephrine, and possibly other compounds.

Sympathetic input to ganglia of the guinea pig sphincter of Oddi.

Intracellular recording and immunohistochemical staining techniques were used to establish whether sphincter of Oddi (SO) ganglia are a target of sympathetic input to this region. Norepinephrine (0.01-10.0 microM) decreased the amplitude of the nicotinic fast excitatory postsynaptic potential (EPSP) evoked by stimulation of interganglionic fiber tracts, with a half-maximal inhibitory concentration (EC50) of 300 nM. Norepinephrine did not alter the responsiveness of the neurons to acetylcholine. The alpha 2-adrenoreceptor agonist UK-14304 mimicked the norepinephrine-induced effect with a EC50 of 2.5 nM, whereas alpha 1- and beta-adrenoreceptor agonists had no effect on the EPSP. The alpha 2-adrenoreceptor antagonist idazoxan (1.0 microM) inhibited the UK-14304 response, with a dissociation constant of 1.0 nM. Release of endogenous catecholamines, by the addition of tyramine (100 microM) to the bath, caused an idazoxan-sensitive decrease in the amplitude of the fast EPSP. In the minority of SO neurons that exhibited inhibitory postsynaptic potentials (IPSPs), norepinephrine caused a hyperpolarization of the membrane potential. The IPSP and the norepinephrine-induced hyperpolarization were inhibited by alpha 2-adrenoreceptor antagonists. Desipramine (1.0 microM), an uptake inhibitor, reversibly increased the amplitude of the IPSP. Immunoreactivities for tyrosine hydroxylase and dopamine beta-hydroxylase were coexistent in nerve fibers and nonexistent in cell bodies in the ganglionated plexus of the SO. The results of this study indicate that norepinephrine acts pre- and postsynaptically as an inhibitory neurotransmitter in SO ganglia.

Physiological and morphological properties of neurons in sphincter of Oddi region of the guinea pig.

Intracellular recordings and dye injections were used to investigate neurons located in ganglia of the sphincter of Oddi (SO) region in guinea pigs. Four types of neurons were encountered based on physiological properties. The two most abundant types, tonic and phasic, had similar membrane characteristics and morphologies but yet could be differentiated by their spiking characteristics. Tonic cells spiked throughout a depolarizing current pulse and were sometimes spontaneously active. Phasic cells fired only a single action potential at the onset of a current pulse regardless of stimulus amplitude or duration. Both tonic and phasic cells had Dogiel type I morphologies. They typically had a single long process and several very short processes emanating from the soma. NADPH diaphorase activity was demonstrated in cells with similar morphologies, indicating that nitric oxide may be an intrinsic transmitter in some of these cells. Cells with a prolonged afterhyperpolarization (AH cells), similar to the type 1/AH cells of the gut, were rarely encountered. This finding was consistent with the observation that very few calbindin D-immunoreactive neurons exist in this region. Action potentials could not be generated in the fourth type of neuron, called nonspiking neurons, even though they did receive synaptic input. Most tonic and phasic cells received at least one nicotinic fast excitatory postsynaptic potential (EPSP). In addition, both slow EPSPs and inhibitory postsynaptic potentials were observed. Most AH cells received only slow excitatory synaptic input.

Platelet-leukocyte plasmapheresis attenuates the deleterious effects of cardiopulmonary bypass.

A method of harvesting a high yield of concentrated platelet- and leukocyte-rich plasma was developed with the goal of attenuating some of the deleterious effects of cardiopulmonary bypass. The study involved 32 patients who underwent coronary artery bypass grafting with plasmapheresis before cardiopulmonary bypass and a control group of 32 patients who did not have plasmapheresis. A volume of 857 +/- 359 mL of platelet- and leukocyte-rich plasma was concentrated from 4.6 +/- 1.5 L of blood, and red cells and plasma were returned to the patient. The platelet- and leukocyte-rich plasma contained yields of 3.5 +/- 1.4 x 10(11) platelets and 3.4 +/- 1.9 x 10(9) leukocytes. There were no differences in age, sex, duration of cardiopulmonary bypass, and major risk factors between groups. However, total mediastinal chest tube drainage was 788 +/- 542 mL in the controls and 425 +/- 207 mL in the plasmapheresis group (p less than 0.01). Homologous units transfused were 3.9 +/- 2 in controls and 1.6 +/- 2 in the plasmapheresis group (p less than 0.01). Arterial oxygen tension on extubation was 94 +/- 32 mm Hg in controls and 119 +/- 25 mm Hg in the plasmapheresis group (p less than 0.01). This technique of platelet and leukocyte protection results in reduced postoperative bleeding, a decreased need for homologous blood products, and improved pulmonary function.

Isomorphic activation of astrocytes in the somatosensory thalamus.

Structural recovery in the rat somatosensory thalamus after the loss of one of its major inputs provided a model for studying the changes in astrocytes associated with reactive synaptogenesis. The temporal separation of the initiation of Wallerian degeneration and reactive synaptogenesis permitted astrocytic changes to be correlated either with the removal of degeneration, early in the recovery sequence, or with synaptogenesis, later in recovery. Over a period of post-lesion times ranging from 3 days to 13.5 months, GFAP-positive astrocytic fibers were quantified and the population density of S-100-positive astrocytic cell bodies was determined in the ventral posterolateral nucleus (VPL). The relative area of astrocytic cell bodies was measured at an early peak of the increased GFAP immunoreactivity (4-5 days post-lesion). The normal side of VPL (c-VPL) was compared to the deafferented side of VPL (d-VPL) and the ratio d-VPL/c-VPL was determined. Astrocytes in d-VPL underwent a minimal isomorphic activation with little or no hypertrophy or proliferation but with a large increase in GFAP immunoreactivity. Prior to the initiation of synaptogenesis, there was a decrease both in GFAP immunoreactivity and in the population density of VPL astrocytes. The decreases in the recovery curves suggested that a suppression of the influence of astrocytes may have been important for sprouting and/or synaptogenesis. In other systems, where synaptogenesis was initiated early in the recovery sequence, the suppression of astrocytes that was related to synaptogenesis may have been masked by astrocytic changes related to the removal of degeneration.