G. G. Skibo

A Synthetic Neural Cell Adhesion Molecule Mimetic Peptide Promotes Synaptogenesis, Enhances Presynaptic Function, and Facilitates Memory Consolidation

The neural cell adhesion molecule (NCAM) plays a critical role in development and plasticity of the nervous system and is involved in the mechanisms of learning and memory. Here, we show that intracerebroventricular administration of the FG loop (FGL), a synthetic 15 amino acid peptide corresponding to the binding site of NCAM for the fibroblast growth factor receptor 1 (FGFR1), immediately after training rats in fear conditioning or water maze learning, induced a long-lasting improvement of memory. In primary cultures of hippocampal neurons, FGL enhanced the presynaptic function through activation of FGFR1 and promoted synapse formation. These results provide the first evidence for a memory-facilitating effect resulting from a treatment that mimics NCAM function. They suggest that increased efficacy of synaptic transmission and formation of new synapses probably mediate the cognition-enhancing properties displayed by the peptide.

Synaptic potentiation induces increased glial coverage of excitatory synapses in CA1 hippocampus

Patterns of activity that induce synaptic plasticity at excitatory synapses, such as long‐term potentiation, result in structural remodeling of the postsynaptic spine, comprising an enlargement of the spine head and reorganization of the postsynaptic density (PSD). Furthermore, spine synapses represent complex functional units in which interaction between the presynaptic varicosity and the postsynaptic spine is also modulated by surrounding astroglial processes. To investigate how activity patterns could affect the morphological interplay between these three partners, we used an electron microscopic (EM) approach and 3D reconstructions of excitatory synapses to study the activity‐related morphological changes underlying induction of synaptic potentiation by theta burst stimulation or brief oxygen/glucose deprivation episodes in hippocampal organotypic slice cultures. EM analyses demonstrated that the typical glia‐synapse organization described in in vivo rat hippocampus is perfectly preserved and comparable in organotypic slice cultures. Three‐dimensional reconstructions of synapses, classified as simple or complex depending upon PSD organization, showed significant changes following induction of synaptic potentiation using both protocols. The spine head volume and the area of the PSD significantly enlarged 30 min and 1 h after stimulation, particularly in large synapses with complex PSD, an effect that was associated with a concomitant enlargement of presynaptic terminals. Furthermore, synaptic activity induced a pronounced increase of the glial coverage of both pre‐ and postsynaptic structures, these changes being prevented by application of the NMDA receptor antagonist D‐2‐amino‐5‐phosphonopentanoic acid. These data reveal dynamic, activity‐dependent interactions between glial processes and pre‐ and postsynaptic partners and suggest that glia can participate in activity‐induced structural synapse remodeling.

A synthetic NCAM-derived peptide, FGL, protects hippocampal neurons from ischemic insult both in vitro and in vivo.

There is a major unmet need for development of innovative strategies for neuroprotection against ischemic brain injury. Here we show that FGL, a neural cell adhesion molecule (NCAM)‐derived peptide binding to and inducing phosphorylation of the fibroblast growth factor receptor (FGFR), acts neuroprotectively after an ischemic insult both in vitro and in vivo. The neuroprotective activity of FGL was tested in vitro on dissociated rat hippocampal neurons and hippocampal slice cultures, using a protocol of oxygen–glucose deprivation (OGD). FGL protected hippocampal neurons from damage and maintained or restored their metabolic and presynaptic activity, both if employed as a pretreatment alone to OGD, and if only applied after the insult. In vivo 24 h pretreatment with a single suboccipital injection of FGL significantly protected hippocampal CA1 neurons from death in a transient global ischemia model in the gerbil. We conclude that FGL promotes neuronal survival after ischemic brain injury.

Tenascin-R–deficient mice show structural alterations of symmetric perisomatic synapses in the CA1 region of the hippocampus

Accumulating evidence suggests that extracellular matrix (ECM) molecules play important roles in formation of synapses. Our previous electrophysiologic study of mice deficient in the extracellular matrix glycoprotein tenascin‐R (TN‐R) showed an impaired γ‐aminobutyric acid release at perisomatic inhibitory synapses in the CA1 pyramidal cell layer of the hippocampus. The present study investigated possible ultrastructural correlates of abnormal perisomatic inhibition. Topographic, morphometric, and stereologic methods were applied at the light and electron microscopic levels to quantify the density and spatial arrangement of cell bodies of CA1 pyramidal neurons and density and architecture of symmetric synapses formed on them in TN‐R−/− and wild‐type mice of different ages. The spatial arrangement of neuronal cell bodies in the CA1 pyramidal cell layer was found more diffuse and disordered in TN‐R−/− mice than in wild‐type animals. The coverage of the plasma membrane of pyramidal cell bodies by active zones of symmetric synapses was reduced by at least 40% in TN‐R−/− animals compared with control animals. Further, the length of active zone profiles of perisomatic inhibitory synapses in the CA1 pyramidal cell layer was 8–14% smaller, whereas the number of active zones calculated per length unit of cell body profile was 30–40% smaller in TN‐R mutants than in wild‐type animals. The density and spatial arrangement of synaptic vesicles in the synaptic terminals provided ultrastructural evidence for reduced synaptic activity in TN‐R mutants. Thus, TN‐R appears to play an important role in the regulation of the number and architecture of perisomatic inhibitory synapses, which play crucial roles in the synchronization of neuronal activity and modulation of synaptic plasticity in the hippocampus. J. Comp. Neurol. 456:338–349, 2003.

Early reaction of astroglial cells in rat hippocampus to streptozotocin-induced diabetes

Diabetic patients show impaired brain functions, although underlying mechanisms remain unclear. Little is known as well about early diabetes-related changes in a brain tissue. To investigate them we analyzed the reaction of astroglial cells in the hippocampus of rats rendered diabetic by a single injection of streptozotocin (STZ). Astrocyte count, size and shape as well as levels of glial fibrillary acidic protein (GFAP) and S100b protein were assessed 3, 7 and 14 days after the STZ injection using immunohistochemistry, immuno-enzyme assay and computer-assisted image analysis. The reduced GFAP-positive cell count was found on day 3 when these cells were significantly smaller and less arborized with respect to the control. This tendency reversed on day 7 when more numerous GFAP-positive cells grew in size and became more ramified. S100b-positive cell count changes followed a contrasting pattern, elevating on days 3 and 7 and dropping on day 14. The level of cytoskeletal GFAP changed in parallel with GFAP expression revealed by immunocytochemistry, while cytosolic GFAP level started to increase only 7 days after the STZ injection. At the same time S100b level experienced an elevation on day 3 reaching the peak on day 7 and decreasing afterwards. These results suggest that the reaction of astroglial cells may be the earliest response of the brain tissue to an altered glucose metabolism playing presumably the critical role in the mechanisms underlying diabetes-related impairments of brain functions.

Blockade of endogenous neuraminidase leads to an increase of neuronal excitability and activity-dependent synaptogenesis in the rat hippocampus

Polysialic acids are widely distributed in neuronal tissue. Due to their position on glycoproteins and gangliosides on the outer cell membranes and anionic nature, polysialic acids are involved in multiple cell signaling events. The level of sialylation of the cellular surface is regulated by endogenous neuraminidase (NEU), which catalyses the hydrolysis of terminal sialic acid residues. Using the specific blocker of endogenous NEU, N‐acetyl‐2,3‐dehydro‐2‐deoxyneuraminic acid (NADNA), we show that downregulation of the endogenous NEU activity causes a significant increase in the level of hippocampal tissue sialylation. Acute application of NADNA increased the firing frequency and amplitude of spontaneous synchronous oscillations, and frequency of multiple unit activity in cultured hippocampal slices. The tonic phase of seizure‐like activity in the low‐magnesium model of ictogenesis was significantly increased in slices pretreated with NADNA. These data indicate that the degree of synchronization is influenced by the amount of active NEU in cultured hippocampal slices. Pretreatment with NADNA led to an increase of the density of simple and perforated synapses in the hippocampal CA1 stratum radiatum region. Co‐incubation of slices with NADNA and high concentrations of calcium eliminated the effect of the NEU blocker on synaptic density, suggesting that synaptogenesis observed following downregulation of the endogenous NEU activity is an activity‐dependent process.

Post-tetanic depression of GABAergic synaptic transmission in rat hippocampal cell cultures.

The effect of tetanic stimulation (30 Hz, 4 s) on evoked GABAergic inhibitory postsynaptic currents (IPSCs) was studied in cell cultures of dissociated hippocampal neurons with established synaptic connections. It was found that tetanic stimulation elicited post-tetanic depression (PTD) of the evoked IPSCs with a duration of more than 50 s in about 60% of the connections tested; post-tetanic potentiation was induced in 25% of the connections. We propose that the opposite effects of tetanization on IPSC amplitude are due to differences in the type of the interneuron that was tetanized. Since PTD in our experiments was usually accompanied by changes in the IPSC coefficient of variation and changes of a paired pulse depression, which are thought to reflect presynaptic mechanisms of modulation, we suggest that part of the PTD is due to a presynaptic mechanism(s).

Distribution of microglia and astrocytes in different regions of the normal adult rat brain

The distribution of glial cells (microglia and astrocytes) in different regions of normal adult rat brain was studied using immunohistochemical techniques and computer analysis. Antibodies against lipocortin 1 (LC1) and phosphotyrosine (PT), as well as an isolectin, GSA B4 (GSA), were used for identification of microglial units, while antibodies against protein S100β allowed us to identify astrocytes. If LC1 was used as a marker, more microglial cells were detected than with the use of PT or GSA. The highest density of LC1-positive microglial cells (on average, 130±5 cells/mm2 of the brain section area) was found in the neostriatum, while the lowest density (51±4 cells/mm2) was observed in the medulla oblongata. In general, the density of an LC1-positive microglial population was higher in the forebrain and lower in the midbrain, and the smallest number of these cells was detected in the brainstem and cerebellum. The number of astrocytes was, on average, 2–3 times as large as the number of microglial cells. High density of astrocytes, was found in the hypothalamus and hippocampus (more than 260 cells/mm2); they were more, numerous in the white matter than in the gray matter. Lower densities of this type cells were observed in the cerebral cortex, neostriatum, midbrain, medulla oblongata, and cerebellum (less than 200 cells/mm2).

HIF-1α-mediated upregulation of SERCA2b: The endogenous mechanism for alleviating the ischemia-induced intracellular Ca2+ store dysfunction in CA1 and CA3 hippocampal neurons

Pyramidal neurons of the hippocampus possess differential susceptibility to the ischemia-induced damage with the highest vulnerability of CA1 and the lower sensitivity of CA3 neurons. This damage is triggered by Ca(2+)-dependent excitotoxicity and can result in a delayed cell death that might be potentially suspended through activation of endogenous neuroprotection with the hypoxia-inducible transcription factors (HIF). However, the molecular mechanisms of this neuroprotection remain poorly understood. Here we show that prolonged (30min) oxygen and glucose deprivation (OGD) in situ impairs intracellular Ca(2+) regulation in CA1 rather than in CA3 neurons with the differently altered expression of genes coding Ca(2+)-ATPases: the mRNA level of plasmalemmal Ca(2+)-ATPases (PMCA1 and PMCA2 subtypes) was downregulated in CA1 neurons, whereas the mRNA level of the endoplasmic reticulum Ca(2+)-ATPases (SERCA2b subtype) was increased in CA3 neurons at 4h of re-oxygenation after prolonged OGD. These demonstrate distinct susceptibility of CA1 and CA3 neurons to the ischemic impairments in intracellular Ca(2+) regulation and Ca(2+)-ATPase expression. Stabilization of HIF-1α by inhibiting HIF-1α hydroxylation prevented the ischemic decrease in both PMCA1 and PMCA2 mRNAs in CA1 neurons, upregulated the SERCA2b mRNA level and eliminated the OGD-induced Ca(2+) store dysfunction in these neurons. Cumulatively, these findings reveal the previously unknown HIF-1α-driven upregulation of Ca(2+)-ATPases as a mechanism opposing the ischemic impairments in intracellular Ca(2+) regulation in hippocampal neurons. The ability of HIF-1α to modulate expression of genes coding Ca(2+)-ATPases suggests SERCA2b as a novel target for HIF-1 and may provide potential implications for HIF-1α-stabilizing strategy in activating endogenous neuroprotection.

Contribution of protease-activated receptor 1 in status epilepticus-induced epileptogenesis.

Clinical observations and studies on different animal models of acquired epilepsy consistently demonstrate that blood-brain barrier (BBB) leakage can be an important risk factor for developing recurrent seizures. However, the involved signaling pathways remain largely unclear. Given the important role of thrombin and its major receptor in the brain, protease-activated receptor 1 (PAR1), in the pathophysiology of neurological injury, we hypothesized that PAR1 may contribute to status epilepticus (SE)-induced epileptogenesis and that its inhibition shortly after SE will have neuroprotective and antiepileptogenic effects. Adult rats subjected to lithium-pilocarpine SE were administrated with SCH79797 (a PAR1 selective antagonist) after SE termination. Thrombin and PAR1 levels and neuronal cell survival were evaluated 48h following SE. The effect of PAR1 inhibition on animal survival, interictal spikes (IIS) and electrographic seizures during the first two weeks after SE and behavioral seizures during the chronic period was evaluated. SE resulted in a high mortality rate and incidence of IIS and seizures in the surviving animals. There was a marked increase in thrombin, decrease in PAR1 immunoreactivity and hippocampal cell loss in the SE-treated rats. Inhibition of PAR1 following SE resulted in a decrease in mortality and morbidity, increase in neuronal cell survival in the hippocampus and suppression of IIS, electrographic and behavioral seizures following SE. These data suggest that the PAR1 signaling pathway contributes to epileptogenesis following SE. Because breakdown of the BBB occurs frequently in brain injuries, PAR1 inhibition may have beneficial effects in a variety of acquired injuries leading to epilepsy.