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 processin

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

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

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

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

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