Distance-dependent differences in synapse number and AMPA receptor expression in hippocampal CA1 pyramidal neurons.

The ability of synapses throughout the dendritic tree to influence neuronal output is crucial for information processing in the brain. Synaptic potentials attenuate dramatically, however, as they propagate along dendrites toward the soma. To examine whether excitatory axospinous synapses on CA1 pyramidal neurons compensate for their distance from the soma to counteract such dendritic filtering, we evaluated axospinous synapse number and receptor expression in three progressively distal regions: proximal and distal stratum radiatum (SR), and stratum lacunosum-moleculare (SLM). We found that the proportion of perforated synapses increases as a function of distance from the soma and that their AMPAR, but not NMDAR, expression is highest in distal SR and lowest in SLM. Computational models of pyramidal neurons derived from these results suggest that they arise from the compartment-specific use of conductance scaling in SR and dendritic spikes in SLM to minimize the influence of distance on synaptic efficacy.

Contacts between medial and lateral perforant pathway fibers and parvalbumin expressing neurons in the subiculum of the rat.

The entorhinal cortex (EC) projects via the perforant pathway to all subfields in the hippocampal formation. One can distinguish medial and lateral components in the pathway, originating in corresponding medial and lateral subdivisions of EC. We analyzed the innervation by medial and lateral perforant pathway fibers of parvalbumin-expressing neurons in the subiculum. A neuroanatomical tracer (biotinylated dextran amine, BDA) was stereotaxically injected in the medial or lateral entorhinal cortex, thus selectively labeling either perforant pathway component. Transport was allowed for 1 week. Transported BDA was detected with streptavidin-Alexa Fluor 488. Parvalbumin neurons were visualized via immunofluorescence histochemistry, using the fluorochrome Alexa Fluor 594. Via a random systematic sampling scheme using a two-channel, sequential-mode confocal laser scanning procedure, we obtained image series at high magnification from the molecular layer of the subiculum. Labeled entorhinal fibers and parvalbumin-expressing structures were three dimensionally (3D) reconstructed using computer software. Further computer analysis revealed that approximately 16% of the 3D objects ('boutons') of BDA-labeled fibers was engaged in contacts with parvalbumin-immunostained dendrites in the subiculum. Both medial and lateral perforant pathway fibers and their boutons formed such appositions. Contacts are suggestive for synapses. We found no significant differences between the medial and lateral components in the relative numbers of contacts. Thus, the medial and lateral subdivisions of the entorhinal cortex similarly tune the firing of principal neurons in the subiculum by way of parvalbumin positive interneurons in their respective terminal zones.

Autophagy mediates the degradation of synaptic vesicles: A potential mechanism of synaptic plasticity injury induced by microwave exposure in rats.

To explore how autophagy changes and whether autophagy is involved in the pathophysiological process of synaptic plasticity injury caused by microwave radiation, we established a 30 mW/cm2 microwave-exposure in vivo model, which caused reversible injuries in rat neurons. Microwave radiation induced cognitive impairment in rats and synaptic plasticity injury in rat hippocampal neurons. Autophagy in rat hippocampal neurons was activated following microwave exposure. Additionally, we observed that synaptic vesicles were encapsulated by autophagosomes, a phenomenon more evident in the microwave-exposed group. Colocation of autophagosomes and synaptic vesicles in rat hippocampal neurons increased following microwave exposure. CONCLUSION: microwave exposure led to the activation of autophagy in rat hippocampal neurons, and excessive activation of autophagy might damage synaptic plasticity by mediating synaptic vesicle degradation.

Plasticity of synaptopodin and the spine apparatus organelle in the rat fascia dentata following entorhinal cortex lesion.

Synaptopodin is an actin-associated molecule essential for the formation of a spine apparatus in telencephalic spines. To study whether synaptopodin and the spine apparatus organelle are regulated under conditions of lesion-induced plasticity, synaptopodin and the spine apparatus were analyzed in granule cells of the rat fascia dentata following entorhinal denervation. Confocal microscopy was employed to quantify layer-specific changes in synaptopodin-immunoreactive puncta densities. Electron microscopy was used to quantify layer-specific changes in spine apparatus organelles. Within the denervated middle and outer molecular layers, the layers of deafferentation-induced spine loss, synaptogenesis, and spinogenesis, the density of synaptopodin puncta and the number of spine apparatuses decreased by 4 days postlesion and slowly recovered in parallel with spinogenesis by 180 days postlesion. Within the nondenervated inner molecular layer, the zone without deafferentation-induced spine loss, a rapid loss of synaptopodin puncta and spine apparatuses was also observed. In this layer, spine apparatus densities recovered by 14 days postlesion, in parallel with plastic remodeling at the synaptic level and the postlesional recovery of granule cell activity. These data demonstrate layer-specific changes in the distribution of synaptopodin and the spine apparatus organelle following partial denervation of granule cells: in the layer of spine loss, spine apparatus densities follow spine densities; in the layer of spine maintenance, however, spine apparatus densities appear to be regulated by other signals.

The transient receptor potential vanilloid-1 is localized at excitatory synapses in the mouse dentate gyrus.

The transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel that plays an important role in pain perception and modulates neurotransmitter release and synaptic plasticity in the brain. TRPV1 function must lay on its anatomical distribution in the peripheral and central nervous system regions involved in the physiological roles of the channel. However, the anatomical localization of TRPV1 is well established in the periphery, but in the brain it is a matter of debate. While some studies support the presence of TRPV1 in several brain regions, recent evidences suggest a restricted distribution of the channel in the central nervous system. To investigate to what extent central TRPV1 function stands on a precise brain distribution of the channel, we examined the mouse hippocampal dentate molecular layer (ML) where TRPV1 mediates long-term synaptic plasticity. Using pre-embedding immunocytochemistry for high resolution electron microscopy, we show that TRPV1 immunoparticles are highly concentrated in postsynaptic dendritic spines to asymmetric perforant path synapses in the outer 2/3 of the ML. However, TRPV1 is poorly expressed at the excitatory hilar mossy cell synapses in the inner 1/3 of this layer. Importantly, the TRPV1 pattern distribution disappeared in the ML of TRPV1-knockout mice. Taken together, these findings support the notion of the presence of TRPV1 in a brain region where the channel has been shown to have a functional role, such as the perforant path synapses in the hippocampal dentate ML.

An increased expression of the mGlu5 receptor protein following LTP induction at the perforant path-dentate gyrus synapse in freely moving rats.

The involvement of metabotropic glutamate (mGlu) receptors in the induction of long-term potentiation (LTP) in vivo has been consistently documented. We have investigated whether LTP induction in the dentate gyrus of rats leads to changes in expression of mGlu2/3 or -5 receptor subtypes in the hippocampus. LTP was induced at the medial perforant path-dentate gyrus synapses, and mGlu receptor expression was examined by Western blot or in situ hybridization. An up-regulation of mGlu5 receptors was observed in the hippocampus both 24 and 48 h following LTP induction. This effect was restricted to the dentate gyrus and CA1 region, whereas no changes in mGlu5 receptor protein (but an increase in mRNA levels) were observed in the CA3 region. The increased expression of mGlu5 receptors was directly related to the induction of LTP, because it was not observed when tetanic stimulation was carried out in animals treated with the NMDA receptor antagonist, 2-amino-5-phosphonopentanoate (AP5). Western blot analysis also showed a reduced expression of mGlu2/3 receptors in the whole hippocampus 24 h after LTP induction, indicating that the increased expression of mGlu5 receptors was specific. These data suggest that an up-regulation of mGlu5 receptors is a component of the plastic changes that follow the induction of LTP at the perforant path-dentate gyrus synapse.

Tracing of the entorhinal-hippocampal pathway in vitro.

In vitro tract tracing allowing for continuous observation of the perforant path is a crucial prerequisite for experimental studies on the entorhinal-hippocampal interaction in an organotypic slice culture containing the entorhinal cortex, the perforant path, and the dentate gyrus (OEHSC). We prepared horizontal slices of the temporal entorhinal-hippocampal region of the rat on a vibratome, and the perforant path axons were traced by application of the fluorescent tracer Mini Ruby on the entorhinal cortex. After 2 days in vitro (div), the perforant path became visible in most cultures. Entorhinal neurons and single perforant fibers could be followed to the outer molecular layers of the dentate gyrus by in vitro fluorescence microscopy and it was possible to monitor the perforant path directly over a period of 25 div. Moreover, ultrastructural analysis proved the existence of traced perforant path boutons forming synapses with spines and dendritic shafts in the outer molecular layers of the dentate gyrus. Transsection of the prelabelled perforant path in vitro resulted in anterograde degeneration and subsequent phagocytosis of axonal material by activated microglial cells in the zone of denervation. In conclusion, in vitro tracing demonstrates the maintenance of the entorhinal-hippocampal pathway in OEHSCs and permits monitoring of dynamic changes in the prelabeled perforant path after various lesion paradigms, e.g., transsection or neurotoxin treatment. This approach permits further studies on the efficacy of neuroprotectants, cytokines, and growth factors in the treatment of lesion-induced neuronal degeneration.

Immunoelectron microscopic localization of the neural recognition molecules L1, NCAM, and its isoform NCAM180, the NCAM-associated polysialic acid, beta1 integrin and the extracellular matrix molecule tenascin-R in synapses of the adult rat hippocampus.

We have investigated the possibility that morphologically different excitatory glutamatergic synapses of the "trisynaptic circuit" in the adult rodent hippocampus, which display different types of long-term potentiation (LTP), may express the immunoglobulin superfamily recognition molecules L1 and NCAM, the extracellular matrix molecule tenascin-R, and the extracellular matrix receptor constituent beta1 integrin in a differential manner. The neural cell adhesion molecules L1, NCAM (all three major isoforms), NCAM180 (the largest major isoform with the longest cytoplasmic domain), beta1 integrin, polysialic acid (PSA) associated with NCAM, and tenascin-R were localized by pre-embedding immunostaining procedures in the CA3/CA4 region (mossy fiber synapses) and in the dentate gyrus (spine synapses) of the adult rat hippocampus. Synaptic membranes of mossy fiber synapses where LTP is expressed presynaptically did not show detectable levels of immunoreactivity for any of the molecules/epitopes studied. L1, NCAM, and PSA, but not NCAM180 or beta1 integrin, were detectable on axonal membranes of fasciculating mossy fibers. In contrast to mossy fiber synapses, spine synapses in the outer third of the molecular layer of the dentate gyrus, which display postsynaptic expression mechanisms of LTP, were both immunopositive and immunonegative for NCAM, NCAM180, beta1 integrin, and PSA. Those spine synapses postsynaptically immunoreactive for NCAM or PSA also showed immunoreactivity on their presynaptic membranes. NCAM180 was not detectable presynaptically in spine synapses. L1 could not be found in spine synapses either pre- or postsynaptically. Also, the extracellular matrix molecule tenascin-R was not detectable in synaptic clefts of all synapses tested, but was amply present between fasciculating axons, axon-astrocyte contact areas, and astrocytic gap junctions. Differences in expression of the membrane-bound adhesion molecules at both types of synapses may reflect the different mechanisms for induction and/or maintenance of synaptic plasticity.