Purification and characterization of rat hippocampal CA3-dendritic spines associated with mossy fiber terminals.
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We report a revised and improved isolation procedure for CA3-dendritic spines, most of them still in association with mossy fiber terminals resulting in a 7.5-fold enrichment over nuclei and a 29-fold enrichment over myelin. Additionally, red blood cells, medullated fibers, mitochondria and small synaptosomes were significantly depleted. We show by high resolution electron microscopy that this subcellular fraction contains numerous dendritic spines with a rich ultrastructure, e.g. an intact spine apparatus, membranous organelles, free and membrane-bound polyribosomes, endocytic structures and mitochondria. This improved experimental system will allow us to study aspects of post-synaptic functions at the biochemical and molecular level. (+info)
Somatostatin acts in CA1 and CA3 to reduce hippocampal epileptiform activity.
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Although the peptide somatostatin (SST) has been speculated to function in temporal lobe epilepsy, its exact role is unclear, as in vivo studies have suggested both pro- and anticonvulsant properties. We have shown previously that SST has multiple inhibitory cellular actions in the CA1 region of the hippocampus, suggesting that in this region SST should have antiepileptic actions. To directly assess the effect of SST on epileptiform activity, we studied two in vitro models of epilepsy in the rat hippocampal slice preparation using extracellular and intracellular recording techniques. In one, GABA-mediated neurotransmission was inhibited by superfusion of the GABAA receptor antagonist bicuculline. In the second, we superfused Mg2+-free artificial cerebrospinal fluid to remove the Mg2+ block of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. We show here that SST markedly reduces the intensity of evoked epileptiform afterdischarges and the frequency of spontaneous bursts in both CA1 and CA3. SST appears to act additively in the two regions to suppress the transmission of epileptiform events through the hippocampus. We further examined SST's actions in CA3 and found that SST dramatically reduced the frequency of paroxysmal depolarizing shifts (PDSs) recorded intracellularly in current clamp, as well as increasing the threshold for evoking "giant" excitatory postsynaptic currents (EPSCs), large polysynaptically mediated EPSCs that are the voltage-clamp correlate of PDSs. We also examined the actions of SST on pharmacologically isolated EPSCs generated at both mossy fiber (MF) and associational/commissural (A/C) synapses. SST appears to act specifically to reduce recurrent excitation between CA3 neurons because it depresses A/C- but not MF-evoked EPSCs. SST also increased paired-pulse facilitation of A/C EPSCs, suggesting a presynaptic site of action. Reciprocal activation of CA3 neurons through A/C fibers is critical for generation of epileptiform activity in hippocampus. Thus SST reduces feedforward excitation in rat hippocampus, acting to "brake" hyperexcitation. This is a function unique from that described for other hippocampal neuropeptides, which affect more standard neurotransmission. Our results suggest that SST receptors could be a unique, selective clinical target for treatment of limbic seizures. (+info)
Recurrent mossy fiber pathway in rat dentate gyrus: synaptic currents evoked in presence and absence of seizure-induced growth.
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A common feature of temporal lobe epilepsy and of animal models of epilepsy is the growth of hippocampal mossy fibers into the dentate molecular layer, where at least some of them innervate granule cells. Because the mossy fibers are axons of granule cells, the recurrent mossy fiber pathway provides monosynaptic excitatory feedback to these neurons that could facilitate seizure discharge. We used the pilocarpine model of temporal lobe epilepsy to study the synaptic responses evoked by activating this pathway. Whole cell patch-clamp recording demonstrated that antidromic stimulation of the mossy fibers evoked an excitatory postsynaptic current (EPSC) in approximately 74% of granule cells from rats that had survived >10 wk after pilocarpine-induced status epilepticus. Recurrent mossy fiber growth was demonstrated with the Timm stain in all instances. In contrast, antidromic stimulation of the mossy fibers evoked an EPSC in only 5% of granule cells studied 4-6 days after status epilepticus, before recurrent mossy fiber growth became detectable. Notably, antidromic mossy fiber stimulation also evoked an EPSC in many granule cells from control rats. Clusters of mossy fiber-like Timm staining normally were present in the inner third of the dentate molecular layer at the level of the hippocampal formation from which slices were prepared, and several considerations suggested that the recorded EPSCs depended mainly on activation of recurrent mossy fibers rather than associational fibers. In both status epilepticus and control groups, the antidromically evoked EPSC was glutamatergic and involved the activation of both AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors. EPSCs recorded in granule cells from rats with recurrent mossy fiber growth differed in three respects from those recorded in control granule cells: they were much more frequently evoked, a number of them were unusually large, and the NMDA component of the response was generally much more prominent. In contrast to the antidromically evoked EPSC, the EPSC evoked by stimulation of the perforant path appeared to be unaffected by a prior episode of status epilepticus. These results support the hypothesis that recurrent mossy fiber growth and synapse formation increases the excitatory drive to dentate granule cells and thus facilitates repetitive synchronous discharge. Activation of NMDA receptors in the recurrent pathway may contribute to seizure propagation under depolarizing conditions. Mossy fiber-granule cell synapses also are present in normal rats, where they may contribute to repetitive granule cell discharge in regions of the dentate gyrus where their numbers are significant. (+info)
Inhibition of dentate granule cell neurogenesis with brain irradiation does not prevent seizure-induced mossy fiber synaptic reorganization in the rat.
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Aberrant reorganization of dentate granule cell axons, the mossy fibers, occurs in human temporal lobe epilepsy and rodent epilepsy models. Whether this plasticity results from the remodeling of preexisting mossy fibers or instead reflects an abnormality of developing dentate granule cells is unknown. Because these neurons continue to be generated in the adult rodent and their production increases after seizures, mossy fibers that arise from either developing or mature granule cells are potential substrates for this network plasticity. Therefore, to determine whether seizure-induced, mossy fiber synaptic reorganization arises from either developing or mature granule cell populations, we used low-dose, whole-brain x-irradiation to eliminate proliferating dentate granule cell progenitors in adult rats. A single dose of 5 Gy irradiation blocked cell proliferation and eliminated putative progenitor cells in the dentate subgranular proliferative zone. Irradiation 1 d before pilocarpine-induced status epilepticus significantly attenuated dentate granule cell neurogenesis after seizures. Two irradiations, 1 d before and 4 d after status epilepticus, essentially abolished dentate granule cell neurogenesis but failed to prevent mossy fiber reorganization in the dentate molecular layer. These results indicate that dentate granule cell neurogenesis in the mature hippocampal formation is vulnerable to the effects of low-dose ionizing irradiation. Furthermore, the development of aberrant mossy fiber remodeling in the absence of neurogenesis suggests that mature dentate granule cells contribute substantially to seizure-induced network reorganization. (+info)
Immunohistochemical evidence of seizure-induced activation of trk receptors in the mossy fiber pathway of adult rat hippocampus.
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Recent work suggests that limiting the activation of the trkB subtype of neurotrophin receptor inhibits epileptogenesis, but whether or where neurotrophin receptor activation occurs during epileptogenesis is unclear. Because the activation of trk receptors involves the phosphorylation of specific tyrosine residues, the availability of antibodies that selectively recognize the phosphorylated form of trk receptors permits a histochemical assessment of trk receptor activation. In this study the anatomy and time course of trk receptor activation during epileptogenesis were assessed with immunohistochemistry, using a phospho-specific trk antibody. In contrast to the low level of phosphotrk immunoreactivity constitutively expressed in the hippocampus of adult rats, a striking induction of phosphotrk immunoreactivity was evident in the distribution of the mossy fibers after partial kindling or kainate-induced seizures. The anatomic distribution, time course, and threshold for seizure-induced phosphotrk immunoreactivity correspond to the demonstrated pattern of regulation of BDNF expression by seizure activity. These results provide immunohistochemical evidence that trk receptors undergo activation during epileptogenesis and suggest that the mossy fiber pathway is particularly important in the pro-epileptogenic effects of the neurotrophins. (+info)
Selective disruption of "late onset" sagittal banding patterns by ectopic expression of engrailed-2 in cerebellar Purkinje cells.
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To explore the role of Engrailed proteins in development of the cerebellum, Engrailed-2 (En-2) was ectopically expressed in cerebellar Purkinje cells from the late embryonic stage into adulthood. The fundamental organization of Purkinje cell sagittal zones as revealed by the "early onset" markers L7-beta-gal and cadherin-8 was found to be virtually identical to that in wild type. In contrast, "late onset" sagittal banding patterns revealed by Purkinje cell markers zebrin I, zebrin II, and 9-O-acetyl GD3 Ganglioside (P-Path), and the granule cell marker NADPH-diaphorase, were disrupted. In general, although some evidence of banding was still detectable, boundaries defined by the latter markers were poorly defined, and the patterns overall took on a diffuse appearance. In parallel with the changes in late onset markers, anterograde tracing of spinocerebellar axons revealed a general diffusion of the mossy fiber projection pattern in lobule VIII and the anterior lobe. These observations suggest that at least two separate mediolateral boundary systems exist in the cerebellum, and these are differentially affected by ectopic En-2 expression. Alternatively, one boundary system exists that remains primarily intact in the mutant, but recognition of this system by a set of late developmental events is perturbed. (+info)
Actions of brain-derived neurotrophic factor in slices from rats with spontaneous seizures and mossy fiber sprouting in the dentate gyrus.
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This study examined the acute actions of brain-derived neurotrophic factor (BDNF) in the rat dentate gyrus after seizures, because previous studies have shown that BDNF has acute effects on dentate granule cell synaptic transmission, and other studies have demonstrated that BDNF expression increases in granule cells after seizures. Pilocarpine-treated rats were studied because they not only have seizures and increased BDNF expression in granule cells, but they also have reorganization of granule cell "mossy fiber" axons. This reorganization, referred to as "sprouting," involves collaterals that grow into novel areas, i.e., the inner molecular layer, where granule cell and interneuron dendrites are located. Thus, this animal model allowed us to address the effects of BDNF in the dentate gyrus after seizures, as well as the actions of BDNF on mossy fiber transmission after reorganization. In slices with sprouting, BDNF bath application enhanced responses recorded in the inner molecular layer to mossy fiber stimulation. Spontaneous bursts of granule cells occurred, and these were apparently generated at the site of the sprouted axon plexus. These effects were not accompanied by major changes in perforant path-evoked responses or paired-pulse inhibition, occurred only after prolonged (30-60 min) exposure to BDNF, and were blocked by K252a. The results suggest a preferential action of BDNF at mossy fiber synapses, even after substantial changes in the dentate gyrus network. Moreover, the results suggest that activation of trkB receptors could contribute to the hyperexcitability observed in animals with sprouting. Because human granule cells also express increased BDNF mRNA after seizures, and sprouting can occur in temporal lobe epileptics, the results may have implications for understanding temporal lobe epilepsy. (+info)
Dual mechanism for presynaptic modulation by axonal metabotropic glutamate receptor at the mouse mossy fibre-CA3 synapse.
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1. To investigate mechanisms responsible for the presynaptic inhibitory action mediated by the axonal group II metabotropic glutamate receptor (mGluR) at the mossy fibre-CA3 synapse, we used a quantitative fluorescence measurement of presynaptic Ca2+ in mouse hippocampal slices. 2. Bath application of the group II mGluR-specific agonist (2S,1'R,2'R,3'R)-2-(2, 3-dicarboxycyclopropyl)glycine (DCG-IV, 1 microM) reversibly suppressed the presynaptic Ca2+ influx (to 55.2 +/- 4.6 % of control, n = 5) as well as field EPSPs recorded simultaneously (to 3.1 +/- 2.0%). Presynaptic fibre volley was not affected by 1 microM DCG-IV. 3. A quantitative analysis of the inhibition of presynaptic Ca2+ influx and field EPSP suggested that DCG-IV suppressed the field EPSP to a greater extent than would be expected if the suppression were solely due to a decrease in the presynaptic Ca2+ influx. 4. DCG-IV at 1 microM suppressed the mean frequency (to 73.8 +/- 3.9% of control, n = 11), but not the mean amplitude (to 97.0 +/- 3.5%), of miniature EPSCs recorded from CA3 neurones using the whole-cell patch-clamp technique. 5. These results suggest that group II mGluR-mediated suppression is due both to a reduction of presynaptic Ca2+ influx and downregulation of the subsequent exocytotic machinery. (+info)