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.

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.

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 time-lapse 2-photon-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 spines that underwent pruning first lost SP before disappearing. Whereas the survival time courses of SP-positive (SP+) spines followed conditional two-phase decay functions, SP-negative (SP-) spines and all spines of SP-deficient animals showed single exponential decays. 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.

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 TNFdeficient 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.

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