The psychiatric disease risk factors DISC1 and TNIK interact to regulate synapse composition and function.

Disrupted in schizophrenia 1 (DISC1), a genetic risk factor for multiple serious psychiatric diseases including schizophrenia, bipolar disorder and autism, is a key regulator of multiple neuronal functions linked to both normal development and disease processes. As these diseases are thought to share a common deficit in synaptic function and architecture, we have analyzed the role of DISC1 using an approach that focuses on understanding the protein-protein interactions of DISC1 specifically at synapses. We identify the Traf2 and Nck-interacting kinase (TNIK), an emerging risk factor itself for disease, as a key synaptic partner for DISC1, and provide evidence that the DISC1-TNIK interaction regulates synaptic composition and activity by stabilizing the levels of key postsynaptic density proteins. Understanding the novel DISC1-TNIK interaction is likely to provide insights into the etiology and underlying synaptic deficits found in major psychiatric diseases.

Direct interaction of CASK/LIN-2 and syndecan heparan sulfate proteoglycan and their overlapping distribution in neuronal synapses.

CASK, the rat homolog of a gene (LIN-2) required for vulval differentiation in Caenorhabditis elegans, is expressed in mammalian brain, but its function in neurons is unknown. CASK is distributed in a punctate somatodendritic pattern in neurons. By immunogold EM, CASK protein is concentrated in synapses, but is also present at nonsynaptic membranes and in intracellular compartments. This immunolocalization is consistent with biochemical studies showing the presence of CASK in soluble and synaptosomal membrane fractions and its enrichment in postsynaptic density fractions of rat brain. By yeast two-hybrid screening, a specific interaction was identified between the PDZ domain of CASK and the COOH terminal tail of syndecan-2, a cell surface heparan sulfate proteoglycan (HSPG). The interaction was confirmed by coimmunoprecipitation from heterologous cells. In brain, syndecan-2 localizes specifically at synaptic junctions where it shows overlapping distribution with CASK, consistent with an interaction between these proteins in synapses. Cell surface HSPGs can bind to extracellular matrix proteins, and are required for the action of various heparin-binding polypeptide growth/differentiation factors. The synaptic localization of CASK and syndecan suggests a potential role for these proteins in adhesion and signaling at neuronal synapses.

A beta2 adrenergic receptor signaling complex assembled with the Ca2+ channel Cav1.2.

The existence of a large number of receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) raises the question of how a particular receptor selectively regulates specific targets. We provide insight into this question by identifying a prototypical macromolecular signaling complex. The beta(2) adrenergic receptor was found to be directly associated with one of its ultimate effectors, the class C L-type calcium channel Ca(v)1.2. This complex also contained a G protein, an adenylyl cyclase, cyclic adenosine monophosphate-dependent protein kinase, and the counterbalancing phosphatase PP2A. Our electrophysiological recordings from hippocampal neurons demonstrate highly localized signal transduction from the receptor to the channel. The assembly of this signaling complex provides a mechanism that ensures specific and rapid signaling by a G protein-coupled receptor.

CRIPT, a novel postsynaptic protein that binds to the third PDZ domain of PSD-95/SAP90.

The synaptic protein PSD-95/SAP90 binds to and clusters a variety of membrane proteins via its two N-terminal PDZ domains. We report a novel protein, CRIPT, which is highly conserved from mammals to plants and binds selectively to the third PDZ domain (PDZ3) of PSD-95 via its C terminus. While conforming to the consensus PDZ-binding C-terminal sequence (X-S/T-X-V-COOH), residues at the -1 position and upstream of the last four amino acids of CRIPT determine its specificity for PDZ3. In heterologous cells, CRIPT causes a redistribution of PSD-95 to microtubules. In brain, CRIPT colocalizes with PSD-95 in the postsynaptic density and can be coimmunoprecipitated with PSD-95 and tubulin. These findings suggest that CRIPT may regulate PSD-95 interaction with a tubulin-based cytoskeleton in excitatory synapses.

Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin.

NMDA receptors are linked to intracellular cytoskeletal and signaling molecules via the PSD-95 protein complex. We report a novel family of postsynaptic density (PSD) proteins, termed Shank, that binds via its PDZ domain to the C terminus of PSD-95-associated protein GKAP. A ternary complex of Shank/GKAP/PSD-95 assembles in heterologous cells and can be coimmunoprecipitated from rat brain. Synaptic localization of Shank in neurons is inhibited by a GKAP splice variant that lacks the Shank-binding C terminus. In addition to its PDZ domain, Shank contains a proline-rich region that binds to cortactin and a SAM domain that mediates multimerization. Shank may function as a scaffold protein in the PSD, potentially cross-linking NMDA receptor/PSD-95 complexes and coupling them to regulators of the actin cytoskeleton.

Novel anchorage of GluR2/3 to the postsynaptic density by the AMPA receptor-binding protein ABP.

We report the cloning of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-binding protein (ABP), a postsynaptic density (PSD) protein related to glutamate receptor-interacting protein (GRIP) with two sets of three PDZ domains, which binds the GluR2/3 AMPA receptor subunits. ABP exhibits widespread CNS expression and is found at the postsynaptic membrane. We show that the protein interactions of the ABP/GRIP family differ from the PSD-95 family, which binds N-methyl-D-aspartate (NMDA) receptors. ABP binds to the GluR2/3 C-terminal VKI-COOH motif via class II hydrophobic PDZ interactions, distinct from the class I PSD-95-NMDA receptor interaction. ABP and GRIP also form homo- and heteromultimers through PDZ-PDZ interactions but do not bind PSD-95. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors.

Shaping excitation at glutamatergic synapses.

Glutamatergic synapses vary, exhibiting EPSCs of widely different magnitudes and timecourses. The main contributors to this variability are: presynaptic factors, including release probability, quantal content and vesicle composition; factors that modulate the concentration and longevity of glutamate in the cleft, including diffusion and the actions of glutamate transporters; and postsynaptic factors, including the types and locations of ionotropic glutamate receptors, their numbers, and the nature and locations of associated intracellular signalling systems.

Spatial organization of cofilin in dendritic spines.

Synaptic plasticity is associated with morphological changes in dendritic spines. The actin-based cytoskeleton plays a key role in regulating spine structure, and actin reorganization in spines is critical for the maintenance of long term potentiation. To test the hypothesis that a stable pool of F-actin rests in the spine "core," while a dynamic pool lies peripherally in its "shell," we performed immunoelectron microscopy in the stratum radiatum of rat hippocampus to elucidate the subcellular distribution of cofilin, an actin-depolymerizing protein that mediates reorganization of the actin cytoskeleton. We provide direct evidence that cofilin in spines avoids the core, and instead concentrates in the shell and within the postsynaptic density. These data suggest that cofilin may link synaptic plasticity to the actin remodeling that underlies changes in spine morphology.

Laminar organization of the NMDA receptor complex within the postsynaptic density.

The NR2 subunit is an essential component of the NMDA receptor. Recent biochemical research has identified a number of molecules that can bind directly or indirectly to its cytoplasmic tail. These postsynaptic density (PSD) proteins play a role in intracellular signal transduction, and are implicated in synaptic plasticity and memory mechanisms. We performed systematic electron microscopic immunogold analysis in rat neocortex to determine the spatial organization of NR2, in relation to six other proteins thought to be involved in the NMDA receptor complex. Peak concentrations of each protein were within the PSD but in different "layers" of the density. In the axodendritic axis, gold particles coding for PSD-95 lay an average of 12 nm cytoplasmic to the extracellular face of the plasma membrane, very close to the C terminal of NR2. Nitric oxide synthase lay 18 nm inside the membrane; the scaffolding proteins guanylate kinase-associated protein and Shank lay 24-26 nm inside the membrane; and CRIPT and dynein light chain, proteins that may link the complex to cytoskeletal elements, lay on the cytoplasmic side of the PSD, 29-32 nm inside the plasma membrane and extending into the spine cytoplasm. The supramolecular organization of these molecules may modulate intracellular transduction of NMDA-mediated signals.

Differential cellular and subcellular localization of ampa receptor-binding protein and glutamate receptor-interacting protein.

Excitatory synaptic currents in the mammalian brain are typically mediated by the neurotransmitter glutamate, acting at AMPA receptors. We used immunocytochemistry to investigate the distribution of AMPA receptor-binding protein (ABP) in the cerebral neocortex. ABP was most prominent in pyramidal neurons, although it was also present (at lower levels) in interneurons. ABP and its putative binding partners, the GluR2/3 subunits of the AMPA receptor, exhibited prominent cellular colocalization. Under appropriate processing conditions, colocalization could also be documented in puncta, many of which could be recognized as dendritic spines. However, a sizable minority of GluR2/3-positive puncta were immunonegative for ABP. Because glutamate receptor-interacting protein (GRIP) may also anchor GluR2, we studied the relative distribution of ABP and GRIP. There was extensive colocalization of these two antigens at the cellular level, although GRIP, unlike ABP, was strongest in nonpyramidal neurons. Different parts of a single dendrite could stain selectively for ABP or GRIP. To further characterize this heterogeneity, we investigated punctate staining of neuropil using synaptophysin and the membrane tracer DiA to identify probable synapses. Some puncta were comparably positive for both ABP and GRIP, but the majority were strongly positive for one antigen and only weakly positive or immunonegative for the other. This heterogeneity could be seen even within adjacent spines of a single dendrite. These data suggest that ABP may act as a scaffold for AMPA receptors either in concert with or independently from GRIP.

Brainstem projections to the rat cuneate nucleus.

Neurons in the pontomedullary tegmentum have been proposed as a final common pathway subserving descending inhibition in the dorsal column nuclei. To investigate the anatomical substrate for these descending effects, brainstem projections to the cuneate nucleus of rats were studied with injections of lectin-conjugated horseradish peroxidase. In rats with iontophoretic tracer injections in this nucleus, many labeled neurons were detected near the injection site, especially ventral and caudal to it. Intrinsic reciprocal projections were observed after injections in caudal, middle, or rostral levels of the cuneate nucleus. Neurons were labeled in the red nucleus, in agreement with previous anatomical studies, and also in the trigeminal, vestibular, and cochlear nuclei. An ipsilateral dorsomedial group of neurons was labeled in the upper cervical segments and scattered neurons were also labeled bilaterally near the central canal. Sparse retrograde labeling in the tegmentum was focused in the lateral paragigantocellular nucleus and caudal raphe. Consistent with the retrograde experiments, anterograde labeling after pressure injections of lectin-conjugated horseradish peroxidase in the pontomedullary tegmentum was very sparse within the dorsal column nuclei; labeling was dense, however, in the region immediately ventral to these nuclei. These results confirm previous work indicating that the activity of cuneate neurons is modulated by brainstem sensory nuclei. However, it appears that direct projections to the cuneate nucleus from pontine and rostral medullary regions are sparser than previously suggested. The last link of a polysynaptic descending inhibitory pathway may include GABAergic neurons immediately adjacent to the dorsal column nuclei and/or intrinsic to these nuclei.

Immunogold localization of AMPA and NMDA receptors in somatic sensory cortex of albino rat.

We performed an electron microscopic study of S-1 cortex by using postembedding immunogold histochemistry to examine the subcellular distribution of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors (assessed with an antibody recognizing the glutamate receptor 2 and 3 [GluR2 and GluR3] subunits) and to compare this distribution with that of N-methyl-D-aspartate (NMDA) receptors (assessed with an antibody for the NR1 subunit). Both receptors were concentrated at active zones of asymmetric synapses, often directly apposed to presynaptic dense bodies. GluR2/3 showed a bias for long active zones, whereas short active zones expressed GluR2/3 at substantially lower levels; in contrast, labeling for NR1 was independent of synaptic size. Particle counts suggested that synaptic labeling was Poisson distributed and implied that the majority of synapses express both receptors. Quantitative analysis indicates that approximately one-half of synapses express high levels of GluR2/3 and that the remainder express GluR2/3 at a much lower level. Approximately three-fourths of synapses express NR1 at a uniform level; the remainder, which may lack NR1 completely, include synapses with especially large active zones. The present results suggest that the smallest active zones may play a special role in synaptic plasticity.

NADPH diaphorase in the spinal cord of rats.

To identify spinal neurons that may synthesize nitric oxide, cells and fibers histochemically stained for NADPH diaphorase (a nitric oxide synthase) were studied in the spinal cord of rats. The histochemical reaction gave an image similar to the best Golgi impregnations, staining cells down to their finest processes. Transverse, horizontal, and parasagittal 50 and 100 microns sections were used to follow dendritic and axonal arborizations of stained neurons. Major cell groups were identified in the superficial dorsal horn and around the central canal (at all spinal levels), and in the intermediolateral cell column (at thoracic and sacral levels). Scattered positive cells were also found in deeper dorsal horn, ventral horn, and white matter. In some cases, axons of cells in the dorsal horn could be traced into the white matter; many of these cells resembled neurons projecting to various supraspinal targets. Stained cells in the intermediolateral column, which sent their axons into the ventral root, were presumed to be preganglionic autonomic neurons. Dense plexes of fibers were stained in laminae I and II and in the intermediolateral column. A large number of NADPH diaphorase-positive neurons in the spinal cord appear to be involved in visceral regulation. Fibers of the intermediolateral system had a special relationship with vasculature, suggesting that nitric oxide may help to couple neural activity with regional blood flow in the spinal cord. The abundance of NADPH diaphorase-positive neurons and fibers in the superficial dorsal horn suggests that nitric oxide may also be involved in spinal sensory processing.

Single fiber studies of ascending input to the cuneate nucleus of cats: I. Morphometry of primary afferent fibers.

The morphology of afferent fibers ascending to the cuneate nucleus has been examined in this and the subsequent paper in order to quantify the pattern of arborization and bouton arrangement of selected classes of primary afferents and to compare these data with data from postsynaptic fibers ascending to the cuneate nucleus. Electrophysiologically identified G hair and Ia muscle afferent fibers in the cuneate fasciculus were intraaxonally injected with horseradish peroxidase. Cutaneous afferents terminated dorsal to proprioceptive afferents, especially at middle levels of the cuneate nucleus. The spacing of collaterals along G hair fibers was variable, but averaged 1.46 collaterals per mm; collateral density was higher at middle cuneate levels than in the rest of the nucleus. Collateral density of Ia fibers was lower than for G hair fibers and was lowest at caudal levels of the nucleus. Branches of G hair collaterals, though often initially diverging, usually converged to terminate in a single focus in the dorsal part of the nucleus. The probability of bifurcation of Ia collaterals decreased steadily at successive branch points. These collaterals branched less symmetrically than G hair collaterals, and terminated in the ventral cuneate with less dense arbors, stretched mediolaterally, but of comparable cross-sectional area. Individual G hair collaterals gave rise to more boutons than Ia collaterals; in both cases they were mostly of the en passant type. Boutons were restricted to distal branches of G hair collaterals, whereas boutons of Ia collaterals were also located on proximal branches. Bouton size was similar for the two classes of collaterals. The data reported here, in combination with the published literature, suggest that the collaterals of roughly 300 G hair fibers overlap at any given point at middle levels of the cuneate nucleus. This high degree of anatomical convergence is not predicted by the functional segregation described with electrophysiological mapping, implying the presence of intrinsic nuclear mechanisms enhancing response specificity.

Single fiber studies of ascending input to the cuneate nucleus of cats: II. Postsynaptic afferents.

The morphology of single postsynaptic afferent fibers terminating in the feline cuneate nucleus was investigated by using transport of Phasolus vulgaris leucoagglutinin from the cervical spinal cord and intraaxonal injections of horseradish peroxidase into identified postsynaptic fibers in the cuneate fasciculus. Injections of Phaseolus in C5 and C6 of both rhizotomized and non-rhizotomized cats gave similar results and confirmed previous observations with other techniques. In one animal with the smallest injection and the fewest labeled fibers in the cuneate nucleus, ten individual collaterals were reconstructed from serial sections. Most of these collaterals were at middle levels of the cuneate (from obex to about 4 mm caudal to it); they were largely confined to the rim and ventral regions of the nucleus, and their terminal fields were restricted rostrocaudally. Electrophysiologically identified fibers stained with horseradish peroxidase had large receptive fields on the ipsilateral forepaw, and latencies suggesting an oligosynaptic link to the periphery. Most of the collaterals from these fibers were also at middle cuneate levels and terminated mainly at the periphery of the nucleus but gave rise to larger terminal arbors, including sparse terminal branches to the core of the nucleus. Individual postsynaptic fibers differed in several respects from primary afferent fibers. While the spacing of collaterals of postsynaptic fibers was intermediate between that of G hair and Ia fibers, their arbors were larger than either, and could extend through the dorsoventral extent of the cuneate nucleus. The pattern of bifurcation of postsynaptic fibers resulted in stringier arbors which encompassed larger and less dense terminal fields than those of primary afferents. The number of boutons per collateral was intermediate between G hair and Ia fibers, but boutons of postsynaptic fibers were substantially smaller. These morphological differences are consistent with distinct functional roles for the two main ascending afferent systems, as suggested by electrophysiological data.

Expression of NR2 receptor subunit in rat somatic sensory cortex: synaptic distribution and colocalization with NR1 and PSD-95.

Functional N-methyl-D-aspartate (NMDA) receptors comprise heteromeric combinations of NR1 and NR2 subunits. In the present study, we employed light and electron microscopic immunocytochemistry to study the expression of NR2A and NR2B (NR2A/B) protein in somatic sensory cortex of adult rats. To relate this distribution to that of NR1 and to the NMDA receptor anchoring protein PSD-95, we documented extensive cellular colocalization of NR2A/B with NR1 at the light microscopic level. In contrast, PSD-95 exhibited little somatic staining, being restricted mainly to dendrites and neuropil. We employed postembedding immunocytochemistry to study the ultrastructural expression of NR2A/B. Labeling in neuronal perikarya was associated with rough endoplasmic reticulum and Golgi apparatus; in dendrites, gold particles labeled microtubules. The preponderance of labeling was associated with asymmetric synapses. Double immunolabeling revealed that NR2 colocalized in many synapses with NR1 and with PSD-95. Quantitative measurements revealed that density of gold particles coding for both NR2 and PSD-95 was highest just inside the postsynaptic membrane. Tangentially along the membrane, gold particles were concentrated at the synaptic specialization. These data provide structural evidence in neocortex for heteromeric NMDA receptors anchored at the postsynaptic membrane.

GluR1-immunopositive interneurons in rat neocortex.

Recent in vitro studies suggest that inhibitory interneurons in cortex may express the GluR1 glutamate receptor subunit in the absence of GluR2, leading to calcium-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) channels. We performed a study of rat somatic sensory cortex to confirm and extend these observations, using quantitative immunocytochemistry for multiple antigens. A morphologically distinct subpopulation of nonpyramidal neurons in neocortex was intensely immunoreactive for GluR1. Electron microscopic analysis of these cells revealed somatic staining for GluR1, mainly in the rough endoplasmic reticulum. Dendritic staining was concentrated at the synaptic active zone and in the adjacent subsynaptic cytoplasm. Double immunostaining revealed that the large majority of intensely GluR1-positive cells contained gamma-aminobutyric acid or its synthetic enzyme, glutamic acid decarboxylase, but little or no GluR2. Thus, AMPA receptors on a subpopulation of inhibitory interneurons in cortex are likely to be calcium permeable. This calcium permeability is likely to influence functional properties of these neurons; it may underlie the high levels of calcium-binding proteins they contain; and may render them liable to excitotoxic injury

Immunohistochemical localization of nitric oxide synthase and soluble guanylyl cyclase in the ventral cochlear nucleus of the rat.

The diffusible messenger nitric oxide (NO) is implicated in auditory processing. It acts in the brain largely through activation of soluble guanylyl cyclase (sGC), a heterodimer comprised of alpha and beta subunits. The authors used immunohistochemistry to study the NO/guanosine 3',5'-cyclic monophosphate (cGMP) pathway in the cochlear nucleus of Sprague-Dawley rats. Central fibers of the cochlear nerve were stained for neuronal nitric oxide synthase (NOS-I) but not for sGCbeta. Within the ventral cochlear nucleus, a large fraction of principal cells were immunopositive for both NOS-I and sGCbeta; these cells could be seen at times receiving contacts from NOS-I-positive fibers. sGC staining of somatic cytoplasm extended into the distal dendritic tree. At variance with this pattern, NOS-I was concentrated mainly in somata. Double-labeling experiments showed that most of the principal neurons expressed both antigens. By contrast, in the granule cell domain, small cells that were immunopositive for NOS-I rarely corresponded to those that were immunopositive for sGC. To assess whether NOS-I and sGC immunoreactivities colocalize with their respective catalytic activities, the authors performed multiple labeling with L-citrulline (a by-product of the formation of NO from L-arginine) and cGMP, respectively. L-citrulline was restricted to NOS-I-positive elements, and the large majority of NOS-expressing neurons were positive for citrulline. Multiple labeling revealed that almost all sGC-positive neurons also accumulated cGMP both in the ventral cochlear nucleus and in the granule cell domain. These data suggest that NO is a signaling molecule in the cochlear nucleus, perhaps functioning in both a paracrine manner and an autocrine manner.

Substance P and nitric oxide signaling in cerebral cortex: anatomical evidence for reciprocal signaling between two classes of interneurons.

Parvalbumin-containing fast-spiking interneurons in the cerebral cortex exhibit widespread electrical coupling, as do somatostatin-containing low-threshold spiking interneurons. Besides the classical neurotransmitter gamma-aminobutyric acid, these cortical interneurons may also release various neuropeptides including substance P (SP), as well as the freely diffusible messenger nitric oxide (NO). To investigate whether these two networks of interneurons might interact via these nonclassical messengers, we performed immunocytochemistry for SP and NO signaling pathways in rat somatic sensory cortex. SP was found in a subset of parvalbumin-positive cells concentrated in layers IV and V, whereas its receptor, NK1, was found in a subset of somatostatin-containing neurons (and also, at much lower levels, in a disjoint subset of parvalbumin-containing neurons). Only 4% of SP-containing axon terminals were apposed to NK1-positive dendrites, suggesting that in the cerebral cortex, SP may act predominantly as a paracrine neuromediator. Nitric oxide synthase-I (NOS-I), the synthetic enzyme for NO, was found almost exclusively in NK1-positive neurons; 95% of intensely somatostatin/NK1-positive neurons were also positive for NOS-I, and 94% of NOS-positive neurons were also positive for NK1. Immunoreactivity for soluble guanylyl cyclase (the NO receptor) was at high levels in the apical dendrites of layer V pyramidal neurons and in parvalbumin/SP-positive neurons. These data point to a novel reciprocal chemical interaction between two inhibitory networks in the rat neocortex.

Ultrastructural and immunocytochemical characterization of primary afferent terminals in the rat cuneate nucleus.

The cuneate nucleus is a relay center for somatosensory information by receiving tactile and proprioceptive inputs from primary afferent fibers that ascend in the dorsal funiculus. The morphology, synaptic contacts, and neurochemical content of primary afferent terminals in the cuneate nucleus of rats were investigated by combining anterograde transport of horseradish peroxidase conjugated to wheat-germ agglutinin or to cholera toxin (injected in cervical dorsal root ganglia) with postembedding immunogold labeling for glutamate and GABA. Both tracers gave similar results. Two types of terminals were labeled: type I terminals were irregularly shaped, had a mean area of 4.0 microns 2, synapsed on several dendrites, and were contacted by other terminals, some of which were GABA positive. Type II terminals were dome-shaped, had a mean area of 2.18 microns 2, and made synaptic contact on a single dendrite. All the anterogradely labeled terminals (interpreted as endings of primary afferents) were enriched in glutamate but not in GABA. The finding that identified primary afferent terminals are enriched in glutamate with respect to other tissue profiles strongly suggests a neurotransmitter role for glutamate in this afferent pathway to the rat cuneate nucleus.