Selective expression of Narp in primary nociceptive neurons: role in microglia/macrophage activation following nerve injury.

Neuronal activity regulated pentraxin (Narp) is a secreted protein implicated in regulating synaptic plasticity via its association with the extracellular surface of AMPA receptors. We found robust Narp immunostaining in dorsal root ganglia (DRG) that is largely restricted to small diameter neurons, and in the superficial layers of the dorsal horn of the spinal cord. In double staining studies of DRG, we found that Narp is expressed in both IB4- and CGRP-positive neurons, markers of distinct populations of nociceptive neurons. Although a panel of standard pain behavioral assays were unaffected by Narp deletion, we found that Narp knockout mice displayed an exaggerated microglia/macrophage response in the dorsal horn of the spinal cord to sciatic nerve transection 3days after surgery compared with wild type mice. As other members of the pentraxin family have been implicated in regulating innate immunity, these findings suggest that Narp, and perhaps other neuronal pentraxins, also regulate inflammation in the nervous system.

Organization of NMDA receptors at extrasynaptic locations.

NMDA receptors are found in neurons both at synapses and in extrasynaptic locations. Extrasynaptic locations are poorly characterized. Here we used preembedding immunoperoxidase and postembedding immunogold electron microscopy and fluorescence light microscopy to characterize extrasynaptic NMDA receptor locations in dissociated hippocampal neurons in vitro and in the adult and postnatal hippocampus in vivo. We found that extrasynaptic NMDA receptors on neurons in vivo and in vitro were usually concentrated at points of contact with adjacent processes, which were mainly axons, axon terminals, or glia. Many of these contacts were shown to contain adhesion factors such as cadherin and catenin. We also found associations of extrasynaptic NMDA receptors with the membrane associated guanylate kinase (MAGUKs), postsynaptic density (PSD)-95 and SAP102. Developmental differences were also observed. At postnatal day 2 in vivo, extrasynaptic NMDA receptors could often be found at sites with distinct densities whereas dense material was seen only rarely at sites of extrasynaptic NMDA receptors in the adult hippocampus in vivo. This difference probably indicates that many sites of extrasynaptic NMDA receptors in early postnatal ages represent synapse formation or possibly sites for synapse elimination. At all ages, as suggested in both in vivo and in vitro studies, extrasynaptic NMDA receptors on dendrites or the sides of spines may form complexes with other proteins, in many cases, at stable associations with adjacent cell processes. These associations may facilitate unique functions for extrasynaptic NMDA receptors.

Activity-dependent secretion of neuronal activity regulated pentraxin from vasopressin neurons into the systemic circulation.

Neuronal activity regulated pentraxin (Narp) is a secreted, synaptic protein that has been implicated in modulating synaptic transmission. However, it is unclear how Narp secretion is regulated. Since we noted prominent Narp immunostaining in vasopressin neurons of the hypothalamus and in the posterior pituitary, we assessed whether it, like vasopressin, is released into the systemic circulation in an activity-dependent fashion. Consistent with this hypothesis, electron microscopic studies of the posterior pituitary demonstrated that Narp is located in secretory vesicles containing vasopressin. Using affinity chromatography, we detected Narp in plasma and found that these levels are markedly decreased by hypophysectomy. In addition, we confirmed that injection of a viral Narp construct into the hypothalamus restores plasma Narp levels in Narp knockout mice. In checking for activity-dependent secretion of Narp from the posterior pituitary, we found that several stimuli known to trigger vasopressin release, i.e. hypovolemia, dehydration and endotoxin, elevate plasma Narp levels. Taken together, these findings provide compelling evidence that Narp is secreted from vasopressin neurons in an activity-dependent fashion.

Glutamate receptor targeting in the postsynaptic spine involves mechanisms that are independent of myosin Va.

Targeting of glutamate receptors (GluRs) to synapses involves rapid movement of intracellular receptors. This occurs in forms of synaptic upregulation of receptors, such as long-term potentiation. Thus, many GluRs are retained in a cytoplasmic pool in dendrites, and are transported to synapses for upregulation, presumably via motor proteins such as myosins travelling along cytoskeletal elements that extend up into the spine. In this ultrastructural immunogold study of the cerebellar cortex, we compared synapses between normal rats/mice and dilute lethal mutant mice. These mutant mice lack myosin Va, which has been implicated in protein trafficking at synapses. The postsynaptic spine in the cerebellum lacks the inositol trisphosphate receptor (IP3R) -laden reticular tubules that are found in normal mice and rats (Takagishi et al., Neurosci. Lett., 1996, 215, 169). Thus, we tested the hypothesis that myosin Va is necessary for transport of GluRs and associated proteins to spine synapses. We found that these spines retain a normal distribution of (i) GluRs (delta 1/2, GluR2/3 and mGluR1alpha), (ii) at least one associated MAGUK (membrane-associated guanylate kinase) protein, (iii) Homer (which interacts with mGluR1alpha and IP3Rs), (iv) the actin cytoskeleton, (v) the reticulum-associated protein BiP, and (vi) the motor-associated protein, dynein light chain. Thus, while myosin Va may maintain the IP3R-laden reticulum in the spine for proper calcium regulation, other mechanisms must be involved in the delivery of GluRs and associated proteins to synapses. Other possible mechanisms include diffusion along the extrasynaptic membrane and delivery via other motors running along the spine's actin cytoskeleton.

Immunoprecipitation, immunoblotting, and immunocytochemistry studies suggest that glutamate receptor delta subunits form novel postsynaptic receptor complexes.

An antibody was made to a C-terminus peptide of the glutamate receptor delta 2 subunit and used to study the distribution, biochemical properties, and developmental expression of the delta receptor in rat brain. The antibody recognizes both delta 1 and delta 2 but not AMPA, kainate, NMDA, and mGluR1 alpha glutamate receptor subunits based on Western blot analysis of transfected HEK-293 cells. Western blot analysis of brain showed a single immunoreactive band, migrating at M(r) = 114,000. Immunoprecipitation of detergent-solubilized cerebellar membranes was done to determine if delta is associated with other glutamate receptor subunits and if it binds any of the common excitatory amino acid ligands. Based on results of these studies, AMPA, kainate, NMDA, and mGluR1 alpha subunits do not coassemble with delta subunits, and 3H-glutamate, 3H-AMPA and 3H-kainate do not bind to the delta receptor complex. Western blot and immunocytochemical analyses showed marked expression of delta in the cerebellum while lower levels were detected in other regions of the brain. A dramatic increase of delta 1/2 immunoreactivity was observed in the cerebellum between the ages of 10 and 15 d postnatal. Light and electron microscopy, respectively, demonstrated dense immunostaining in Purkinje cells and in postsynaptic densities of the adult parallel fiber-Purkinje spine synapse. The prominent delta 1/2 immunoreactivity found in the parallel fiber-Purkinje spine synapse, and the temporal correlation of the development of this synapse with the major increase in delta 1/2 immunoreactivity, suggest a major functional role for the delta subunits in cerebellar circuitry.

Light and electron microscope distribution of the NMDA receptor subunit NMDAR1 in the rat nervous system using a selective anti-peptide antibody.

NMDA receptors play key roles in synaptic plasticity and neuronal development, and may be involved in learning, memory, and compensation following injury. A polyclonal antibody that recognizes four of seven splice variants of NMDAR1 was made using a C-terminus peptide (30 amino acid residues). NMDAR1 is the major NMDA receptor subunit, found in most or all NMDA receptor complexes. On immunoblots, this antibody labeled a single major band migrating at M(r) = 120,000. The antibody did not cross-react with extracts from transfected cells expressing other glutamate receptor subunits, nor did it label non-neuronal tissues. Immunostained vibratome sections of rat tissue showed labeling in many neurons in most structures in the brain, as well as in the cervical spinal cord, dorsal root and vestibular ganglia, and in pineal and pituitary glands. Staining was moderate to dense in the olfactory bulb, neocortex, striatum, some thalamic and hypothalamic nuclei, the colliculi, and many reticular, sensory, and motor neurons of the brainstem and spinal cord. The densest stained cells included the pyramidal and hilar neurons of the CA3 region of the hippocampus, Purkinje cells of the cerebellum, supraoptic and magnocellular paraventricular neurons of the hypothalamus, inferior olive, red nucleus, lateral reticular nucleus, peripheral dorsal cochlear nucleus, and motor nuclei of the lower brainstem and spinal cord. Ultrastructural localization of immunostaining was examined in the hippocampus, cerebral cortex, and cerebellar cortex. The major staining was in postsynaptic densities apposed by unstained presynaptic terminals with round or mainly round vesicles, and in associated dendrites. The pattern of staining matched that of previous in situ hybridization but differed somewhat from that of binding studies, implying that multiple types of NMDA receptors exist. Comparison with previous studies of localization of other glutamate receptor types revealed that NMDAR1 may colocalize with these other types in many neurons throughout the nervous system.

Biochemical and assembly properties of GluR6 and KA2, two members of the kainate receptor family, determined with subunit-specific antibodies.

To examine subunit assembly and biochemical properties of two members of the kainate family of glutamate receptors (GluR), antibodies were made to synthetic peptides corresponding to the carboxyl termini of GluR6 and KA2. Immunoblot analysis of membranes from human embryonic kidney cells transfected with glutamate receptor cDNAs showed that these antibodies are selective for their respective receptor subunit except that the antibody to GluR6 also recognizes GluR7, which is expected due to the sequence homology between the two subunits at the carboxyl terminus. In transfected cell membranes, immunoblot analysis with the antibody to GluR6 showed a major immunoreactive band at 118 kDa and minor bands at 103 and 28 kDa. The 103-kDa band appears to be a deglycosylated form of GluR6 since deglycosylation eliminates staining at 118 kDa and increases staining of the 103-kDa band. Immunoblot analysis of KA2 transfected cell membranes shows a major band at 123 kDa and minor bands at 109 and 37 kDa. Deglycosylation converts the 123-kDa band into a 109-kDa band. Analysis of brain tissues shows that both antibodies label single major bands which migrate at the same molecular masses as those from transfected cell membranes, 118 and 123 kDa for GluR6 and KA2, respectively. Immunoprecipitation studies showed that antibodies to GluR6 and KA2 selectively immunoprecipitated [3H]kainate binding activity, but not 3H-labeled alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) binding activity, from Triton X-100-solubilized rat brain membranes. Furthermore, each antibody coimmunoprecipitated GluR6 and KA2 from cells co-transfected with GluR6 and KA2 cDNAs and from detergent-solubilized rat brain membranes, indicating that these two subunits can coassemble into a molecular complex. Interestingly, GluR1 and GluR2, subunits of the AMPA receptor, also co-immunoprecipitated with GluR6 in cells co-transfected with GluR6 and GluR1 or GluR2 cD-NAs. Such complexes appear to be present to a limited extent in the brain.

Ultrastructural evidence that early synapse formation on central vestibular sensory neurons is independent of peripheral vestibular influences.

Migration and early differentiation of neurons of the tangential vestibular nucleus of the chick take place between embryonic days 5 and 8. In the absence of primary vestibular afferents (otocyst-ablation), a previous light microscope study documented that early developmental events still occurred, but the neurons failed to complete differentiation and to survive. In order to understand why these neurons undergo normal early development, we have repeated the vestibular deafferentation paradigm followed by ultrastructural observations on these neurons. We found that the ultrastructural events associated with migration and differentiation in the deafferented tangential nucleus were essentially normal from 5 to 8 days. Most important, longitudinal fibers, presumably of central, nonvestibular origins, formed the first synapses at the same time and sequence as observed in normal embryos. Thus vestibular sensory neurons receive their first input from central fibers, initiating events in the formation of a central vestibular circuitry without the influence of peripheral vestibular fibers or endorgan.