Developmental switch in axon guidance modes of hippocampal mossy fibers in vitro

Hippocampal mossy fibers (MFs), axons of dentate granule cells, run through a narrow strip, called the stratum lucidum, and make synaptic contacts with CA3 pyramidal cells. This stereotyped pathfinding is assumed to require a tightly controlled guidance system, but the responsible mechanisms have not been proven directly. To clarify the cellular basis for the MF pathfinding, microslices of the dentate gyrus (DG) and Ammon's horn (AH) were topographically arranged in an organotypic explant coculture system. When collagen gels were interposed between DG and AH slices prepared from postnatal day 6 (P6) rats, the MFs passed across this intervening gap and reached CA3 stratum lucidum. Even when the recipient AH was chemically pre-fixed with paraformaldehyde, the axons were still capable of accessing their normal target area only if the DG and AH slices were directly juxtaposed without a collagen bridge. The data imply that diffusible and contact cues are both involved in MF guidance. To determine how these different cues contribute to MF pathfinding during development, a P6 DG slice was apposed simultaneously to two AH slices prepared from P0 and P13 rats. MFs projected normally to both the host slices, whereas they rarely invaded P0 AH when the two hosts were fixed. Early in development, therefore, the MFs are guided mainly by a chemoattractant gradient, and thereafter, they can find their trajectories by a contact factor, probably via fasciculation with pre-established MFs. The present study proposes a dynamic paradigm in CNS axon pathfinding, that is, developmental changes in axon guidance cues.     

Presynaptic glycine receptors on hippocampal mossy fibers

Presynaptic glycine receptors (GlyRs) have been implicated in the regulation of glutamatergic synaptic transmission. Here, we characterized presynaptic GlyR-mediated currents by patch-clamp recording from mossy fiber boutons (MFBs) in rat hippocampal slices. In MFBs, focal puff-application of glycine-evoked chloride currents that were blocked by the GlyR antagonist strychnine. Their amplitudes declined substantially during postnatal development, from a mean conductance per MFB of ∼600 pS in young to ∼130 pS in adult animals. Single-channel analysis revealed multiple conductance states between ∼20 and ∼120 pS, consistent with expression of both homo- and hetero-oligomeric GlyRs. Accordingly, estimated GlyRs densities varied between 8-17 per young, and 1-3 per adult, MFB. Our results demonstrate that functional presynaptic GlyRs are present on hippocampal mossy fiber terminals and suggest a role of these receptors in the regulation of glutamate release during the development of the mossy fiber - CA3 synapse.     

Zinc transporter 3 immunohistochemical tracing of sprouting mossy fibres

Zinc transporter 3 (ZNT3) has been shown to transport zinc ions from the cytosol into presynaptic vesicles in the mammalian brain. Several studies have stated that the zinc ion containing synaptic vesicles of zinc-enriched neurons (ZEN) are loaded with ZNT3 proteins in their membranes. This fact makes it possible to trace sprouting mossy fibres in the temporal lobe epileptic hippocampus. In the present study, we examined the expression and distribution patterns of ZNT3 protein and chelatable zinc ions in the mouse hippocampus after pilocarpine treatment. Our results demonstrate that both ZNT3 immunostaining and autometallography reveal identical patterns of sprouting mossy fibres in the inner molecular layer in the mouse hippocampus. Using ZNT3 immuno-electron microscopic analysis we confirmed the presence of ectopic mossy fibre terminals in the inner molecular layer and found additionally by immuno-blotting a significant increase of ZNT3 in the pilocarpine-treated mouse hippocampi compared to age-matched controls. The increase of ZNT3 after pilocarpine treatment was time-dependent. The results support the notion that ZNT3 immunohistochemistry provides an excellent tool for tracing sprouting of ZEN terminals. The progressive increase of ZNT3 immunostaining in the temporal lobe epileptic hippocampus may relate to the increased levels of vesicular zinc ions during seizure.     

Enhancement of hippocampal mossy fiber activity in zinc deficiency and its influence on behavior

The extracellular concentration of glutamate in the hippocampus is increased by hippocampal perfusion with CaEDTA, a membrane-impermeable zinc chelator, suggesting that the activity of glutamatergic neurons in the hippocampus are influenced by the extracellular concentrations of zinc. In the present study, the relationship between the extracellular concentrations of zinc and mossy fiber activity in the hippocampus was examined in mice and rats fed a zinc-deficient diet for 4 weeks. Timm's stain, by which histochemically reactive zinc in the presynaptic vesicles is detected, was attenuated in the hippocampus in zinc deficiency. The extracellular signal of ZnAF-2, a membrane-impermeable zinc indicator, was also lower in the hippocampal CA3, suggesting that the basal extracellular concentrations of zinc are lower maintained in zinc deficiency. To check mossy fiber activity after 4-week zinc deprivation, the decrease in the signal of FM4-64, an indicator of presynaptic activity (exocytosis), at mossy fiber synapses was measured under the condition of spontaneous depolarization. The decrease was significantly facilitated by zinc deficiency, suggesting that the basal exocytosis at mossy fiber synapses is enhanced by zinc deficiency. On the other hand, the increase in anxiety-like behavior was observed in the open-field test after 4-week zinc deprivation. The present study demonstrates that the decrease in the basal extracellular concentrations of zinc may be linked to the enhancement of the basal mossy fiber activity in zinc deficiency. This decrease seems to be also involved in neuropsychological behavior in zinc deficiency.     

Temperature effects on evoked potentials of hippocampal slices from euthermic chipmunks, hamsters and rats

1. Neural activity was recorded in hippocampal slices from euthermic chipmunks, hamsters and rats. 2. While recording the evoked potentials, the temperature of the Ringer's solution bathing the slice was varied by controlling the temperature of an outer chamber jacketing the recording chamber. 3. The temperature just below that at which a population spike could be evoked, Tt, was 10.4 +/- 0.3 degrees C (mean +/- SEM) for chipmunk slices, 14.1 +/- 0.4 degrees C for rat slices and 14.8 +/- 0.4 degrees C for hamster slices. Tt was significantly lower in the chipmunk slices (P<0.01) than in the rat and hamster slices. 4. Data were interpreted as consistent with the hypothesis that chipmunk hippocampal neurons are intrinsically cold resistant.     

Parvalbumin Immunoreactivity and Protein Level are Altered in the Gerbil Hippocampus During Normal Aging

Hippocampal interneurons are local circuit neurons which are responsible for inhibitory activity in the hippocampus. Parvalbumin (PV) is one of useful markers for GABAergic interneurons, not for principle cells, in the hippocampus. In the present study, we investigated age-related changes in PV immunoreactive neurons and protein levels in the gerbil hippocampus during normal aging. PV immunoreactive neurons were detected in all hippocampal subregions of all groups. PV immunoreactive neurons, which innervated principal neurons, were non-pyramidal neurons in the hippocampal CA1-3 regions, and were polymorphic neurons in the dentate gyrus. In the hippocampal CA1 region, the number of PV immunoreactive neurons was significantly reduced in the postnatal month 3 (PM 3) group, which was sustained by PM 18, and, at PM 24, the number of PV immunoreactive neurons was significantly decreased. In the CA2/3 region and dentate gyrus, the number of PV immunoreactive neurons was significantly decreased at PM 6: Thereafter, the number of PV immunoreactive neurons was sustained until PM 24. In addition, changes in PV protein levels in the gerbil hippocampus were similar to immunohistochemical changes during normal aging: PV protein levels were significantly decreased with age by PM 6: Thereafter, PV protein levels were sustained by PM 24. These results suggest that PV immunoreactive interneurons were decreased in the hippocampus with age in gerbils.     

Postnatal maturation of gamma-aminobutyric acidA and B-mediated inhibition in the CA3 hippocampal region of the rat

In the adult central nervous system, GABAergic synaptic inhibition is known to play a crucial role in preventing the spread of excitatory glutamatergic activity. This inhibition is achieved by a membrane hyperpolarization through the activation of postsynaptic γ-aminobutyric acid_A (GABA_A) and GABA_B receptors. In addition, GABA also depress transmitter release acting through presynaptic GABA_B receptors. Despite the wealth of data regarding the role of GABA in regulating the degree of synchronous activity in the adult, little is known about GABA transmission during early stages of development. In the following we report that GABA mediates most of the excitatory drive at early stages of development in the hippocampal CA3 region. Activation of GABA_A receptors induces a depolarization and excitation of immature CA3 pyramidal neurons and increases intracellular Ca²⁺ ([Ca²⁺]_i) during the first postnatal week of life. During the same developmental period, the postsynaptic GABA_B-mediated inhibition is poorly developed. In contrast, the presynaptic GABA_B-mediated inhibition is well developed at birth and plays a crucial role in modulating the postsynaptic activity by depressing transmitter release at early postnatal stages. We have also shown that GABA plays a trophic role in the neuritic outgrowth of cultured hippocampal neurons. © 1995 John Wiley & Sons, Inc.     

Mossy fiber pathfinding in multilayer organotypic cultures of rat hippocampal slices

1. Using a novel technique of organotypic cultures, in which two hippocampal slices were cocultured in a bilayer style, we found that the mossy fibers arising from the dentate gyrus grafted onto another dentate tissue grew along the CA3 stratum lucidum of the host hippocampal slice. The same transplantation of a CA1 microslice failed to form a network with the host hippocampus.2. Thus, the type of grafted neurons is important to determine whether they can form an appropriate network after transplantation.     

Temperature effects on evoked potentials of hippocampal slices from noncold-acclimated, cold-acclimated and hibernating hamsters

1. 1.|Neural activity was recorded in hippocampal slices from noncold-acclimated, cold-acclimated and hibernating hamsters.

2. 2.|Action potentials from a population of hippocampal pyramidal neurons were evoked by stimulating an afferent fiber tract, the Schaffer collaterals. The temperature of the artificial cerebrospinal fluid bathing the slice was varied by controlling the temperature of a water chamber jacketing the recording chamber.

3. 3.|The temperature just below that at which a population spike could be evoked, T_t, was 15.8 ± 0.9°C (mean ± SEM) for noncold-acclimated hamsters, 13.9 ± 0.3°C for cold-acclimated hamsters and 12.3 ± 0.3°C for hibernating hamsters.

4. 4.|These thresholds for evoked activity were significantly different in noncold-acclimated, cold-acclimated and hibernating hamsters, and may reflect acclimation of hippocampal neurons to cold.

Effect of long-term administration of the antidepressant drug milnacipran on serotonergic and noradrenergic neurotransmission in the rat hippocampus

The effect of a long-term administration of the antidepressant milnacipran on the function of the serotonergic (5-HT) and noradrenergic (NE) systems was studied using single cell recording of CA3 hippocampal pyramidal cells in chloral hydrate-anesthetized male Sprague-Dawley rats, and in vitro [3H]5-HT release measurement from hippocampal slices. The sensitivity of neither the extrasynaptic nor that of the postsynaptic 5-HT1A receptors of the pyramidal neurons was altered, as indicated by their unchanged responsiveness to the microiontophoretic application of 5-HT, and by the unchanged effect of the electrical stimulation at low frequency of the ascending 5-HT bundle, respectively. Increasing the frequency of stimulation (from 1 to 5 Hz) decreased its efficacy in control rats; the milnacipran treatment abolished this phenomenon. This cannot be attributed to a desensitisation of the terminal 5-HT1B autoreceptor, since the suppressive effect of 5-HT agonist 5-carboxyamidotryptamine on [3H]5-HT release was enhanced in milnacipran-treated rats. As for the NE system, the unchanged suppressing effect of microiontophoretic applications of NE and that of the 5 Hz stimulation in the locus coeruleus (LC) on the firing activity of pyramidal neurons indicates that the milnacipran treatment not altered the sensitivity of extrasynaptic alpha2- and postsynaptic alpha1-adrenergic receptors on pyramidal cells, as well as that of the presynaptic alpha2-autoreceptor on NE terminals. The decreased inhibitory effect of NE on the [3H]5-HT release in milnacipran-treated rats revealed that this treatment results in a desensitisation of the presynaptic alpha2-heteroreceptor located on serotonergic terminals. Taken together with the decreased suppressive effect of a low frequency of stimulation of the NE tract, the present results suggest that long-term milnacipran treatment enhances the efficacy of the 5-HT and reduces that of the NE neurotransmission.     

Inhibition of NMDA-induced outward currents by interleukin-1beta in hippocampal neurons

There is increasing evidence that a functional interaction exists between interleukin-1β (IL-1β) and N-methyl-d-aspartate (NMDA) receptors. The present study attempted to elucidate the effect of IL-1β on the NMDA-induced outward currents in mechanically dissociated hippocampal neurons using a perforated patch recording technique. IL-1β (30-100 ng/ml) inhibited the mean amplitude of the NMDA-induced outward currents that were mediated by charybdotoxin (ChTX)-sensitive Ca²⁺-activated K⁺ (K_Ca) channels. IL-1β (100 ng/ml) also significantly increased the mean ratio of the NMDA-induced inward current amplitudes measured at the end to the beginning of a 20-s application of NMDA. In hippocampal neurons from acute slice preparations, IL-1β significantly inhibited ChTX-sensitive K_Ca currents induced by a depolarizing voltage-step. IL-1 receptor antagonist antagonized effects of IL-1β. These results strongly suggest that IL-1β increases the neuronal excitability by inhibition of ChTX-sensitive K_Ca channels activated by Ca²⁺ influx through both NMDA receptors and voltage-gated Ca²⁺ channels.     

Activation of Mitogen-Activated Protein Kinase by Epidermal Growth Factor in Hippocampal Neurons and Neuronal Cell Lines

Abstract: Epidermal growth factor (EGF) functions in a bimodal capacity in the nervous system, acting as a mitogen in neuronal stem cells and a neurotrophic factor in differentiated adult neurons. Thus, it is likely that EGF signal transduction, as well as receptor expression, differs among various cell types and possibly in the same cell type at different stages of development. We used hippocampal neuronal cell lines capable of terminal differentiation to investigate changes in EGF receptor expression, DNA synthesis, and stimulation of mitogen-activated protein (MAP) kinase by EGF before and after differentiation. H19-7, the line that was most representative of hippocampal neurons, was mitogenically responsive to EGF only before differentiation and increased in EGF binding after differentiation. MAP kinase was stimulated by EGF in both undifferentiated and differentiated cells, as well as in primary hippocampal cultures treated with either EGF or glutamate. These results indicate that the activation of MAP kinase by EGF is an early signaling event in both mitotic and postmitotic neuronal cells. Furthermore, these studies demonstrate the usefulness of hippocampal cell lines as a homogeneous neuronal system for studies of EGF signaling or other receptor signaling mechanisms in the brain.     

Lithium inhibits caspase 3 activation and dephosphorylation of PKB and GSK3 induced by K+ deprivation in cerebellar granule cells

Lithium protects cerebellar granule cells from apoptosis induced by low potassium, and also from other apoptotic stimuli. However, the precise mechanism by which this occurs is not understood. When cerebellar granule cells were switched to low potassium medium, the activation of caspase 3 was detected within 6 h, suggesting a role of caspase 3 in mediating apoptosis under conditions of low potassium. In the same conditions, lithium (5 mM) inhibited the activation of caspase 3 induced by low potassium. As lithium did not inhibit caspase 3 activity in vitro, these results suggest that this ion inhibits an upstream component that is required for caspase 3 activation. Lithium is known to inhibit a kinase termed glycogen sythase kinase 3 (GSK3), which is implicated in the survival pathway of phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB). Here we demonstrate that low potassium in the absence of lithium induces the dephosphorylation, and therefore the activation, of GSK3. However, when lithium was present, GSK3 remained phosphorylated at the same level as observed under conditions of high potassium. Low potassium induced the dephosphorylation and inactivation of PKB, whereas when lithium was present PKB was not dephosphorylated. Our results allow us to propose a new hypothesis about the action mechanism of lithium, this ion could inhibit a serine-threonine phosphatase induced by potassium deprivation.     

Immunohistochemical analysis of Disc1 expression in the developing and adult hippocampus

In recent years, Disrupted-In-Schizophrenia 1 (DISC1) has emerged as one of the most promising candidate genes whose disruption confers an increased risk for schizophrenia. Cell biology studies have implicated DISC1 in key neurodevelopmental processes including neurite outgrowth and neuronal migration. In situ hybridization analysis has revealed that Disc1 is expressed in the hypothalamus, olfactory bulbs, the developing cerebral cortex and the hippocampus. The hippocampus is of particular interest because abnormalities in hippocampal volume and function have been consistently reported in schizophrenics. Moreover, DISC1 mutations have been associated with abnormal activation of the hippocampus in humans. Given the involvement of the hippocampus in the pathophysiology of schizophrenia, there is an intriguing possibility that disruption of DISC1 may increase schizophrenia susceptibility by altering the normal development and function of the hippocampus. In order to contribute to our understanding of DISC1's role in the hippocampus, we have performed a detailed analysis of the Disc1 expression pattern in the mouse hippocampus throughout development. We report that Disc1 is expressed throughout the hippocampus during embryonic development, with expression becoming increasingly specialized in Ammon's horn and dentate gyrus granule cells within the first postnatal week. This expression pattern remains consistent into adulthood, with a noted decrease in Disc1 expression in the adult CA1. Disc1 is also expressed in proliferating cells in the adult subgranular zone, as well as in a subset of GABAergic interneurons. Our results are the first report of a detailed immunohistochemical analysis of the ontogeny of Disc1 expression within the hippocampus.     

Regulation of passive potassium transport of normal and transformed 3T3 mouse cell cultures by external calcium concentration and temperature

Summary Regulation of passive potassium ion transport by the external calcium concentration and temperature was studied on cell cultures of 3T3 mouse cells and their DNA-virus transformed derivatives. Upon lowering of external calcium concentration, passive potassium efflux generally exhibits a sharp increase at about 0.1mm. The fraction of calcium-regulated potassium efflux is largely independent of temperature in the cases of the transformed cells, but shows a sharp increase for 3T3 cells upon increasing temperature above 32°C. In the same range of temperature, the 3T3 cells exhibit the phenomenon of high-temperature inactivation of the residual potassium efflux at 1mm external calcium. At comparable cellular growth densities, the transformed cell lines do not show high-temperature inactivation of residual potassium efflux. These results are consistent with the notion of a decisive role of the internal K⁺ concentration in the cell-density dependent regulation of cell proliferation. In particular, the growth-inhibiting effect of lowering the external Ca²⁺ concentrations is considered as largely due to a rise of passive K⁺ efflux and a subsequent decrease of internal K⁺ concentration. The experimental data on the Ca²⁺ dependence of passive K⁺ flux are quantitatively described by a theoretical model based on the constant field relations including negative surface charges on the external face of the membrane, which cooperatively bind Ca²⁺ ions and may concomitantly undergo a lateral redistribution. The present evidence is consistent with acidic phospholipids as representing these negative surface charges.This work is dedicated to the memory of Max Delbrück (deceased March 10, 1981), in whose laboratory in 1966 the earlier version of the present theoretical model was developed by one of the authors.     

Co-activation of the phosphatidylinositol-3-kinase/Akt signaling pathway by N-methyl-D-aspartate and TrkB receptors in cerebellar granule cell neurons

Summary.  Neuroprotective concentrations of N-methyl-D-aspartate (NMDA) promote survival of cerebellar granule cell neurons against glutamate excitotoxicity through a TrkB receptor-mediated brain-derived neurotrophic factor (BDNF) autocrine loop. However, the intracellular signaling pathway(s) are not clear. Our results show that PI-3 kinase/Akt is activated by either NMDA or BDNF displaying differential kinetics. BDNF and NMDA increased Akt phosphorylation within 5 minutes but maximal activation by NMDA was observed at 3 hours. Akt phosphorylation was completely blocked by the PI-3 kinase inhibitor LY294002. NMDA-mediated activation of Akt was completely blocked by MK-801 and partially blocked by the TrkB receptor inhibitor, K252a, indicating the requirement of TrkB receptors for maximal activation by NMDA. In contrast, BDNF-induced Akt phosphorylation was abolished by K252a, but not by the addition of MK-801. Therefore, the PI-3 kinase/Akt pathway is co-activated by NMDA and TrkB receptors. The kinetics of BDNF and NMDA-mediated activation of PI-3 kinase/Akt suggests that they have different roles in intraneuronal time-related events. Received June 29, 2001 Accepted August 6, 2001 Published online June 3, 2002     

Epigallocatechin-3-O-gallate disrupts stress fibers and the contractile ring by reducing myosin regulatory light chain phosphorylation mediated through the target molecule 67 kDa laminin receptor

Epigallocatechin-3-O-gallate (EGCG), a major polyphenol of green tea, has been shown to inhibit the growth of various cancer cell lines. We show here that EGCG induced the disruption of stress fibers and decreased the phosphorylation of the myosin II regulatory light chain (MRLC) at Thr18/Ser19, which is necessary for both contractile ring formation and cell division. Indirect immunofluorescence analysis revealed that EGCG inhibited the concentration of both F-actin and the phosphorylated MRLC in the cleavage furrow at the equator of dividing cells. In addition, EGCG increased the percentages of cells in the G(2)/M phase and inhibited cell growth. Recently, we have demonstrated that the anticancer activity of EGCG is mediated by the metastasis-associated 67kDa laminin receptor (67LR). To explore whether the effect of EGCG is mediated by the 67LR, we transfected cells with short hairpin RNA (shRNA) expression vector to downregulate 67LR expression. When the 67LR was silenced, the suppressive effect of EGCG on the MRLC phosphorylation was significantly attenuated. These results suggest that EGCG inhibits the cell growth by reducing the MRLC phosphorylation and this effect is mediated by the 67LR.     

Molecular cloning and expression pattern of a Cubitus interruptus homologue from the mulberry silkworm Bombyx mori

Mutations in the human Crumbs homologue 1 (CRB1) gene cause severe retinal dystrophies. CRB1 is homologous to Drosophila Crumbs, a protein essential for establishing and maintaining epithelial polarity. We have isolated the mouse orthologue, Crb1, and analyzed its expression pattern in embryonic and post-natal stages. Crb1 is expressed exclusively in the eye, and the central nervous system. In the developing eye, expression of Crb1 is detected in the retinal progenitors, and later on becomes restricted to the differentiated photoreceptor cells where it remains active up to the adult stage. In the developing neural tube, expression of Crb1 is restricted to its most ventral structures, coinciding with the expression domain of Nkx2.2. In the adult brain, Crb1 expression is defined to areas where the production and migration of neurons occurs in adulthood.     

DISC1-related signaling pathways in adult neurogenesis of the hippocampus

Disrupted-in-schizophrenia 1 (DISC1) is a multifunctional scaffold protein which plays an important role in neurogenesis and neural development in the adult brain, especially in the dentate gyrus (DG) of the hippocampus. Accumulated research has unveiled the role of DISC1 in several aspects of neural development and neurogenesis, such as neuronal maturation, proliferation, migration, positioning, differentiation, dendritic growth, axonal outgrowth, and synaptic plasticity. Studies on the function of this protein have explored multiple facets, including variants and missense mutants in genetics, proteins interactivity and signaling pathways in molecular biology, and pathogenesis and treatment targets of major mental illness, and more. In this review, we present several signaling pathways discussed in recent research, such as the AKT signaling pathway, GABA signaling pathway, GSK3β signaling pathway, Wnt signaling pathway, and NMDA-R signaling pathway. DISC1 interacts, directly or indirectly, with these signaling pathways and they co-regulate the process of adult neurogenesis in the hippocampus.