Dynamic structural reorganisation of the actin cytoskeleton provides a mechanism for synaptic plasticity and memory formation. The pathways and mechanism regulating the restructuring of the actin cytoskeleton in dendritic spines are however, as of yet, almost unknown. Myosin II motor activity had been previously identified as the driver of neurite elongation in immature neurons. In their recent paper Rex et al. Error: Reference source not found explore the regulating pathways driving actin dynamics during long term potentiation and memory formation. They identify myosin II motor activity as a crucial driver for the emergence of actin structures necessary to stabilise LTP after initial potentiation. Myosin II activation is demonstrated to be downstream from a signalling cascade initiated by synaptic stimulation induced NMDA receptor activation. An actin polymerization agent is shown to restore stable LTP and memory formation in myosin II inhibited CA1 hippocampal neurons. This review paper aims to put the work done by Rex et al. into context, critically evaluating its key discoveries and commenting on the implications but also limitations of this work.
Introduction and Background
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Memory formation and consolidation is one of the great enigmas in neuroscience. The idea that the physical brain is the substrate for all our memories, experiences and skills has been around since Hippocrates and Plato, roughly 2400 years ago, although the exact mechanisms have yet to be fully understood.
This review paper will attempt provide an overview of the research into one particular pathway linking structural plasticity with memory formation, while attempting to put the emerging model in context against other pathways involved in memory formation and consolidation. The main focus will however remain on evaluating the Rex et al. paper Error: Reference source not found, which provides a model in which myosin IIb motor activity, initiated by a stimulation-induced NMDAR signalling cascade, regulates actin dynamics in dendritic spines in a pathway crucial for synaptic plasticity and memory formation.
To bring the reviewed paper into context a certain amount of background is required, linking memory formation with the mechanisms by which it is manifested in its physical substrate at several levels of description.
Linking memory and learning to LTP
The synaptic plasticity and memory hypothesis (SPM) states that: â€œactivity dependent synaptic plasticity is induced at appropriate synapses during memory formation and is necessary and sufficient for the information storage underlying the type of memory mediated by the brainâ€Â Error: Reference source not found. Recent research has found that long term potentiation (LTP) can indeed account for the initial encoding, early stabilization and storage of memory, but that sufficiency of the model to account for all forms of memory storage has not yet been demonstrated. Further complicating matters, the mechanisms and pathways may vary greatly in different brain regions Error: Reference source not found. Experimentally, LTP induction and maintenance have been linked to memory formation in a variety of brain areas. Examples include fear learning in the amygdala Error: Reference source not found, olfactory memory in cortical neurons Error: Reference source not found and spatial memory in the hippocampus Error: Reference source not found. The dominant cellular model for LTP has been area CA1 of the hippocampus Error: Reference source not found, which has been used to explore the structural changes and other physical mechanisms involved in LTP Error: Reference source not found.
Linking structural dynamics to LTP
Given that structural changes have been associated with long-term potentiation, the locations, mechanisms and pathways underlying structural plasticity are highly important. Dendritic spines have been identified as a locus for connective plasticity leading to functional short- and long-term plasticity Error: Reference source not found, where small spines may be preferential sites for LTP to occur, while large spines may be structural correlates of long term memory Error: Reference source not found and spine pruning could be associated with LTD and memory erasure Error: Reference source not found. The structural plasticity at mature synapses has been found to be guided by mechanisms based on dynamic actin filaments in the cytoskeleton of dendritic spines Error: Reference source not found. The aforementioned actin cytoskeleton dynamics are of course accompanied and guided by a variety of other pathways and signalling cascades such as the release and action of neurotrophins Error: Reference source not found and more importantly receptor trafficking to the postsynaptic terminals on dendritic spines Error: Reference source not found.
Linking Actin Dynamics to Structural Plasticity
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Actin forms the cytoskeleton of cells and plays an integral role in structural support, growth and intracellular transport. The actin cytoskeleton exists in a dynamic equilibrium between the monomer G-actin and the polymer F-actin, which can be modulated bidirectionally by cellular pathways. It has in fact been demonstrated that high frequency stimulation causes a rapid equilibrium shift towards F-actin in dendritic spines of CA1 neurons Error: Reference source not found. This equilibrium shift induces spine enlargement and an increase of their postsynaptic receptor binding capacity Error: Reference source not found, providing a substrate for bidirectional synaptic plasticity Error: Reference source not found.
The signalling cascade leading to this equilibrium shift is dependent on NMDA receptor activation as can be induced experimentally by presynaptic stimulation Error: Reference source not found. This cascade causes inactivation of the actin depolymerising factor cofilin by phosphorylation, as an increased p-cofilin concentration detected within larger synapses predicts . Parallel to cofilin phosphorylation, the cascade induces local actin network contractions, which provide the necessary forces to remodel large F-actin structures Error: Reference source not found. Indications as to what provides the necessary mechanical forces to induce actin network contractions came from the neurite growth cones of immature neurons, in which very similar mechanical dynamics were shown to be driven by the motor activity of myosin II Error: Reference source not found.
Myosin IIs Role in Regulating Actin Dynamics
Myosins are a family of motor proteins, which bind to F-Actin and are driven by ATP hydrolysis. While myosin V and VI are involved in vesicle and neurotransmitter receptor transport Error: Reference source not found, myosin II was shown to directly regulate actin dynamics Error: Reference source not found. Myosin II mediates contractions within actin networks in growth cones leading to actin-bundle severing and simultaneously driving retrograde flow of the resultant G-actin monomers. The globular G-actin molecules are polymerized once more, by being added to the end of the growth cone, driving neurite elongation. Inhibition of myosin II was shown to prevent retrograde actin flow leading to growth cone collapse. Moreover, the level of inhibition was found to be directly proportional to neurite growth, suggesting that myosin II is the main driver of retrograde flow of monomeric G-actin Error: Reference source not found. Many questions regarding the exact function of myosin II activity in regulating F-actin polymerization and its role in memory formation remain unanswered.
Given that F-actin pools are present in dendritic spines of mature neurons Error: Reference source not found and that myosin II is highly prevalent in the postsynaptic densities of mature forebrain synapses Error: Reference source not found, the reviewed paper hypothesises that myosin II and the actin cytoskeleton form a similarly complex system for dynamic activity dependent cytoskeletal reorganization in dendritic spines as they do in neurite growth cones of immature neurons. This hypothesis is already partially supported by experiments demonstrating that disruption of myosin II in cultured neurons alters the growth and morphology of dendritic spines and impairs synaptic transmission Error: Reference source not found, although it is as of yet not confirmed whether this takes place by the same mechanism. Furthermore, myosin IIs role in LTP and memory formation has so far not been addressed. Therefore the main hypothesis put forward by this paper is that the mechanical force imparted on dendritic spines by myosin IIb, is necessary for the emergence of F-actin structures, underlying synaptic plasticity and memory formation.
Rex et al. address a variety of unknowns regarding myosin II activity and function. They demonstrate that myosin II is indeed a crucial component in hippocampal memory formation and synaptic plasticity of the mature nervous system. Additionally they show that myosin II function is upstream of actin polymerization and filament stabilization and triggered by a signalling pathway initiated by NMDAR activation dependent Rho-GTPase release. Overall the results indicate myosin IIb motor activity regulates NMDAR-driven actin network dynamics and establish that this mechanism is necessary for synaptic plasticity and memory consolidation. Each of their hypothesis is given a heading under which their efforts to demonstrate its validity is described.
Myosin IIb is essential for the stability of synaptic plasticity
Figure 1: Myosin IIb is essential for synaptic stability
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(A) Input-Output relationship for synaptic responses in CA1b hippocampal slices for naive and injected hemispheres of rats injected with control and MyH10 shRNA
(B) Synaptic responses to input before and after TBS in control and MyH10 injected hippocampal slices
In order to test the above hypothesis, Rex et al. developed a virus vector, which would selectively inhibit myosin II in the CA1 are of the hippocampus in adult rats. After optimizing the technique by testing different injection locations and volumes, the viral vector rAAV2/5 containing two expression cassettes was injected unilaterally into the dorsal hippocampus of adult rats. The first of the two expression cassettes would drive wild type green fluorescent protein (wtGFP) expression and the second would express either control shRNA or MyH10 specific shRNA. The MyH10 specific shRNA would then selectively target the heavy chain of myosin IIb and reduce its expression by a factor of five relative to control samples. The naive (non-injected) hemisphere would provide internal control samples. Once the delivered nucleic acids were given enough time to reach maximum expression, hippocampal slices were prepared.
There were no recognizable structural differences between the injected and naive hemispheres in both virus groups. The ~80% reduction of myosin IIb heavy chain from the adult CA1 was found not to affect basic synaptic function and importantly, stimulation of Schaffer collateral (SC) afferents elicited equivalent synaptic field potentials in all test groups as can be seen in Figure 1A. The initial potentiation subsequent to LTP induction was also comparable across all slices. In the slices expressing the MyH10 silencing RNA, however, LTP failed to stabilize and synaptic potentiation returned to near base levels within 60 minutes of LTP induction. The naive hemisphere slice and the slices injected with control shRNA validated these findings by displaying normal, long-lasting potentiation (Figure 1B). This suggests that myosin IIb is crucial for LTP stability.
Myosin IIb regulates actin dynamics during LTP
Extending the result and showing that myosin IIb is essential for synaptic stability due to its regulating function of actin dynamics required labelling F-actin synthesis after LTP induction. This was carried out by imaging co-localization of phalloidin stained actin structures and antibody-tagged postsynaptic density protein 95 (PSD-95) after applying high frequency theta burst stimulation (TBS) in the control and MyH10-shRNA virus injected rats.
The results showed that one hour after LTP induction there was no difference in phalloidin labelling in the control virus injected and naive hemisphere. The MyH10-shRNA virus injected slices on the other hand showed significantly depressed levels of newly synthesized F-actin structures (Error: Reference source not found), providing compelling evidence that myosin IIb is in fact responsible for long-term expression of actin filaments and regulating actin dynamics in general, at synapses.
Myosin II is a target of the NMDAR-activated signalling pathway initiated by LTP induction
To test what signalling cascade would induce myosin II activity required a structural correlate for myosin II ATPase activity to be found. Since myosin II is activated through phosphorylation of its regulatory light chain MLC20, p-MLC20 provides this correlate. The initial testing was performed through two distinct approaches. The first involved application of NMDA, which did indeed induce p-MLC20 as well as p-cofilin. The second method was designed to address whether NMDAR activation induced p-MLC20 by a signalling cascade involving Rho-GTPase/Rho Kinase (ROCK), a signalling G protein. This too was confirmed since pre-administration of the ROCK inhibitor, H1152, blocked NMDAR activation induced myosin and cofilin phosphorylation.
The next step was to prove this would hold true not only under chemical administration of NMDA but also under LTP induction through electrical stimulation. By administering TBS and control stimulation at Schaeffer collateral synapses it was shown that TBS stimulation gave rise to much higher p-MLC20 concentrations in stimulated synapses. To eliminate involvement of other pathways, the NMDAR antagonist APV and a ROCK inhibitor were administered in separate experiments. Both APV and the ROCK inhibitor were shown to block TBS-induced synaptic p-MLC. Taken together this data provides evidence for the idea that NMDAR stimulation and LTP induction trigger secondary signalling systems involving ROCK, which activate motor activity of myosin II.
Myosin II force generating activity regulates synaptic plasticity
Figure 2: Phalloidin labelled F-actin structures in CA1 hippocampal slices of MyH10 shRNA injected and naive hemispheres Trying to elucidate the temporal dynamics and function of myosin II motor activity after LTP induction the myosin II ATPase inhibitor Blebbistatin was applied to adult hippocampal slices. Blebbistatin was previously shown to selectively inhibit myosin II ATPase, while leaving other forms of myosin, such as myosin V and VI, which are involved in receptor transport, unaffected Error: Reference source not found. Synaptic responses were then measured following stimulation of SC inputs to CA1 neurons. Initial input-output characteristics, baseline responses and spine morphology were unaffected by Blebb application (Figure 3A). The initial and acute post-TBS potentiation was also found to be comparable in all the samples. Blebb did however cause complete disruption of TBS-induced LTP stabilisation at the SC-CA1 synapses, which can be observed in Figure 3B showing the field EPSP curves in the Blebb infused slices returning to baseline within 15 minutes. This indicates that Blebb disrupts LTP during the postinduction, stabilisation period. Further testing confirmed Blebb was not disrupting presynaptic activity. It could therefore be concluded that Blebb did not affect other pathways and the disruption of LTP stabilisation could be directly attributed to blockage of myosin IIs force generating activity.
To test the temporal dynamics of myosin II motor activity, Blebb was applied at different times after LTP induction. Application of Blebb 30 seconds after TBS disrupted LTP stabilisation, while application 10 minutes afterwards did not. Next, the experiment was repeated this time infusing the slice with the actin filament assembly blocker Latrunculin A (LatA). The temporal dynamics of disruption to LTP stabilisation turned out to be nearly identical for Blebb and LatA application, suggesting that myosin II ATPase activity is involved in regulating postinduction LTP processes involving F-actin synthesis in dendritic spines.
Further comparison of the effects of Blebb and LatA showed that phalloidin labelled F-actin structures exhibited almost identical structuro-temporal dynamics. These results lend weight to the idea that myosin II motor activity is necessary for LTP induced F-actin synthesis.
Myosin II is upstream of actin filament polymerization underlying the stabilisation of synaptic plasticity
Figure 4: JASP restores LTP stability in response to TBS after blebbistatin administration
Figure 3: Blebbistatin induced myosin II ATPase inhibition
(A) Field response input-output characteristics in SC-CA1 synapses after blebbistatin and control administration
(B) Blebbistatin administration blocks stable LTP at SC-CA1 synapses in response to TBSThe next series of experiments were aimed at determining whether myosin II is directly involved in the synthesis of de novo F-actin structures or merely required to set up the necessary conditions. In order to investigate this, the time course of actin polymerisation after LTP induction needed to observed. In the first 30 seconds after TBS no new F-actin structures were detected even though EPSPs were already almost doubled. Changes did however become apparent after two minutes and persisted for the entire time course of the observation lasting 1 hour. Knowing the exact temporal dynamics of F-actin polymerisation, myosin II involvement in the synthesis and stabilisation could be tested for. By applying the inhibitors Blebb and LatA as well as a p-cofilin antiserum at different times, it was shown that Blebb and thus myosin II inhibition does not disrupt actin filament generation directly by blocking an LTP induced primary signalling cascade. The results also indicated that LTP related phosphorylation of cofilin isnâ€™t induced by the pathway downstream of NMDAR activation.
Further evidence was obtained by using a technique called fluorescence recovery after photobleaching (FRAP) in combination with Blebb administration. This experiment demonstrated that Blebb had no direct effect on the treadmilling rate in spines but caused the pool of stable actin in spines to increase proportionally to the inhibitors concentration. The data therefore suggests that myosin II is involved in the dynamic turnover of stable actin filaments at synapses. Rather than being directly involved in actin polymerization it is responsible for establishing an equilibrium, in which actin filament elongation can take place.
Indeed by perfusing the slices with Jasplakinolide (JASP), which induces actin filament synthesis, the effect of Blebb on LTP stability could be entirely eliminated (Figure 4).
Myosin II expression and motor activity are essential for long-term memory consolidation
The final experiments of the paper attempted to link myosin IIs expression and activity back to memory consolidation. Previous experiments had established that LTP occurs at CA1 synapses during associative learning Error: Reference source not found and that disruption of actin polymerization in this region prevents memory consolidation Error: Reference source not found. Having already demonstrated myosin IIb involvement in LTP stabilisation, Rex et al. hypothesized that myosin II activity may be involved in mechanisms underlying memory formation in the hippocampus.
Figure 5: Effect of myosin II inhibition on memory formation and consolidation
(A) Timeline of rAAV-shRNA viral vector injection, contextual fear conditioning training and LTM testing
(B) Freezing behaviour in control and myosin II inhibited animals
(C) Immediate freezing behaviour and exploratory behaviour during contextual fear conditioning for control and myosin IIb inhibited animals To investigate this hypothesis, the rAAV2/5 viral vector, containing either the control or MyH10 shRNA, was injected into CA1 of rats, which were then to undergo single-trial contextual fear conditioning. To ensure that the transgene introduction and injection procedure itself had no effect on the fear response, the rats, expressing the highest levels of GFP 30 days after the viral injection, were trained under the standard contextual fear conditioning paradigm. This involved placing the animals in a novel environment where they would be subjected to footshock, removing them and measuring the rats freezing response upon being returned to that environment. The same procedures were carried out on mock- and non-injected rats. The injection procedure and transgene expression were thereby demonstrated not to have any effect on contextual memory formation.
In a separate trial, the animals expressing MyH10 shRNA were shown to have deficits in their freezing behaviour, during a 24-hour long-term memory (LTM) test, as compared to animals injected with the control shRNA (Figure 5B). The same animals did however acquire the immediate context-shock association normally and exhibited similar exploratory behaviour (Figure 5C). This data fits the hypothesis, demonstrating that although myosin IIb does not regulate learning, it is the driver of an essential mechanism for the stabilisation of acquired contextual association memories.
To narrow down the temporal dynamics, the experiments were then repeated, replacing the viral vector with injection of (-)-isomer Blebbistatin and the inactive (+)-enantiomer as control at 30 minutes prior to contextual fear conditioning. Both trial groups exhibited normal freezing behaviour during associative training. The animals treated with active Blebb did however present highly significant deficits in freezing behaviour in 24 hour LTM tests (Figure 6A). To narrow down myosin IIs role in memory formation, the next experiments focused on determining whether application of Blebb disrupted memory formation by interfering with contextual fear memory acquisition or consolidation. This time the animals received the same injections 30 minutes prior to training but were tested for short term memory (STM) after 90 minutes. The two groups performed similarly indicating that myosin II inhibition did not interfere with memory formation (Figure 6B STM). LTM testing on the same animals a day later showed the same level of impairment as in the previous experiment (Figure 6B LTM). Repeating the same procedures but injecting the two different compounds 30 minutes after training demonstrated that there was no effect on LTM (Figure 6C). Overall this suggests that myosin II inhibition is not impairing memory consolidation by altering neuronal firing dynamics during contextual training but is having an effect on memory stabilisation in the non-immediate aftermath of initial LTP induced potentiation.
Figure 6: Time dependent effects of Blebb on LTM and STM
(A) Freezing behaviour in Blebb and control injected animals
(B) Freezing behaviour in LTM and STM tests
(C) Freezing behaviour in LTM test in post-training injected animals
The final hypothesis, that myosin II motor activity drives actin dynamics underlying memory consolidation was tested by applying the actin polymerising agent JASP together with Blebb. LTM tests indeed confirmed that simultaneous application Blebb and JASP blocked Blebbs ability to disrupt LTM. To ensure JASP was not simply making amnesia induction in the dorsal CA1 impossible, animals were simultaneously injected with JASP and the amnesia inducing agent MK-801, a compound found to inhibit NMDAR function during memory acquisition. Results showed that MK-801 still caused amnesia win these animals, confirming that the effects of JASP occur downstream from NMDAR activation.
The paper manages to put forward convincing arguments for its model of myosin IIb role in motor activity. Until now research had provided evidence for a crucial function of myosin IIb in actin dynamics underlying LTP and memory formation. In this section the authorsâ€™ experimental work and proposed model will be critically evaluated and assessed for its consistency with previous research. The first step will be to discuss what the proposed model actually says.
The experiments carried out as part of this paper support a model by which NMDAR activation in response to a learning task induces the release of the signalling G-protein Rho-GTPase. Rho-GTPAse activated kinase then causes phosphorylation of the depolymerising agent cofilin and the myosin light chain (MLC), thereby activating myosin IIb motor activity. This motor activity then provides the mechanical force necessary for actin bundle shearing, which frees G-actin monomers for use in F-actin polymerisation and therefore actin filament elongation, insuring stability of synaptic plasticity. Thus myosin II serves a crucial function in LTP stabilisation just after the initial potentiation phase but prior to the final long term storage. This model is summarised by the diagram in Error: Reference source not found.
Figure 7: Effect of JASP on memory formation
(A) Effect of JASP, Blebb and combined application on freezing behaviour in LTM test
(B) Effect of JASP, amnesia inducing agent MK-801 and combined application on freezing behaviour in LTM testIn order to critically assess the robustness of the model as a whole, the evidence for causative element within the model will be evaluated independently; starting downstream from NMDAR induced Rho-GTPAse activation. In experiments, the ROCK inhibitor, H1152, was shown to completely block myosin and cofilin phosphorylation and further testing using NMDAR antagonist APV confirmed this effects dependence on NMDAR activation. These results provide compelling evidence that NMDAR activation results in Rho-GTPAse signalling required for the activation of myosin IIb, although not completely ruling out the necessity of additional signalling steps between NMDAR activation and myosin IIb motor activation.
Downstream in the proposed model are the phosphorylation of the myosin IIb heavy chain MLC and the depolymerising cofilin. Past experiments showed that LTP induction causes cofilin phosphorylation , thereby disrupting actin polymerization. Further experimentation as part of this paper extended this idea by demonstrating that ROCK inhibition blocked both cofilin and MLC phosphorylation, confirming that both are part of the same pathway to induce actin filament synthesis. While experiments showed that myosin IIb inhibition on its own disrupted LTP stabilisation, no experiment was carried out selectively blocking p-cofilin. So although previous experiments have shown that p-cofilin is necessary since it causes cofilin-F-actin dissociation, further study is required to confirm that p-cofilin is crucial for stable actin dynamics.
Getting to the heart of myosin IIb function, the model states that myosin IIb activity is upstream of F-actin polymerisation and LTP stabilisation. Both, MyH10 shRNA and Blebb application showed that myosin II inhibition and filament synthesis disruption were highly correlated. Interestingly JASP restored filament synthesis in both scenarios confirming that myosin II function in relation to LTP stabilisation must be upstream from actin polymerisation.
The most important element of the model is of course the exact function of myosin IIb motor activity in stabilising LTP and maintaining long term memory. The paper initially attempted to narrow the problem down by trying to establish the temporal dynamics of its action. The experiments involving perfusion of the CA1 with Blebb prior to and after LTP induction and associative training narrowed down the criticality of its function to the time period 30 minutes after LTP induction or associative training. Additionally, all their experiments confirmed normal performance in short-term memory tests and normal initial potentiation after TBS. Taken together these results provide compelling evidence for myosin II to be responsible for stabilisation of early LTP and memory formation. Evidence from neuronal growth cones show myosin II to be critical for F-actin reorganisation Error: Reference source not found. Rex et al. therefore propose that the actin reorganisation mechanisms, including myosin II function, are almost identical to those in growth cones Error: Reference source not found. Evidence from growth cones suggest that the mechanical forces imparted by myosin II motor activity causes shearing of actin bundles, which depolymerises them into G-actin monomers. These monomers are then free to be repolymerised at the growing edge of the growth cone. In this model, inhibition of myosin II stops the retrograde flow of actin, causing impaired actin dynamics and growth cone collapse. In support of this idea Rex et al. demonstrated that Blebb perfusion resulted in increases in the stable F-actin pool relative to Blebbs concentration. In this particular model of myosin II function, its motor action is necessary to make the building blocks for rapid filament elongation available. They do however concede that an alternative possibility exists wherein the severing of F-actin bundles by myosin II provides the locus at which new filaments can polymerise. As of now, there is insufficient evidence for either but whatever the exact function of myosin IIb turns out to be, Rex et al. provide conclusive evidence that a) myosin IIb is not directly involved in de novo F-actin synthesis b) that its function is necessary for the stabilisation but not the initiation of LTP.
Although it is tempting to lump LTP and memory formation together it is important to remember sufficiency of the former to explain the latter has not yet been demonstrated. Nonetheless the close alignment of the results during the LTP and contextual fear association experiments, especially the ability of JASP to restore both after Blebb application makes such speculation tempting. Even the authors concede however, that myosin II may have unknown functions at the systems level, which disrupt hippocampal memory formation. Given that LTP in CA1 occurs during hippocampus dependent associative learning Error: Reference source not found and reversing LTP causes memory disruption Error: Reference source not found, this seems less likely.
It is also important to point out that, although myosin II has been shown to be crucial for LTP and memory consolidation in CA1, it is only a small part of one of many different mechanisms giving rise to memory formation in the brain or even the hippocampus. The activity of myosin IIb may however not only be necessary to stabilise early LTP but also provide the prerequisites for other processes involved in long term memory storage to occur. This may include processes as wide range ranging as receptor trafficking and de novo protein synthesis.
Nonetheless, disrupted actin dynamics may be the underlying cause of a variety of memory disorders and a better understanding of its dynamics might provide targets for novel drugs to improve neural plasticity and enhance memory consolidation.
In conclusion, the model presented by the reviewed paper provides a mechanism to explain the emergence of F-actin structures underlying the stabilisation of early LTP at CA1 synapses, necessary for long term memory expression. At the same time it leaves many questions unanswered, which will have to be addressed in future experiments.
Most importantly the paper failed to narrow down by what exact process myosin IIbs motor activity allows new F-actin structures to form following LTP induction. This may be addressed in future studies by making G-actin pools available in myosin II inhibited CA1 synapses and testing whether this would restore LTP and improve long term memory test performance. As mentioned previously further testing of the importance of cofilin phosphorylation would also be of further interest. To test this further, an experiment would have to be devised to selectively inhibit cofilin phosphorylation and study its effects on actin dynamics, LTP persistence and memory consolidation. Other experiments of interest include testing of the effects of altered not merely inhibited myosin II motor activity, investigations into other signalling pathways which may regulate myosin II function.
Branching out to elucidate the entire dynamics of LTP and memory formation future studies should focus on the study of the mechanisms that cause potentiation immediately after high frequency stimulation before actin dynamics have a chance to stabilise potentiation and the pathways which follow once the dendritic spine has been enlarged.
Since memory formation is such a complex problem, which in all likelihood involves a host of different pathways and mechanisms dependent on the brain area involved there is a huge range of experiments still to be done. This paper has however advanced general understanding of associative memory formation in the hippocampus and may prove to be applicable more generally, in different forms of memory and learning across different brain structures.