Myosin V regulates synaptopodin clustering and localization in the dendrites of hippocampal neurons.

The spine apparatus (SA) is an endoplasmic reticulum-related organelle that is present in a subset of dendritic spines in cortical and pyramidal neurons, and plays an important role in Ca2+ homeostasis and dendritic spine plasticity. The protein synaptopodin is essential for the formation of the SA and is widely used as a maker for this organelle. However, it is still unclear which factors contribute to its localization at selected synapses, and how it triggers local SA formation. In this study, we characterized development, localization and mobility of synaptopodin clusters in hippocampal primary neurons, as well as the molecular dynamics within these clusters. Interestingly, synaptopodin at the shaft-associated clusters is less dynamic than at spinous clusters. We identify the actin-based motor proteins myosin V (herein referring to both the myosin Va and Vb forms) and VI as novel interaction partners of synaptopodin, and demonstrate that myosin V is important for the formation and/or maintenance of the SA. We found no evidence of active microtubule-based transport of synaptopodin. Instead, new clusters emerge inside spines, which we interpret as the SA being assembled on-site.

Selective Inactivation of Reelin in Inhibitory Interneurons Leads to Subtle Changes in the Dentate Gyrus But Leaves Cortical Layering and Behavior Unaffected.

Reelin is an extracellular matrix protein, known for its dual role in neuronal migration during brain development and in synaptic plasticity at adult stages. During the perinatal phase, Reelin expression switches from Cajal-Retzius (CR) cells, its main source before birth, to inhibitory interneurons (IN), the main source of Reelin in the adult forebrain. IN-derived Reelin has been associated with schizophrenia and temporal lobe epilepsy; however, the functional role of Reelin from INs is presently unclear. In this study, we used conditional knockout mice, which lack Reelin expression specifically in inhibitory INs, leading to a substantial reduction in total Reelin expression in the neocortex and dentate gyrus. Our results show that IN-specific Reelin knockout mice exhibit normal neuronal layering and normal behavior, including spatial reference memory. Although INs are the major source of Reelin within the adult stem cell niche, Reelin from INs does not contribute substantially to normal adult neurogenesis. While a closer look at the dentate gyrus revealed some unexpected alterations at the cellular level, including an increase in the number of Reelin expressing CR cells, overall our data suggest that Reelin derived from INs is less critical for cortex development and function than Reelin expressed by CR cells.

Loss of tumor necrosis factor (TNF)-receptor 1 and TNF-receptor 2 partially replicate effects of TNF deficiency on dendritic spines of granule cells in mouse dentate gyrus.

The cytokine tumor necrosis factor (TNF) is involved in the regulation of physiological and pathophysiological processes in the central nervous system. In previous work, we showed that mice lacking constitutive levels of TNF exhibit a reduction in spine density and changes in spine head size distribution of dentate granule cells. Here, we investigated which TNF-receptor pathway is responsible for this phenotype and analyzed granule cell spine morphology in TNF-R1-, TNF-R2-, and TNF-R1/R2-deficient mice. Single granule cells were filled with Alexa568 in fixed hippocampal brain slices and immunostained for the actin-modulating protein synaptopodin (SP), a marker for strong and stable spines. An investigator blind to genotype investigated dendritic spines using deconvolved confocal image stacks. Similar to TNF-deficient mice, TNF-R1 and TNF-R2 mutants showed a decrease in the size of small spines (SP-negative) with TNF-R1/R2-KO mice exhibiting an additive effect. TNF-R1 mutants also showed an increase in the size of large spines (SP-positive), mirroring the situation in TNF-deficient mice. Unlike the TNF-deficient mouse, none of the TNF-R mutants exhibited a reduction in their granule cell spine densities. Since TNF tunes the excitability of networks, lack of constitutive TNF reduces network excitation. This may explain why we observed alterations in spine head size distributions in TNF- and TNF-R-deficient granule cells. The changes in spine density observed in the TNF-deficient mouse could not be linked to canonical TNF-R-signaling. Instead, noncanonical pathways or unknown developmental functions of TNF may cause this phenomenon.

Constitutive tumor necrosis factor (TNF)-deficiency causes a reduction in spine density in mouse dentate granule cells accompanied by homeostatic adaptations of spine head size.

The majority of excitatory synapses terminating on cortical neurons are found on dendritic spines. The geometry of spines, in particular the size of the spine head, tightly correlates with the strength of the excitatory synapse formed with the spine. Under conditions of synaptic plasticity, spine geometry may change, reflecting functional adaptations. Since the cytokine tumor necrosis factor (TNF) has been shown to influence synaptic transmission as well as Hebbian and homeostatic forms of synaptic plasticity, we speculated that TNF-deficiency may cause concomitant structural changes at the level of dendritic spines. To address this question, we analyzed spine density and spine head area of Alexa568-filled granule cells in the dentate gyrus of adult C57BL/6J and TNF-deficient (TNF-KO) mice. Tissue sections were double-stained for the actin-modulating and plasticity-related protein synaptopodin (SP), a molecular marker for strong and stable spines. Dendritic segments of TNF-deficient granule cells exhibited ∼20% fewer spines in the outer molecular layer of the dentate gyrus compared to controls, indicating a reduced afferent innervation. Of note, these segments also had larger spines containing larger SP-clusters. This pattern of changes is strikingly similar to the one seen after denervation-associated spine loss following experimental entorhinal denervation of granule cells: Denervated granule cells increase the SP-content and strength of their remaining spines to homeostatically compensate for those that were lost. Our data suggest a similar compensatory mechanism in TNF-deficient granule cells in response to a reduction in their afferent innervation.

Proper layering is important for precisely timed activation of hippocampal mossy cells.

The mammalian cortex exhibits a laminated structure that may underlie optimal synaptic connectivity and support temporally precise activation of neurons. In 'reeler' mice, the lack of the extracellular matrix protein Reelin leads to abnormal positioning of cortical neurons and disrupted layering. To address how these structural changes impact neuronal function, we combined electrophysiological and neuroanatomical techniques to investigate the synaptic activation of hippocampal mossy cells (MCs), the cell type that integrates the output of dentate gyrus granule cells (GCs). While somatodendritic domains of wild-type (WT) MCs were confined to the hilus, the somata and dendrites of reeler MCs were often found in the molecular layer, where the perforant path (PP) terminates. Most reeler MCs received aberrant monosynaptic excitatory input from the PP, whereas the disynaptic input to MCs via GCs was decreased and inhibition was increased. In contrast to the uniform disynaptic discharge of WT MCs, many reeler cells discharged with short, monosynaptic latencies, while others fired with long latencies over a broad temporal window in response to PP activation. Thus, disturbed lamination results in aberrant synaptic connectivity and altered timing of action potential generation. These results highlight the importance of a layered cortical structure for information processing.

Crossed Entorhino-Dentate Projections Form and Terminate With Correct Layer-Specificity in Organotypic Slice Cultures of the Mouse Hippocampus.

The entorhino-dentate projection, i.e., the perforant pathway, terminates in a highly ordered and laminated fashion in the rodent dentate gyrus (DG): fibers arising from the medial entorhinal cortex (MEC) terminate in the middle molecular layer, whereas fibers arising from the lateral entorhinal cortex (LEC) terminate in the outer molecular layer of the DG. In rats and rabbits, a crossed entorhino-dentate projection exists, which originates from the entorhinal cortex (EC) and terminates in the contralateral DG. In contrast, in mice, such a crossed projection is reportedly absent. Using single and double mouse organotypic entorhino-hippocampal slice cultures, we studied the ipsi- and crossed entorhino-dentate projections. Viral tracing revealed that entorhino-dentate projections terminate with a high degree of lamina-specificity in single as well as in double cultures. Furthermore, in double cultures, entorhinal axons arising from one slice freely intermingled with entorhinal axons originating from the other slice. In single as well as in double cultures, entorhinal axons exhibited a correct topographical projection to the DG: medial entorhinal axons terminated in the middle and lateral entorhinal axons terminated in the outer molecular layer. Finally, entorhinal neurons were virally transduced with Channelrhodopsin2-YFP and stimulated with light, revealing functional connections between the EC and dentate granule cells. We conclude from our findings that entorhino-dentate projections form bilaterally in the mouse hippocampus in vitro and that the mouse DG provides a permissive environment for crossed entorhinal fibers.

The actin-modulating protein synaptopodin mediates long-term survival of dendritic spines.

Large spines are stable and important for memory trace formation. The majority of large spines also contains synaptopodin (SP), an actin-modulating and plasticity-related protein. Since SP stabilizes F-actin, we speculated that the presence of SP within large spines could explain their long lifetime. Indeed, using 2-photon time-lapse imaging of SP-transgenic granule cells in mouse organotypic tissue cultures we found that spines containing SP survived considerably longer than spines of equal size without SP. Of note, SP-positive (SP+) spines that underwent pruning first lost SP before disappearing. Whereas the survival time courses of SP+ spines followed conditional two-stage decay functions, SP-negative (SP-) spines and all spines of SP-deficient animals showed single-phase exponential decays. This was also the case following afferent denervation. These results implicate SP as a major regulator of long-term spine stability: SP clusters stabilize spines, and the presence of SP indicates spines of high stability.

A role for synaptopodin and the spine apparatus in hippocampal synaptic plasticity.

Spines are considered sites of synaptic plasticity in the brain and are capable of remodeling their shape and size. A molecule thathas been implicated in spine plasticity is the actin-associated protein synaptopodin. This article will review a series of studies aimed at elucidating the role of synaptopodin in the rodent brain. First, the developmental expression of synaptopodin mRNA and protein were studied; secondly, the subcellular localization of synaptopodin in hippocampal principal neurons was analyzed using confocal microscopy as well as electron microscopy and immunogold labelling; and, finally, the functional role of synaptopodin was investigated using a synaptopodin-deficient mouse. The results of these studies are: (1) synaptopodin expression byhippocampal principal neurons develops during the first postnatal weeks and increases in parallel with the maturation of spines in the hippocampus. (2) Synaptopodin is sorted to the spine compartment, where it is tightly associated with the spine apparatus, an enigmatic organelle believed to be involved in calcium storage or local protein synthesis. (3) Synaptopodin-deficient mice generated by gene targeting are viable but lack the spine apparatus organelle. These mice show deficitsin synaptic plasticity as well as impaired learning and memory. Taken together, these data implicate synaptopodin and the spine apparatus in the regulation of synaptic plasticity in the hippocampus. Future studies will be aimed at finding the molecular link between synaptopodin, the spine apparatus organelle, and synaptic plasticity.

Abnormal positioning of granule cells alters afferent fiber distribution in the mouse fascia dentata: morphologic evidence from reeler, apolipoprotein E receptor 2-, and very low density lipoprotein receptor knockout mice.

The fascia dentata of the hippocampal formation is characterized by the nonoverlapping and lamina-specific termination of afferent fibers: entorhinal fibers terminate in the outer molecular layer and commissural/associational fibers terminate in the inner molecular layer. It has been proposed that this fiber lamination depends on the presence of the correct postsynaptic partner at the time of fiber ingrowth during development. Pioneer neurons that guide afferent fibers to their correct layers as well as signals located on granule cells have both been implicated. To study the role of granule cells for the lamina-specific ingrowth of afferents, the cyto- and fiberarchitecture of three mouse mutants (very low density lipoprotein receptor knockout mouse, apolipoprotein E receptor 2 knockout mouse, and reeler mouse) that show different degrees of granule cell migration defects were analyzed. Anterograde tracing with Phaseolus vulgaris-leucoagglutinin was used to visualize the afferent fiber systems, and immunohistochemistry was used to determine the position of their putative target cells. In controls, granule cells are packed in a single layer. This laminar organization is mildly altered in very low density lipoprotein receptor knockout mice, moderately disturbed in apolipoprotein E receptor 2 knockout mice, and severely disrupted in reeler mice. These changes in granule cell distribution are mirrored by the distribution of commissural fibers. In contrast, changes in granule cell distribution do not severely affect the laminar termination of entorhinal fibers. These data provide further evidence for a role of granule cells in the laminar termination of commissural/associational afferents to the fascia dentata.

Laminar distribution of synaptopodin in normal and reeler mouse brain depends on the position of spine-bearing neurons.

Synaptopodin is the first member of a novel class of proline-rich actin-associated proteins. In brain, it is present in the neck of a subset of mature telencephalic spines and is associated closely with the spine apparatus, a Ca(2+) storing organelle within the spine compartment. The characteristic region- and lamina-specific distribution of synaptopodin in rat brain suggested that the distribution pattern of synaptopodin depends on the cytoarchitectonic arrangement of spine-bearing neurons. To test this hypothesis, synaptopodin was studied in the cortex, striatum, and hippocampus of normal and reeler mice, in which developmental cell migration defects have disrupted the normal array of cells. In situ hybridization histochemistry as well as light- and electron microscopic immunocytochemistry were used. In brain of normal mice, the pattern of synaptopodin mRNA-expressing cells corresponds to that of spine-bearing neurons and synaptopodin protein is found in a region- and lamina-specific distribution pattern. It is specifically sorted to the spine neck where it is associated closely with the spine apparatus. In brain of reeler mice, the pattern of synaptopodin mRNA-expressing cells corresponds to that of the abnormally positioned spine-bearing neurons and the region- and lamina-specific distribution pattern is absent or altered. Nevertheless, synaptopodin was specifically sorted to the spine neck, as in controls. These data demonstrate that the light microscopic distribution pattern of synaptopodin protein depends on the position and orientation of the spine-bearing neurons. The intracellular sorting process, however, is independent of positional cues.

New ways of looking at synapses.

Current concepts of synaptic fine-structure are derived from electron microscopic studies of tissue fixed by chemical fixation using aldehydes. However, chemical fixation with glutaraldehyde and paraformaldehyde and subsequent dehydration in ethanol result in uncontrolled tissue shrinkage. While electron microscopy allows for the unequivocal identification of synaptic contacts, it cannot be used for real-time analysis of structural changes at synapses. For the latter purpose advanced fluorescence microscopy techniques are to be applied which, however, do not allow for the identification of synaptic contacts. Here, two approaches are described that may overcome, at least in part, some of these drawbacks in the study of synapses. By focusing on a characteristic, easily identifiable synapse, the mossy fiber synapse in the hippocampus, we first describe high-pressure freezing of fresh tissue as a method that may be applied to study subtle changes in synaptic ultrastructure associated with functional synaptic plasticity. Next, we propose to label presynaptic mossy fiber terminals and postsynaptic complex spines on CA3 pyramidal neurons by different fluorescent dyes to allow for the real-time monitoring of these synapses in living tissue over extended periods of time. We expect these approaches to lead to new insights into the structure and function of central synapses.

Synaptopodin-deficient mice lack a spine apparatus and show deficits in synaptic plasticity.

The spine apparatus is a cellular organelle that is present in many dendritic spines of excitatory neurons in the mammalian forebrain. Despite its discovery >40 years ago, the function of the spine apparatus is still unknown although calcium buffering functions as well as roles in synaptic plasticity have been proposed. We have recently shown that the 100-kDa protein synaptopodin is associated with the spine apparatus. Here, we now report that mice homozygous for a targeted deletion of the synaptopodin gene completely lack spine apparatuses. Interestingly, this absence of the spine apparatus is accompanied by a reduction in hippocampal long-term potentiation (LTP) in the CA1 region of the hippocampus and by an impairment of spatial learning in the radial arm maze test. This genetic analysis points to a role of the spine apparatus in synaptic plasticity.

Dentate granule cells in reeler mutants and VLDLR and ApoER2 knockout mice.

We have studied the organization and cellular differentiation of dentate granule cells and their axons, the mossy fibers, in reeler mutant mice lacking reelin and in mutants lacking the reelin receptors very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). We show that granule cells in reeler mice do not form a densely packed granular layer, but are loosely distributed throughout the hilar region. Immunolabeling for calbindin and calretinin revealed that the sharp border between dentate granule cells and hilar mossy cells is completely lost in reeler mice. ApoER2/VLDLR double-knockout mice copy the reeler phenotype. Mice deficient only in VLDLR showed minor alterations of dentate organization; migration defects were more prominent in ApoER2 knockout mice. Tracing of the mossy fibers with Phaseolus vulgaris leukoagglutinin and calbindin immunolabeling revealed an irregular broad projection in reeler mice and ApoER2/VLDLR double knockouts, likely caused by the irregular wide distribution of granule cell somata. Mutants lacking only one of the lipoprotein receptors showed only minor changes in the mossy fiber projection. In all mutants, mossy fibers respected the CA3-CA1 border. Retrograde labeling with DiI showed that malpositioned granule cells also projected as normal to the CA3 region. These results indicate that ( 1 ) reelin signaling via ApoER2 and VLDLR is required for the normal positioning of dentate granule cells and (2) the reelin signaling pathway is not involved in pathfinding and target recognition of granule cell axons.