Stabilization of spine Synaptopodin by mGluR1 is required for mGluR-LTD

Dendritic spines, actin-rich protrusions forming the postsynaptic sites of excitatory synapses, undergo activity-dependent molecular and structural remodeling. Activation of group 1 metabotropic glutamate receptors - mGluR1 and mGluR5 - by synaptic or pharmacological stimulation, induces LTD but whether this is accompanied with spine elimination remains unresolved. A subset of telencephalic mushroom spines contains the spine apparatus (SA), an enigmatic organelle composed of stacks of smooth endoplasmic reticulum, whose formation depends on the expression of the actin-bundling protein Synaptopodin. Allocation of Synaptopodin to spines appears governed by cell-intrinsic mechanisms as the relative frequency of spines harboring Synaptopodin is conserved in vivo and in vitro. Here we show that expression of Synaptopodin/SA in spines is required for induction of mGluR-LTD at Schaffer collateral-CA1 synapses. Post-mGluR-LTD, mushroom spines lacking Synaptopodin/SA are selectively lost whereas spines harboring it are preserved, a process dependent on activation of mGluR1 but not mGluR5. Mechanistically, we find that mGluR1 supports physical retention of Synaptopodin within excitatory spine synapses during LTD while triggering lysosome-dependent degradation of the protein residing in dendritic shafts. Together, these results reveal a cellular mechanism, dependent on mGluR1, which enables selective preservation of stronger spines containing Synaptopodin/SA while eliminating weaker ones and potentially countering spurious strengthening by de novo recruitment of Synaptopodin. Overall our results identify spines with Synaptopodin/SA as the locus of mGluR-LTD and underscore the importance of the molecular microanatomy of spines in synaptic plasticity.

Prenatal treatment with rapamycin restores enhanced hippocampal mGluR-LTD and mushroom spine size in a Down’s syndrome mouse model

Down syndrome (DS) is the most frequent genetic cause of intellectual disability including hippocampal-dependent memory deficits. We have previously reported hippocampal mTOR (mammalian target of rapamycin) hyperactivation, and related plasticity as well as memory deficits in Ts1Cje mice, a DS experimental model. Here we characterize the proteome of hippocampal synaptoneurosomes (SNs) from these mice, and found a predicted alteration of synaptic plasticity pathways, including long term depression (LTD). Accordingly, mGluR-LTD (metabotropic Glutamate Receptor-LTD) is enhanced in the hippocampus of Ts1Cje mice and this is correlated with an increased proportion of a particular category of mushroom spines in hippocampal pyramidal neurons. Remarkably, prenatal treatment of these mice with rapamycin has a positive pharmacological effect on both phenotypes, supporting the therapeutic potential of rapamycin/rapalogs for DS intellectual disability.

Sex differences in dendritic spine density and morphology in auditory and visual cortices in adolescence and adulthood

Dendritic spines are small protrusions on dendrites that endow neurons with the ability to receive and transform synaptic input. Dendritic spine number and morphology are altered as a consequence of synaptic plasticity and circuit refinement during adolescence. Dendritic spine density (DSD) is significantly different based on sex in subcortical brain regions associated with the generation of sex-specific behaviors. It is largely unknown if sex differences in DSD exist in auditory and visual brain regions and if there are sex-specific changes in DSD in these regions that occur during adolescent development. We analyzed dendritic spines in 4-week-old (P28) and 12-week-old (P84) male and female mice and found that DSD is lower in female mice due in part to fewer short stubby, long stubby and short mushroom spines. We found striking layer-specific patterns including a significant age by layer interaction and significantly decreased DSD in layer 4 from P28 to P84. Together these data support the possibility of developmental sex differences in DSD in visual and auditory regions and provide evidence of layer-specific refinement of DSD over adolescent brain development.

Microglial depletion disrupts normal functional development of adult-born neurons in the olfactory bulb

Microglia play key roles in regulating synapse development and refinement in the developing brain, but it is unknown whether they are similarly involved during adult neurogenesis. By transiently depleting microglia from the healthy adult mouse brain, we show that microglia are necessary for the normal functional development of adult-born granule cells (abGCs) in the olfactory bulb. Microglial depletion reduces the odor responses of developing, but not preexisting GCs in vivo in both awake and anesthetized mice. Microglia preferentially target their motile processes to interact with mushroom spines on abGCs, and when microglia are absent, abGCs develop smaller spines and receive weaker excitatory synaptic inputs. These results suggest that microglia promote the development of excitatory synapses onto developing abGCs, which may impact the function of these cells in the olfactory circuit.

Impact of JNK and Its Substrates on Dendritic Spine Morphology

The protein kinase JNK1 exhibits high activity in the developing brain, where it regulates dendrite morphology through the phosphorylation of cytoskeletal regulatory proteins. JNK1 also phosphorylates dendritic spine proteins, and Jnk1-/- mice display a long-term depression deficit. Whether JNK1 or other JNKs regulate spine morphology is thus of interest. Here, we characterize dendritic spine morphology in hippocampus of mice lacking Jnk1-/- using Lucifer yellow labelling. We find that mushroom spines decrease and thin spines increase in apical dendrites of CA3 pyramidal neurons with no spine changes in basal dendrites or in CA1. Consistent with this spine deficit, Jnk1-/- mice display impaired acquisition learning in the Morris water maze. In hippocampal cultures, we show that cytosolic but not nuclear JNK, regulates spine morphology and expression of phosphomimicry variants of JNK substrates doublecortin (DCX) or myristoylated alanine-rich C kinase substrate-like protein-1 (MARCKSL1), rescue mushroom, thin, and stubby spines differentially. These data suggest that physiologically active JNK controls the equilibrium between mushroom, thin, and stubby spines via phosphorylation of distinct substrates.

Sex-specific Difference of Hippocampal Synaptic Plasticity in Response to Sex Neurosteroids

Numerous studies provide increasing evidence, which supports the ideas that every cell in the brain of males may differ from those in females due to differences in sex chromosome complement as well as in response to hormonal effects. In this study, we address the question as to whether actions of neurosteroids, thus steroids, which are synthesized and function within the brain, contribute to sex-specific hippocampal synaptic plasticity. We have previously shown that predominantly in the female hippocampus, does inhibition of the conversion of testosterone to estradiol affect synaptic transmission. In this study, we show that testosterone and its metabolite dihydrotestosterone are essential for hippocampal synaptic transmission specifically in males. This also holds true for the density of mushroom spines and of spine synapses. We obtained similar sex-dependent results using primary hippocampal cultures of male and female animals. Since these cultures originated from perinatal animals, our findings argue for sex-dependent differentiation of hippocampal neurons regarding their responsiveness to sex neurosteroids up to birth, which persist during adulthood. Hence, our in vitro findings may point to a developmental effect either directly induced by sex chromosomes or indirectly by fetal testosterone secretion during the perinatal critical period, when developmental sexual priming takes place.

Gamma-protocadherin localization at the synapse corresponds to parameters of synaptic maturation

Clustered protocadherins (Pcdhs) are a large family of ~60 cadherin-like proteins (divided into the subclasses α, β, and γ) that compose a surface “barcode” in individual neurons. The code is generated through combinatorial expression via epigenetic regulation at a large gene cluster that encodes the molecules. During early neural development, Pcdhs were shown to mediate dendrite self-avoidance in some neuronal types through a still uncharacterized anti-adhesive mechanism. Pcdhs were also shown to be important for dendritic complexity in cortical neurons likely through a pro-adhesive mechanism. Pcdhs have also been postulated to participate in synaptogenesis and the specificity of connectivity. Some synaptic defects were noted in knockout animals, including synaptic number and physiology, but the role of these molecules in synaptic development is not understood. The effects of Pcdh knockout on dendritic patterning may present a confound to studying synaptogenesis. We have shown previously in vivo and in cultures that Pcdh-γs are highly enriched in intracellular compartments located in dendrites and spines with localization at only a few synaptic clefts. To gain insight into how Pcdh-γs might affect synapses, we compared synapses that harbored endogenous Pcdh-γs versus those that did not for parameters of synaptic maturation including pre- and postsynaptic size, postsynaptic perforations, and spine morphology by light microscopy in cultured hippocampal neurons and by serial section immuno-electron microscopy in hippocampal CA1. In mature neurons, synapses immunopositive for Pcdh-γs were found to be larger in diameter with more frequent perforations. Analysis of spines in cultured neurons revealed that mushroom spines were more frequently immunopositive for Pcdh-γs at their tips than thin spines. Taken together, these results suggest that Pcdh-γ function at the synapse may be related to promotion of synaptic maturation and stabilization.

Presenilin 1 Regulates [Ca2+]i and Mitochondria/ER Interaction in Cultured Rat Hippocampal Neurons

Mutations in the presenilin 1 (PS1) gene are a major trigger of familial Alzheimer’s disease (AD), yet the mechanisms affected by mutated PS1 causing cognitive decline are not yet elucidated. In the present study, we compared rat hippocampal neurons in culture, transfected with PS1 or with mutant (M146V) PS1 (mPS1) plasmids in several neuronal functions. Initially, we confirmed earlier observations that mPS1-expressing neurons are endowed with fewer mature “mushroom” spines and more filopodial immature protrusions. The correlation between calcium changes in the cytosol, mitochondria, and endoplasmic reticulum (ER) is mitigated in the mPS1 neurons, tested by the response to an abrupt increase in ambient [Ca2+]o; cytosolic [Ca2+]i is higher in the mPS1 neurons but mitochondrial [Ca2+] is lower than in control neurons. Strikingly, mPS1-transfected neurons express higher excitability and eventual lower survival rate when exposed to the oxidative stressor, paraquat. These results highlight an impaired calcium regulation in mPS1 neurons, resulting in a reduced ability to handle oxidative stress, which may lead to cell death and AD.

Microglia depletion disrupts normal functional development of adult-born neurons in the olfactory bulb

Microglia play key roles in regulating synapse development and refinement in the developing brain, but it is unknown whether they are similarly involved during adult neurogenesis. By transiently ablating microglia from the healthy adult mouse brain, we show that microglia are necessary for the normal functional development of adult-born granule cells (abGCs) in the olfactory bulb. Microglia ablation reduces the odor responses of developing, but not preexisting GCs in vivo in both awake and anesthetized mice. Microglia preferentially target their motile processes to interact with mushroom spines on abGCs, and when microglia are absent, abGCs develop smaller spines and receive weaker excitatory synaptic inputs. These results suggest that microglia promote the development of excitatory synapses onto developing abGCs, which may impact the function of these cells in the olfactory circuit.