Mechanistic target of rapamycin is necessary for changes in dendritic spine morphology associated with long-term potentiation
- Fredrick E. Henry†1, 2, 3,
- William Hockeimer†2, 3,
- Alex Chen1, 2, 3,
- Shreesh P. Mysore4 and
- Michael A. Sutton1, 2, 3, 5Email authorView ORCID ID profile
© The Author(s). 2017
Received: 14 June 2017
Accepted: 18 October 2017
Published: 30 October 2017
Alterations in the strength of excitatory synapses in the hippocampus is believed to serve a vital function in the storage and recall of new information in the mammalian brain. These alterations involve the regulation of both functional and morphological features of dendritic spines, the principal sites of excitatory synaptic contact. New protein synthesis has been implicated extensively in the functional changes observed following long-term potentiation (LTP), and changes to spine morphology have similarly been documented extensively following synaptic potentiation. However, mechanistic links between de novo translation and the structural changes of potentiated spines are less clear. Here, we assess explicitly the potential contribution of new protein translation under control of the mechanistic target of rapamycin (mTOR) to LTP-associated changes in spine morphology. Utilizing genetic and pharmacological manipulations of mTORC1 function in combination with confocal microscopy in live dissociated hippocampal cultures, we demonstrate that chemically-induced LTP (cLTP) requires do novo protein synthesis and intact mTORC1 signaling. We observed a striking diversity in response properties across morphological classes, with mushroom spines displaying a particular sensitivity to altered mTORC1 signaling across varied levels of synaptic activity. Notably, while pharmacological inhibition of mTORC1 signaling significantly diminished glycine-induced changes in spine morphology, transient genetic upregulation of mTORC1 signaling was insufficient to produce spine enlargements on its own. In contrast, genetic upregulation of mTORC1 signaling promoted rapid expansion in spine head diameter when combined with otherwise sub-threshold synaptic stimulation. These results suggest that synaptic activity-derived signaling pathways act in combination with mTORC1-dependent translational control mechanisms to ultimately regulate changes in spine morphology. As several monogenic neurodevelopmental disorders with links to Autism and Intellectual Disability share a common feature of dysregulated mTORC1 signaling, further understanding of the role of this signaling pathway in regulating synapse function and morphology will be essential in the development of novel therapeutic interventions.
Dendritic spines comprise the primary sites of excitatory synaptic contact in the mammalian central nervous system. At mature synapses, these actin-rich protrusions are typically composed of a large head compartment densely packed with proteins of numerous types , and a thin neck region that attaches the head to the dendritic shaft. The high resistance of the neck can significantly boost synaptically-driven depolarization of the associated spine head . The distinct structural characteristics of spines are believed to provide both chemical and electrical compartmentalization of incoming synaptic signals [5, 14].
In mature networks, synaptic connections at dendritic spines can be quite stable, as newly emergent spines generated after motor learning have been shown to persist for months . Yet, individual spines have long been known to be highly dynamic structures . While the distribution of spine size across the dendritic arbor of a single neuron can be quite variable , spine size generally correlates with excitatory synapse strength both in vitro  and in vivo . Though clear mechanistic explanations for this correlation are just beginning to be understood , it is generally accepted that spine head diameter and synapse strength co-vary during the expression of long term potentiation (LTP), for example, because additional volume is required in the spine head to accommodate the insertion of additional AMPA receptors into the postsynaptic density [31, 38, 42].
The molecular mechanisms involved in the regulation of spine size and shape largely involve remodeling of the actin cytoskeleton . Cytoskeletal rearrangement is necessary for the expression of long lasting plasticity at excitatory synapses, as inhibitors of actin polymerization impair LTP in the CA1 region of the hippocampus [25, 26]. The Rho family of small GTPases has been shown to play pivotal roles in the induction and maintenance of altered spine morphology, particularly in the context of long lasting synaptic plasticity . An emerging model of the signaling dynamics involved in spine enlargement during LTP suggests that calcium influx through NMDARs activates CaMKIIα, leading to the subsequent recruitment of multiple RhoGTPases, wherein RhoA is critical for the initial enlargement of spine size and Cdc42 is necessary for sustaining these structural changes over time [29, 39, 40].
In addition to cytoskeletal remodeling, there is also a well-established role for new protein synthesis in the expression of long lasting plasticity at excitatory synapses. While early work focused on the contribution of cell-wide changes in gene expression via altered transcription , more recent evidence has established a role for de novo protein translation operating locally in dendrites during the expression of long term plasticity and memory formation . Despite a clear requirement for actin remodeling as well as new protein synthesis during LTP, relatively little is known about whether these processes influence each other or are otherwise co-regulated for the expression of long lasting changes in synaptic strength.
Insofar as alterations in spine morphology during LTP are indeed bolstered by or are dependent on de novo protein synthesis, it is currently an open question as to the specific signaling pathways that may be involved in linking these processes. Given the previously demonstrated importance of BDNF signaling and protein synthesis in spine enlargement driven by local glutamate uncaging [13, 19, 58], one system of particular interest is the mechanistic target of rapamycin complex 1 (mTORC1) pathway. The mTORC1 pathway is known to be activated by BDNF signaling at excitatory synapses [49, 57] and is a well-characterized regulator of new protein synthesis, operating at the level of translation initiation . mTORC1 signaling is necessary for the induction of LTP in the CA1 region of the hippocampus, where it has been demonstrated to act locally in dendrites to orchestrate the synthesis of new proteins which are crucial for long lasting changes in synaptic strength [8, 56, 61]. mTORC1 has also been shown to play a role in dendritic spine morphology as chronic pharmacological blockade of mTORC1 results in a decrease in spine density in dissociated hippocampal neurons . In addition, animal models which harbor mutations leading to dysregulated mTORC1 signaling display deficits in long term potentiation [11, 53], and commonly exhibit abnormal spine morphology [28, 55]. Collectively, these results suggest that mTORC1 may play an active role in regulating new spine structure, though whether it contributes to morphological changes during long term potentiation remains an open question.
Here, we use a live cell imaging approach to demonstrate a requirement for mTORC1-dependent protein synthesis in the emergence of altered spine morphology after chemically induced LTP. We find that mTORC1 activation is not sufficient for changes in spine morphology, as transient genetic enhancement of mTORC1 activity via overexpression of a constitutively active mutant version of the upstream mTORC1 effector Rheb does not induce increases in spine head volume on its own. However, when paired with a subthreshold dose of glycine, mTORC1 activation results in robust increases in spine volume, suggesting that the combined action of mTORC1 signaling and other synaptically driven signals are required for activity-dependent changes in spine morphology. As dysregulation in spine morphology is a common feature of many neuropsychiatric disorders including autism spectrum disorders (ASD) and schizophrenia , a more precise understanding of the mechanisms that regulate their properties in response to changes in activity will be essential for the development of future therapeutic advances.
Cell culture and transfection
Dissociated postnatal hippocampal neuron cultures, prepared from postnatal day 1–2 rat pups of either sex, were plated at a density of 230–460 mm2 in poly-D-lysine-coated glass bottom Petri dishes (Mattek), as previously described . Cultures were maintained for at least 21 DIV at 37 °C in growth medium [Neurobasal A supplemented with B27 and Glutamax-1 (Invitrogen)] before use. To achieve sparse expression, neurons were transfected with 0.5 μg of total DNA using the Ca2+ phosphate CalPhos Transfection kit (ClonTech) according to the manufacturer’s protocol. Unless otherwise indicated, all experiments were performed 24 h post-transfection.
Under baseline conditions, neurons were incubated in HEPES-buffered saline (HBS) containing (in mM) the following: 119 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 30 Glucose, 10 HEPES, pH 7.4. Pharmacological induction of LTP in cultured hippocampal neurons was achieved via brief (5 min) exposure to a Mg2+ −free HBS solution supplemented with (in mM): 0.4 Glycine (Fisher, Waltham, MA), 0.02 Bicuculline (Tocris), and 0.003 Strychnine (Tocris, Bristol, UK) Neurons were immediately washed with warm HBS after glycine stimulation and imaged.
Neurons were imaged 1–3 days post-transfection. All imaging was performed on an inverted Olympus FV1000 laser-scanning confocal microscope using a Plan-Apochromat 63×/1.4 oil objective with 1× or 2× digital zoom. GFP was excited with the 488 nm line of an argon ion laser and emitted light was typically collected between 500 and 530 nm with a tunable emission filter. Z-stack images of eGFP signal were obtained at 10 min intervals, beginning with a pre-stimulus series of baseline measures, immediately after completion of the 5 min glycine stimulus, then regularly until 45 min post-treatment. During the imaging session, cells were perfused with HBS using a closed-loop perfusion system (Ismatec, Wertheim, Germany) and maintained at 37 °C using an in-line heater (Warner Instrument Corporation, Hamden CT). The perfusion loop was opened after stimulation to empty the system of glycine. During experiments involving treatment with anisomycin or rapamycin, these reagents were added to the HBS and perfused over the cells for the duration of the experiment.
Maximum projected Z-stack images (6 per cell) were first preprocessed in ImageJ (NIH, Bethesda, MD). Series of z-stacks obtained over the course of a 45 min imaging session were registered using the StackReg plugin (EPFL, Lausanne, Switzerland). Images were adjusted for size (1500 × 1500 pixels) and type (8-bit) before further processing. Automated analysis of spine morphology was performed using a custom package (‘SpineZap’) developed in the MATLAB computing environment (Mathworks, Natick, MA). Neurons were imaged so that their cell body was positioned to one corner slightly out of frame, to maximize the length of primary dendrite captured. ROI’s were defined over all visible spines on primary, secondary, and tertiary dendrites beginning immediately adjacent to the cell soma and extending progressively along the length of the primary dendrite. We did not detect obvious differences in the behavior of proximal and distal spines and those spines emanating from primary dendrites vs secondary or tertiary dendrites. The following parameters were automatically generated for each spine: head width, length, neck width and morphological class. Spines were grouped into the following morphological classes based on previously published anatomical studies using electron microscopy [15, 44]: filopodial, mushroom, flat (or “cup-shaped”) thin, and stubby. Filopodia are defined as protrusions with a length greater than or equal to 5 μm. Mushroom spines are defined as having a head to neck ratio greater than or equal to 2.5. Flat spines are defined as having a head width to length ratio greater than or equal to 1. Thin spines are defined as having a length to neck width ratio greater than or equal to 3. A spine that does not satisfy any of these conditions is classified as a stubby spine. The class of each spine is determined by checking against these conditions sequentially (in the order described above). Data analysis was performed in Origin (OriginLab, Northhampton, MA) and MATLAB. Statistical differences between multiple groups were assessed by ANOVA, followed by Tuckey’s HSD post hoc tests. For comparisons of probability distribution using the Kolmogorov–Smirnov test, alpha was set at 0.001. Ideal number of clusters for the dataset in Fig. 2e-i was determined using the NbClust package for R .
Recent work has indicated that mTORC1-mediated phosphorylation of 4E–BP is an indispensable step in the process by which this pathway controls cap-dependent translation . As such, we next addressed whether our previously observed protein synthesis-dependent increases in spine head width were also dependent on mTORC1 activity. Similar to our results using anisomycin, pretreatment with the mTORC1 inhibitor rapamycin (100 nM, administered 30 min prior to glycine treatment), resulted in significantly less pronounced head enlargements than spines treated with glycine alone (Fig. 2a). By 45 min post stimulation, cells treated with glycine + rapamycin displayed a net gain in spine head width of 8.6%, which was not significantly different from HBS-treated controls (Fig. 2a). Like the effects of co-treatment with anisomycin, the cumulative probability distribution of altered head diameter for spines treated with glycine + rapamycin was significantly different from the distribution of size changes in spines treated with glycine alone (Fig. 2b). The finding that both anisomycin and rapamycin attenuate persistent increases in spine head width after glycine treatment collectively support the hypothesis that morphological plasticity after LTP relies on mTORC1-dependent protein synthesis.
We next considered the possibility that starting differences in spine size might contribute to the magnitude of relative changes in spine head area among the various treatment groups assessed above. Figure 2d shows the full distribution of spine head areas at baseline in each of the six relevant treatment conditions. Overall, the distribution of spine head widths is similar among treatments, though the proportion of large- and small-diameter spines is not uniform in all cases, which gives rise to significant differences in mean spine head widths among the conditions (ANOVA, F5,5061 = 18.13, p = 7.00e−18, Fig. 2d). These differences were not systematic according to pre-treatment condition, however, with only one group receiving pretreatment with anisomycin (“Control + aniso”) and one group later receiving glycine (“Glycine alone”) showing significant differences from HBS treated control values by Tukey HSD post hoc tests. Given that these baseline differences exist before treatments were initiated, it is unlikely that they reflect any specific treatment effect. However, in principle, the starting size of spines can impact the assessment of relative changes in spine size over time, as smaller spines are closer to a measurement floor and larger spines closer to a measurement ceiling. Small spines, for example, are limited in the range where they can shrink further, but there is a large dynamic range for these spines to increase in size. Accordingly, mean spine head width will tend to increase across repeated measurements even if an equal number of spines grow and shrink within a group. Likewise, for initially large diameter spines, mean spine head width will adopt a negative trajectory over time given equal proportions of spines that grow and shrink. To account for this potential issue, it was necessary to compare treatment effects on spines of different sizes separately.
To determine ideal threshold values to separate spines in our dataset according to size, we used the ‘NbClust’ package in R which compares multiple indices to determine the ideal number of naturally occurring clusters in a dataset . Out of 5061 total spines across all 6 experimental conditions, we found that our data was best represented by a separation into three groups (Fig. 2e-f). Kmeans clustering revealed groups as follows: group 1 n = 3068 spines, range = 0.15–1.558um; group 2 n = 1504 spines, range = 1.559–2.317um, group 3 n = 489 spines, range = 2.319–4.795um (Fig. 2e-f). When partitioned into these clusters, we observed that the sampling from each group across experimental conditions was non-uniform (Control = 55.757% group 1, 32.575% group 2, and 11.666% group 3; Control + Aniso = 71.374% group 1, 17.748% group 2, and 10.877% group 3; Control + Rap = 56.28% group 1, 32.534% group 2, 11.1776% group 3; Glycine = 65.669% group 1, 27.828% group 2, 6.5019% group 3; Glycine + Aniso = 61.336% group 1, 29.959% group 2, 8.7044% group 3; Glycine + Rap = 53.070% group 1, 35.380% group 2, 11.5497% group 3). A comparison of these populations separated out by group at baseline (minute 0) or 45 min post stimulus are shown in Fig. 2g-i. We find that after clustering, within a particular spine group, mean spine head widths are now highly similar across treatment conditions at baseline (Fig. 2g-i, rightmost panels). For group 1 spines (the most represented in all treatment conditions), the glycine treatment condition exhibits an increase in spine head width that is substantively higher than in controls, despite similar spine head width in the two conditions at baseline. Likewise, for group 2 spines (the next most represented in all treatment conditions) where mean spine head width tends to decrease, the drop is noticeably less in the glycine treatment condition than in the controls. Anisomycin or rapamycin pre-treatment eliminates this effect in both cases, while having little impact when administered alone. Of note, no apparent treatment effects are evident in group 3 spines (the least represented in the data set), likely due to a measurement ceiling effect. Together, this analysis reveals that relative changes in spine head diameter are influenced by basal differences in spine head size. For the majority of spines (groups 1 and 2), glycine stimulation positively regulates spine head area relative to control conditions, an effect that is both protein synthesis- and mTORC1-dependent.
We next assessed the requirements for protein synthesis and mTORC1 signaling during glycine-induced potentiation for each morphological sub-class. In most cases, blocking either protein synthesis (40 μM anisomycin,30 min pretreatment) or mTORC1 kinase activity (100 nM rapamycin, 30 min pre-treatment) tended to produce similar effects on changes in spine head width following cLTP induction within each sub-group. For mushroom spines (Fig. 3b-c, pre-treatment with either anisomycin or rapamycin resulted in a significant decrease in head width compared to spines treated with glycine alone (normalized control mushroom head width 45 min post stim = 100+/−40.68%, Control plus anisomycin = 109.59+/−45.4%, Control plus rapamycin = 103.39+/−36.06%, glycine = 117.27+/−43.59%, glycine + anisomycin = 104.49+/−38.77%, glycine + rapamycin = 108.32+/−43.01%; ANOVA, F5,1181 = 6.54, p = 5.22e−6). A similar effect was observed for stubby spines (Fig. 3e-f); normalized control stubby head width 45 min post stim = 100+/−37.42% Control + Aniso = 101.86+/−40.39%, Control + Rap = 106.74+/−41.27%, glycine = 114.29+/−41.08, glycine + anisomycin = 104.11+/−43.35%, glycine + rapamycin = 108.28+/−37.48%; ANOVA, F5,1679 = 6.86, p = 2.40e−6). Glycine-induced head width changes in thin spines (Fig. 3k-l) were not sensitive to protein synthesis inhibition with anisomycin (normalized control thin head width 45 min post stim = 100+/−45.71%, Control plus aniso = 102.96+/−41.77% of HBA treated controls, Control + Rapamycin = 98.97+/−41.77%, glycine = 113.56+/−47.01%, glycine + anisomycin = 116.55+/−45.49%, glycine + rapamycin = 107.86+/−44.13; ANOVA, F5,1020 = 4.26, p = 7.62e−4) and neither flat spines nor filopodia showed significant differences between any of the groups assessed at 45 min post-glycine stimulation (Fig. 3h-i, n-o).
Here, we examined the role of mTORC1 signaling in structural remodeling of synapses during LTP. We show that cLTP induction caused an expansion in dendritic spine heads in cultured hippocampal neurons (Fig. 1), a specific form of structural plasticity that is widely believed to be necessary for persistent strengthening of excitatory synaptic connections. The maintenance of this altered head diameter at later time points (here, 45 m post-stimulation) is notably dependent on mTORC1-dependent protein synthesis, as it is blocked by concurrent application of anisomycin or rapamycin during glycine-induced cLTP (Fig. 2). This effect appears to be most strongly seen in a sub-population of dendritic spines with a mature, mushroom type morphology (Fig. 3). Despite being necessary to induce these changes, enhanced mTORC1 activity alone is not sufficient to elicit increased spine head diameter (Fig. 4). However, when active mTORC1 signaling is paired with a sub-threshold level of synaptic stimulation, strong changes in spine morphology rapidly emerge, resulting in significant increases in head width by 15 m post stimulation (Fig. 5). Lastly, we show that glycine-dependent changes in spine morphology occurring in the context of elevated mTORC1 signaling are highly volatile, with strong increases in head diameter often occurring between single imaging periods (Fig. 6). Collectively these results suggest that mTORC1 acts in tandem with additional activity-dependent synaptic signals to produce structural changes, and shed new light on the cellular mechanisms needed to induce structural changes underlying long lasting increases in the strength of excitatory synapses.
A role for mTORC1-mediated protein synthesis in maintaining altered spine morphology
Of general interest was our finding that changes in spine morphology during chemically-induced LTP require cap-dependent translation. We found that pharmacological blockade of protein synthesis using anisomycin significantly diminished the extent of spine head enlargement after glycine treatment, an effect which was much stronger at 45 min post stimulation than at 15 min post stimulation (Fig. 2c). Spine head growth was similarly blocked by the mTORC1 inhibitor rapamycin (Fig. 2c-f). Given the well established role of mTORC1 in regulating translation initiation, the inhibitory effect of anisomycin/rapamycin at later time points could indicate that potentiated spines require new proteins for the maintenance of increased head width, possibly as a compensatory response to concurrent activity-dependent degradation of synaptic proteins mediated by the ubiquitin proteasome system [3, 4].
Contribution of particular morphological types
Dendritic spines are often distinguished by morphological classification. Historically, categories have included stubby, thin, and mushroom, flat, and filopodial [15, 44], although some schemes include other categories such as bifurcated . Different spine types are thought to be functionally distinct, with larger, mushroom spines widely presumed to harbor stronger synapses. It has been proposed that thin and mushroom spines contribute to learning and memory in different ways, with thin spines believed to be both more transient and plastic than mushroom spines . The head diameter of thin spines is, by definition, smaller than those of mushroom spines and as such theoretically have more room to grow under LTP. For these reasons some hold that thin spines are converted into mushroom-type spines during the stabilization process of a structural engram after learning has occurred .
We found remarkable separation between the responsiveness of particular morphological spine classes to a glycine-based stimulation paradigm (Fig. 3). Of all types assessed, mushroom spines display particularly large changes in head diameter in response to glycine, and appear to be particularly reliant on new protein synthesis for this effect to be maintained (Fig. 2a-c). Because of their cardinal role in integrating synaptic transmission, dendritic spines are endowed with a variety of organelles and sub-cellular entities that mediate and modulate their role in synaptic plasticity. The smooth endoplasmic reticulum (SER) plays a known role in regulating calcium levels, an ion centrally implicated in LTP. Roughly half of all spines contain a SER stack  and the stack tends to occupy almost 20% of the total spine volume, suggesting a mechanism for regulating the calcium concentration within spines. Additionally, Ca2+ released from the SER can augment stimulus- induced Ca2+ currents . It has also long been known that polyribosomes are capable of localizing to dendritic spines . These dendritic polyribosomes are not uniformly distributed at synapses, but are far more prevalent in mature spines, such as mushroom spines , suggesting they play some specific role in mediating changes in synapse form or function during activity-induced plasticity. Furthermore, after LTP there is an increase in the proportion of spines containing polyribosomes, the presence of which can predict which spine will exhibit potentiation . It will be interesting for future experiments to examine the unique mechanisms which support persistent, protein-synthesis dependent morphological changes after potentiating stimuli.
Combinatorial action of mTOR with synaptically derived signals
Our results regarding cell autonomous mTORC1 activation paired with application of otherwise sub-threshold concentration of glycine (Fig. 5) suggest that synaptically evoked signaling mechanisms operate in conjunction with mTORC1 activity to mediate the morphological changes observed after cLTP. Constitutive mTORC1 activation alone appears to be insufficient to induce these changes (Fig. 3), possibly because they depend on synaptically driven signals to direct mTORC1-dependent translation of particular sets of mRNA to maintain altered spine structure. In such a scenario, synaptic activity would not induce a global increase in the synthesis of dendritically localized mRNAs, but might rather elicit the synthesis of specific sets of ‘LTP proteins’, a scenario that has been previously suggested for mTORC1 signaling at the synapse [1, 2]. Interestingly, we found that a sub-threshold dose of glycine can elicit rapid increases in spine size, provided this stimulus occurs on a background of high mTORC1 signaling (Fig. 5). This result supports the hypothesis that mTORC1 activation provides a context of active translation upon which even slight activity can drive synthesis of the new proteins required for changes in spine morphology. Additionally, the rapid nature of these changes is in agreement with previous reports showing an immediate impact of protein synthesis inhibitors on the extent of initial spine growth after induction of LTP at single spines using 2-photon glutamate uncaging [13, 58].
The question remains, however, as to the nature of the upstream synaptic signals that co-activate mTORC1 signaling alongside signaling pathways involved in actin cytoskeletal rearrangement. Calcium influx after LTP-inducing stimuli at single dendritic spines has been shown to induce a brief, spine-specific increase in the phosphorylation of CaMKII , which subsequently activates members of the Rho family of small GTPases including RhoA and Cdc42 [39, 40]. Transiently autophosphoylated CaMKII also activates the small GTPase Ras in the postsynaptic domain [16, 63]. Ras is a known activator of the PI3K/mTOR pathway , and it remains an intriguing possibility that Ras acts to stimulate mTORC1 directly or perhaps operates in tandem with Wnt signaling  to activate mTORC1 in the context of LTP. Recent work has also highlighted an important role for mTORC2 in directly regulating actin polymerization during LTP in the hippocampus . This molecularly distinct complex of proteins, comprised of mTOR bound to rictor, among other partners, plays a role in regulating the actin cytoskeleton, but is usually insensitive to acute inhibition by rapamycin . However, rapamycin has been shown to exert effects on mTORC2 signaling via disruption of mTOR complex formation with rictor, though only after chronic exposure of 24 h or longer . As such, we find it unlikely that our reported inhibition of spine growth with acute rapamycin pre-treatment (30 min; Fig. 2) is due to an effect on mTORC2-mediated actin polymerization rather than mTORC1-mediated signaling.
Achieving a more detailed understanding of the signaling underlying structural remodeling of dendritic spines provides a window into how information is stored within the mammalian brain. Our results implicate mTORC1 activation as an important combinatorial signal that interacts with other local synaptic events to promote spine enlargement during long-lasting synaptic plasticity. Since dysregulation of mTORC1 has been strongly implicated in neurodevelopmental disorders characterized by disorders of social interaction, intellectual disability, and epileptic seizures, a better understanding of mTORC1’s role in structural plasticity may shed insight into novel therapeutic approaches for such disorders.
We thank Cindy Carruthers and Christian Althaus for generating the cultured hippocampal neurons used in these studies.
This work was supported by NIH grants RO1MH085798 and RO1NS097498 to M.A.S.
Availability of data and materials
Please contact corresponding author for data requests.
FEH carried out experiments, helped conceive the study, analyzed data, and co-wrote the manuscript. WH carried out experiments, analyzed the data, and participated in the design of the study. AC carried out experiments and analyzed data. SPM designed the automated spine analysis and provided input on interpretation of the results. MAS conceived the study, designed the experiments, and co-wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All animal use was in accordance with federal guidelines and was approved by the University of Michigan’s Institutional Animal Care and Use Committee.
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The authors declare that they have no competing interests.
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