Nerve growth factor rapidly suppresses basal, NMDA-evoked, and AMPA-evoked nitric oxide synthase activity in rat hippocampus in vivo

In adult forebrain, nerve growth factor (NGF) influences neuronal maintenance and axon sprouting and is neuroprotective in several injury models through mechanisms that are incompletely understood. Most NGF signaling is thought to occur after internalization and retrograde transport of trkA receptor and be mediated through the nucleus. However, NGF expression in hippocampus is rapidly and sensitively regulated by synaptic activity, suggesting that NGF exerts local effects more dynamically than possible through signaling requiring retrograde transport to distant afferent neurons. Interactions have been reported between NGF and nitric oxide (NO). Because NO affects both neural plasticity and degeneration, and trk receptors can mediate signaling within minutes, we hypothesized that NGF might rapidly modulate NO production. Using in vivo microdialysis we measured conversion of l-[14C]arginine to l-[14C]citrulline as an accurate reflection of NO synthase (NOS) activity in adult rat hippocampus. NGF significantly reduced NOS activity to 61% of basal levels within 20 min of onset of delivery and maintained NOS activity at less than 50% of baseline throughout 3 hr of delivery. This effect did not occur with control protein (cytochrome c) and was not mediated by an effect of NGF on glutamate levels. In addition, simultaneous delivery of NGF prevented significant increases in NOS activity triggered by the glutamate receptor agonists N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Rapid suppression by NGF of basal and glutamate-stimulated NOS activity may regulate neuromodulatory functions of NO or protect neurons from NO toxicity and suggests a novel mechanism for rapidly mediating functions of NGF and other neurotrophins.

Plasticity of the neoplastic phenotype in vivo is regulated by epigenetic factors

Age of host and transplantation-site microenvironment influence the tumorigenic potential of neoplastically transformed liver epithelial cells. Tumorigenic BAG2-GN6TF rat liver epithelial cells consistently form tumors at ectopic sites, but differentially express tumorigenicity or hepatocytic differentiation in the liver depending on host age and route of cell transplantation into the liver. Direct inoculation into host livers concentrates tumor cells locally, resulting in undifferentiated tumors near the transplantation site in both young (3-month-old) and old (18-month-old) rats. Transplantation-site tumors regress within 1 month in the livers of young rats, but grow progressively in old rats. However, inoculation of cells into the spleen distributes transplanted cells individually throughout the liver, resulting in hepatocytic differentiation by tumor cells with concomitant suppression of their tumorigenicity in young rats. When transplanted into livers of old rats by splenic inoculation, or when young hepatic-transplant recipients are allowed to age, hepatocytic progeny of BAG2-GN6TF cells proliferate to form foci, suggesting that the liver microenvironment of old rats incompletely regulates the proliferation and differentiation of tumor cell-derived hepatocytes. Upon removal from the liver, BAG2-GN6TF-derived hepatocytes revert to an undifferentiated, aggressively tumorigenic phenotype. We posit that the spectrum between normal differentiation and malignant potential of these cells reflects the dynamic interaction of the specific transformation-related genotype of the cells and the characteristics of the tissue microenvironment at the transplantation site. Changes in the tissue milieu, such as those that accompany normal aging, may determine the ability of a genetically aberrant cell to produce a tumor.

Preferential synaptic relationships between substance P-immunoreactive boutons and neurokinin 1 receptor sites in the rat spinal cord

Substance P plays an important role in the transmission of pain-related information in the dorsal horn of the spinal cord. Recent immunocytochemical studies have shown a mismatch between the distribution of substance P and its receptor in the superficial laminae of the dorsal horn. Because such a mismatch was not observed by using classical radioligand binding studies, we decided to investigate further the issue of the relationship between substance P and its receptor by using an antibody raised against a portion of the carboxyl terminal of the neurokinin 1 receptor and a bispecific monoclonal antibodies against substance P and horseradish peroxidase. Light microscopy revealed a good correlation between the distributions of substance P and the neurokinin 1 receptor, both being localized with highest densities in lamina I and outer lamina II of the spinal dorsal horn. An ultrastructural double-labeling study, combining preembedding immunogold with enzyme-based immunocytochemistry, showed that most neurokinin 1 receptor immunoreactive dendrites were apposed by substance P containing boutons. A detailed quantitative analysis revealed that neurokinin 1 receptor immunoreactive dendrites received more appositions and synapses from substance P immunoreactive terminals than those not expressing the neurokinin 1 receptor. Such preferential innervation by substance P occurred in all superficial dorsal horn laminae even though neurokinin 1 receptor immunoreactive dendrites were a minority of the total number of dendritic profiles in the above laminae. These results suggest that, contrary to the belief that neuropeptides act in a diffuse manner at a considerable distance from their sites of release, substance P should act on profiles expressing the neurokinin 1 receptor at a short distance from its site of release.

Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs

SNARE [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex. The association of the four α-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. We conclude that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and R-SNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.

Chronic stress alters synaptic terminal structure in hippocampus

Repeated psychosocial or restraint stress causes atrophy of apical dendrites in CA3 pyramidal neurons of the hippocampus, accompanied by specific cognitive deficits in spatial learning and memory. Excitatory amino acids mediate this atrophy together with adrenal steroids and the neurotransmitter serotonin. Because the mossy fibers from dentate granule neurons provide a major excitatory input to the CA3 proximal apical dendrites, we measured ultrastructural parameters associated with the mossy fiber–CA3 synapses in control and 21-day restraint-stressed rats in an effort to find additional morphological consequences of stress that could help elucidate the underlying anatomical as well as cellular and molecular mechanisms. Although mossy fiber terminals of control rats were packed with small, clear synaptic vesicles, terminals from stressed animals showed a marked rearrangement of vesicles, with more densely packed clusters localized in the vicinity of active zones. Moreover, compared with controls, restraint stress increased the area of the mossy fiber terminal occupied by mitochondrial profiles and consequently, a larger, localized energy-generating capacity. A single stress session did not produce these changes either immediately after or the next day following the restraint session. These findings provide a morphological marker of the effects of chronic stress on the hippocampus that points to possible underlying neuroanatomical as well as cellular and molecular mechanisms for the ability of repeated stress to cause structural changes within the hippocampus.

Synaptic and extrasynaptic γ-aminobutyric acid type A receptor clusters in rat hippocampal cultures during development

We have simultaneously measured the expression of postsynaptic γ-aminobutyric acid type A (GABAA) receptor clusters and of presynaptic boutons in neonatal rat hippocampal cultures between days 1 and 30. GABAA receptors were labeled with antibodies recognizing the extracellular domains of β2/3 and γ2 subunits. Boutons were visualized by activity-dependent uptake of the styryl dye FM4-64, or by antibodies against the presynaptic vesicular protein SV2 or the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). GABAA receptor clusters could be seen in living neurons already 6 h after culturing, much before presynaptic markers could be identified in nerve terminals. The densities of receptor clusters that contained the β2/3 subunits were constant between days 10 and 30 in culture, whereas γ2 subunit-containing clusters fluctuated and reached a maximum on day 20. SV2 and GAD staining could be measured from day 2 onwards. Clustering of GAD in presynaptic terminals and FM4-64 uptake were observed only at day 5 and afterward. SV2 staining and FM4-64 uptake increased in parallel between days 5 and 20 and remained constant thereafter. GAD-stained boutons were fewer than those labeled with other, less specific, presynaptic stains. They reached a maximum on day 20 and fell again toward day 30. Double labeling of GABAA receptors and of presynaptic boutons in neurons during differentiation showed that, even after 30 days in culture, large fractions of GABAA receptor clusters containing β2/3 and/or γ2 subunits remained extrasynaptic.

Plasticity of the cochleotopic (frequency) map in specialized and nonspecialized auditory cortices

Auditory conditioning (associative learning) causes reorganization of the cochleotopic (frequency) maps of the primary auditory cortex (AI) and the inferior colliculus. Focal electric stimulation of the AI also evokes basically the same cortical and collicular reorganization as that caused by conditioning. Therefore, part of the neural mechanism for the plasticity of the central auditory system caused by conditioning can be explored by focal electric stimulation of the AI. The reorganization is due to shifts in best frequencies (BFs) together with shifts in frequency-tuning curves of single neurons. In the AI of the Mongolian gerbil (Meriones unguiculatus) and the posterior division of the AI of the mustached bat (Pteronotus parnellii), focal electric stimulation evokes BF shifts of cortical auditory neurons located within a 0.7-mm distance along the frequency axis. The amount and direction of BF shift differ depending on the relationship in BF between stimulated and recorded neurons, and between the gerbil and mustached bat. Comparison in BF shift between different mammalian species and between different cortical areas of a single species indicates that BF shift toward the BF of electrically stimulated cortical neurons (centripetal BF shift) is common in the AI, whereas BF shift away from the BF of electrically stimulated cortical neurons (centrifugal BF shift) is special. Therefore, we propose a hypothesis that reorganization, and accordingly organization, of cortical auditory areas caused by associative learning can be quite different between specialized and nonspecialized (ordinary) areas of the auditory cortex.

Spontaneous corticospinal axonal plasticity and functional recovery after adult central nervous system injury

Although it is believed that little recovery occurs after adult mammalian spinal cord injury, in fact significant spontaneous functional improvement commonly occurs after spinal cord injury in humans. To investigate potential mechanisms underlying spontaneous recovery, lesions of defined components of the corticospinal motor pathway were made in adult rats in the rostral cervical spinal cord or caudal medulla. Following complete lesions of the dorsal corticospinal motor pathway, which contains more than 95% of all corticospinal axons, spontaneous sprouting from the ventral corticospinal tract occurred onto medial motoneuron pools in the cervical spinal cord; this sprouting was paralleled by functional recovery. Combined lesions of both dorsal and ventral corticospinal tract components eliminated sprouting and functional recovery. In addition, functional recovery was also abolished if dorsal corticospinal tract lesions were followed 5 weeks later by ventral corticospinal tract lesions. We found extensive spontaneous structural plasticity as a mechanism correlating with functional recovery in motor systems in the adult central nervous system. Experimental enhancement of spontaneous plasticity may be useful to promote further recovery after adult central nervous system injury.

Estrogen increases synaptic connectivity between single presynaptic inputs and multiple postsynaptic CA1 pyramidal cells: A serial electron-microscopic study

Dendritic spines are sites of the vast majority of excitatory synaptic input to hippocampal CA1 pyramidal cells. Estrogen has been shown to increase the density of dendritic spines on CA1 pyramidal cell dendrites in adult female rats. In parallel with increased spine density, estrogen has been shown also to increase the number of spine synapses formed with multiple synapse boutons (MSBs). These findings suggest that estrogen-induced dendritic spines form synaptic contacts with preexisting presynaptic boutons, transforming some previously single synapse boutons (SSBs) into MSBs. The goal of the current study was to determine whether estrogen-induced MSBs form multiple synapses with the same or different postsynaptic cells. To quantify same-cell vs. different-cell MSBs, we filled individual CA1 pyramidal cells with biocytin and serially reconstructed dendrites and dendritic spines of the labeled cells, as well as presynaptic boutons in synaptic contact with labeled and unlabeled (i.e., different-cell) spines. We found that the overwhelming majority of MSBs in estrogen-treated animals form synapses with more than one postsynaptic cell. Thus, in addition to increasing the density of excitatory synaptic input to individual CA1 pyramidal cells, estrogen also increases the divergence of input from individual presynaptic boutons to multiple postsynaptic CA1 pyramidal cells. These findings suggest the formation of new synaptic connections between previously unconnected hippocampal neurons.

Insulin promotes rapid delivery of N-methyl-d- aspartate receptors to the cell surface by exocytosis

Insulin potentiates N-methyl-d-aspartate receptors (NMDARs) in neurons and Xenopus oocytes expressing recombinant NMDARs. The present study shows that insulin induced (i) an increase in channel number times open probability (nPo) in outside-out patches excised from Xenopus oocytes, with no change in mean open time, unitary conductance, or reversal potential, indicating an increase in n and/or Po; (ii) an increase in charge transfer during block of NMDA-elicited currents by the open channel blocker MK-801, indicating increased number of functional NMDARs in the cell membrane with no change in Po; and (iii) increased NR1 surface expression, as indicated by Western blot analysis of surface proteins. Botulinum neurotoxin A greatly reduced insulin potentiation, indicating that insertion of new receptors occurs via SNARE-dependent exocytosis. Thus, insulin potentiation occurs via delivery of new channels to the plasma membrane. NMDARs assembled from mutant subunits lacking all known sites of tyrosine and serine/threonine phosphorylation in their carboxyl-terminal tails exhibited robust insulin potentiation, suggesting that insulin potentiation does not require direct phosphorylation of NMDAR subunits. Because insulin and insulin receptors are localized to glutamatergic synapses in the hippocampus, insulin-regulated trafficking of NMDARs may play a role in synaptic transmission and plasticity, including long-term potentiation.

Formation of temporal-feature maps by axonal propagation of synaptic learning

Computational maps are of central importance to a neuronal representation of the outside world. In a map, neighboring neurons respond to similar sensory features. A well studied example is the computational map of interaural time differences (ITDs), which is essential to sound localization in a variety of species and allows resolution of ITDs of the order of 10 μs. Nevertheless, it is unclear how such an orderly representation of temporal features arises. We address this problem by modeling the ontogenetic development of an ITD map in the laminar nucleus of the barn owl. We show how the owl's ITD map can emerge from a combined action of homosynaptic spike-based Hebbian learning and its propagation along the presynaptic axon. In spike-based Hebbian learning, synaptic strengths are modified according to the timing of pre- and postsynaptic action potentials. In unspecific axonal learning, a synapse's modification gives rise to a factor that propagates along the presynaptic axon and affects the properties of synapses at neighboring neurons. Our results indicate that both Hebbian learning and its presynaptic propagation are necessary for map formation in the laminar nucleus, but the latter can be orders of magnitude weaker than the former. We argue that the algorithm is important for the formation of computational maps, when, in particular, time plays a key role.

Diacylglycerol kinase ɛ regulates seizure susceptibility and long-term potentiation through arachidonoyl– inositol lipid signaling

Arachidonoyldiacylglycerol (20:4-DAG) is a second messenger derived from phosphatidylinositol 4,5-bisphosphate and generated by stimulation of glutamate metabotropic receptors linked to G proteins and activation of phospholipase C. 20:4-DAG signaling is terminated by its phosphorylation to phosphatidic acid, catalyzed by diacylglycerol kinase (DGK). We have cloned the murine DGKɛ gene that showed, when expressed in COS-7 cells, selectivity for 20:4-DAG. The significance of DGKɛ in synaptic function was investigated in mice with targeted disruption of the DGKɛ. DGKɛ−/− mice showed a higher resistance to eletroconvulsive shock with shorter tonic seizures and faster recovery than DGKɛ+/+ mice. The phosphatidylinositol 4,5-bisphosphate-signaling pathway in cerebral cortex was greatly affected, leading to lower accumulation of 20:4-DAG and free 20:4. Also, long-term potentiation was attenuated in perforant path–dentate granular cell synapses. We propose that DGKɛ contributes to modulate neuronal signaling pathways linked to synaptic activity, neuronal plasticity, and epileptogenesis.

Developmental plasticity of CNS microglia

Microglia arise from CD45+ bone marrow precursors that colonize the fetal brain and play a key role in central nervous system inflammatory conditions. We report that parenchymal microglia are uncommitted myeloid progenitors of immature dendritic cells and macrophages by several criteria, including surface expression of “empty” class II MHC protein and their cysteine protease (cathepsin) profile. Microglia express receptors for stem cell factor and can be skewed toward more dendritic cell or macrophage-like profiles in response to the lineage growth factors granulocyte/macrophage colony-stimulating factor or macrophage colony-stimulating factor. Thus, in contrast to other organs, where terminally differentiated populations of resident dendritic cells and/or macrophages outnumber colonizing precursors, the majority of microglia within the brain remain in an undifferentiated state.