Intrinsic excitability mechanisms of neuronal ensemble formation
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Evaluation Summary:
This manuscript reveals the contribution of intrinsic excitability to the formation of cortical neuronal ensembles. By combining optogenetic and electrophysiological approaches in vitro, the authors provide new insight regarding the role that plasticity of membrane excitability (intrinsic plasticity) plays in synaptic plasticity and the formation of memories.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Neuronal ensembles are coactive groups of cortical neurons, found in spontaneous and evoked activity, that can mediate perception and behavior. To understand the mechanisms that lead to the formation of ensembles, we co-activated layer 2/3 pyramidal neurons in brain slices from mouse visual cortex, in animals of both sexes, replicating in vitro an optogenetic protocol to generate ensembles in vivo. Using whole-cell and perforated patch-clamp pair recordings we found that, after optogenetic or electrical stimulation, coactivated neurons increased their correlated activity, a hallmark of ensemble formation. Coactivated neurons showed small biphasic changes in presynaptic plasticity, with an initial depression followed by a potentiation after a recovery period. Optogenetic and electrical stimulation also induced significant increases in frequency and amplitude of spontaneous EPSPs, even after single-cell stimulation. In addition, we observed unexpected strong and persistent increases in neuronal excitability after stimulation, with increases in membrane resistance and reductions in spike threshold. A pharmacological agent that blocks changes in membrane resistance reverted this effect. These significant increases in excitability can explain the observed biphasic synaptic plasticity. We conclude that cell-intrinsic changes in excitability are involved in the formation of neuronal ensembles. We propose an ‘iceberg’ model, by which increased neuronal excitability makes subthreshold connections suprathreshold, enhancing the effect of already existing synapses, and generating a new neuronal ensemble.
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Author Response:
Reviewer #1:
This is a very interesting manuscript showing the contribution of intrinsic excitability in the formation of cortical neuronal ensembles. Using paired recordings from layer 2/3 neurons from the visual cortex, the authors show that co-activation of neurons by optogenetic or electrical stimuli leads to persistent synaptic potentiation preceded by a transient synaptic depression. The stimulation of neurons induces, in addition, persistent plasticity of intrinsic neuronal excitability that is associated with an enhancement of membrane resistance and a hyperpolarization of the spike threshold. The authors conclude that intrinsic plasticity allows to persistently maintain activated circuits according to an iceberg-like effect, and thus to generate a new neuronal ensemble.
This study is interesting as it …
Author Response:
Reviewer #1:
This is a very interesting manuscript showing the contribution of intrinsic excitability in the formation of cortical neuronal ensembles. Using paired recordings from layer 2/3 neurons from the visual cortex, the authors show that co-activation of neurons by optogenetic or electrical stimuli leads to persistent synaptic potentiation preceded by a transient synaptic depression. The stimulation of neurons induces, in addition, persistent plasticity of intrinsic neuronal excitability that is associated with an enhancement of membrane resistance and a hyperpolarization of the spike threshold. The authors conclude that intrinsic plasticity allows to persistently maintain activated circuits according to an iceberg-like effect, and thus to generate a new neuronal ensemble.
This study is interesting as it integrates synaptic and intrinsic plasticity in the frame of the formation of cortical neuronal ensembles. However, it is unclear whether intrinsic plasticity occurs at pre- and post-synaptic neurons as illustrated in the final iceberg scheme. This has been shown by Ganguly et al., Nat Neurosci 2000 & Li et al., Neuron (2004). Nevertheless, this paper will have a strong impact in the field because of its conceptual clarity and the quality of the data.
We thank the reviewer for the comments and have incorporated the references to the previous work. Also, the synaptic depression and potentiation observed is consistent with the well-described exhaustion and recovery of the ready releasable pool of presynaptic neurotransmitter.
We have clarified our argument and modified the interpretation. In our model, we propose that after the optogenetic or electrical stimulation neurons shift to a more excitable state, therefore, neuronal responses would be amplified. Indeed, our findings show that stimulated pyramidal cells in layer 2/3 from visual cortex, whether they are presynaptic and postsynaptic, become more excitable. This happens even electrically stimulated single neurons. These changes confound the interpretation of potential synaptic plasticity, as EPSPs will be increased in size by the increased excitability.
We also added the follow clarification to the footnote in Figure 8:
" Neurons shift to a more excitable state after stimulation, so neuronal responses are amplified and the circuit now responds to an external input by activating a neuronal ensemble.", "All stimulated neurons become more excitable, ..."
Reviewer #2:
This is a potentially very impactful manuscript. The reason is that plasticity of membrane excitability (intrinsic plasticity) is largely understood as a mechanism that merely aids synaptic plasticity (in cortex: LTP) in its role in the formation of memory engrams and in learning. For example, one prominent hypothesis (Josselyn/Silva) suggests that intrinsic plasticity might enhance the probability for subsequent LTP induction and form/stabilize engrams in this way. A somewhat different view has been presented by Brunel/Hansel, who argue that under some conditions, intrinsic plasticity can integrate neurons into engrams, even when synaptic weights remain frozen. Importantly, the current work might provide evidence for this latter intrinsic theory of learning. However, in this in vitro study, the application of optogenetic or electric protocols to drive correlated neuronal activity in a defined ensemble does not only lead to strong changes in membrane excitability but also causes a biphasic change in synaptic weights. Below, I will make suggestions on how these synaptic and intrinsic effects could be further separated (this should be done if the goal of this study is to show that excitability changes alone can promote ensemble integration).
Major comments:
Line 79 f: It is not clear from this paragraph, which of the cited papers provide experimental details and which one presents the 'alternative hypothesis' (Titley et al., 2017; see above). This should be described more accurately to share the precise status quo of this research field with the audience.
We thank the reviewer for the suggestion. We have adjusted and incorporated the references that explain the alternative hypothesis and the segments corresponding to experimental details.
Figure 2: This is the critical point, and my experimental comments will focus on this: the authors show in this figure that both the optical stimulation as well as the electrical stimulation trigger synaptic plasticity, consisting of an immediate depression, followed after a pause by a potentiation. This potentiation is - in the case of electrical stimulation - significantly different from the baseline values (not significant for the opto group, but the number of recordings is quite low). It thus is conceivable that this effect contributes to the enhanced correlation of activity in the network that is shown in Figure 1.
This is a physiological observation, but attempts should be made to block/prevent the synaptic change and to assess whether enhanced correlation can persist with only excitability changes being available as a cellular mechanism. One way to do this is to perform recordings with physiological calcium and magnesium concentrations in the ACSF. As stated in the methods, the authors used 2mM Calcium and 1mM Magnesium. The physiological concentrations are about 1.2 mM Calcium and 1 mM Magnesium, thus creating an ionic milieu that is likely to be less permissive for LTP. The authors should try whether under these conditions the biphasic synaptic change is gone/reduced and the excitability change persists. If so, they can then test whether the enhanced activity correlation is still seen. Also, these recordings should be performed at physiological temperature.
If this is not successful (or as an alternative to start with), the authors might try pharmacological or genetic LTP blockade (e.g. targeting NMDA receptors or CaMKII) or use weaker stimulation protocols (intrinsic plasticity has a lower induction threshold than LTP).
Thanks for this important point. In order to separate synaptic effects, we measured synaptic activation of neighbor neurons (local circuit, Figure 4) from neurons without opsin expression. Synaptic inputs in non-expressing cells did not change: spontaneous EPSP amplitude or intrinsic excitability were similar before and after (Figure 4C). This can be explained because presynaptic activation did not increase firing probability in non- expressing cells. Neurons only became more excitable state when action potentials were evoked. This explains why we also observed increase in intrinsic excitability under electrically stimulated of single cells.
It would be very interesting to dissect whether synaptic changes were influenced by changes in excitability of presynaptic neuron. However, the number of unitary connections is already small and these connections are weak, and, due to the experimental difficulties, we had very few opportunities to test them. But still, according to our observation, changes in the synapses are temporally independent from intrinsic excitability. While we observed changes in membrane excitability after 3 min of stimulation, recovery from synaptic depression and partial potentiation took at least 20 min.
While the suggestion to repeat these experiments with altered ACSF is a good one, our plan is to explore this phenomenon in vivo. We have recently succeeded in obtaining high- quality whole-cell recordings in vivo and hope to directly reveal the role of increased excitability in the generation of ensembles in a physiological setting.
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Evaluation Summary:
This manuscript reveals the contribution of intrinsic excitability to the formation of cortical neuronal ensembles. By combining optogenetic and electrophysiological approaches in vitro, the authors provide new insight regarding the role that plasticity of membrane excitability (intrinsic plasticity) plays in synaptic plasticity and the formation of memories.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This is a very interesting manuscript showing the contribution of intrinsic excitability in the formation of cortical neuronal ensembles. Using paired recordings from layer 2/3 neurons from the visual cortex, the authors show that co-activation of neurons by optogenetic or electrical stimuli leads to persistent synaptic potentiation preceded by a transient synaptic depression. The stimulation of neurons induces, in addition, persistent plasticity of intrinsic neuronal excitability that is associated with an enhancement of membrane resistance and a hyperpolarization of the spike threshold. The authors conclude that intrinsic plasticity allows to persistently maintain activated circuits according to an iceberg-like effect, and thus to generate a new neuronal ensemble.
This study is interesting as it integrates …
Reviewer #1 (Public Review):
This is a very interesting manuscript showing the contribution of intrinsic excitability in the formation of cortical neuronal ensembles. Using paired recordings from layer 2/3 neurons from the visual cortex, the authors show that co-activation of neurons by optogenetic or electrical stimuli leads to persistent synaptic potentiation preceded by a transient synaptic depression. The stimulation of neurons induces, in addition, persistent plasticity of intrinsic neuronal excitability that is associated with an enhancement of membrane resistance and a hyperpolarization of the spike threshold. The authors conclude that intrinsic plasticity allows to persistently maintain activated circuits according to an iceberg-like effect, and thus to generate a new neuronal ensemble.
This study is interesting as it integrates synaptic and intrinsic plasticity in the frame of the formation of cortical neuronal ensembles. However, it is unclear whether intrinsic plasticity occurs at pre- and post-synaptic neurons as illustrated in the final iceberg scheme. This has been shown by Ganguly et al., Nat Neurosci 2000 & Li et al., Neuron (2004). Nevertheless, this paper will have a strong impact in the field because of its conceptual clarity and the quality of the data.
-
Reviewer #2 (Public Review):
This is a potentially very impactful manuscript. The reason is that plasticity of membrane excitability (intrinsic plasticity) is largely understood as a mechanism that merely aids synaptic plasticity (in cortex: LTP) in its role in the formation of memory engrams and in learning. For example, one prominent hypothesis (Josselyn/Silva) suggests that intrinsic plasticity might enhance the probability for subsequent LTP induction and form/stabilize engrams in this way. A somewhat different view has been presented by Brunel/Hansel, who argue that under some conditions, intrinsic plasticity can integrate neurons into engrams, even when synaptic weights remain frozen. Importantly, the current work might provide evidence for this latter intrinsic theory of learning. However, in this in vitro study, the application …
Reviewer #2 (Public Review):
This is a potentially very impactful manuscript. The reason is that plasticity of membrane excitability (intrinsic plasticity) is largely understood as a mechanism that merely aids synaptic plasticity (in cortex: LTP) in its role in the formation of memory engrams and in learning. For example, one prominent hypothesis (Josselyn/Silva) suggests that intrinsic plasticity might enhance the probability for subsequent LTP induction and form/stabilize engrams in this way. A somewhat different view has been presented by Brunel/Hansel, who argue that under some conditions, intrinsic plasticity can integrate neurons into engrams, even when synaptic weights remain frozen. Importantly, the current work might provide evidence for this latter intrinsic theory of learning. However, in this in vitro study, the application of optogenetic or electric protocols to drive correlated neuronal activity in a defined ensemble does not only lead to strong changes in membrane excitability but also causes a biphasic change in synaptic weights. Below, I will make suggestions on how these synaptic and intrinsic effects could be further separated (this should be done if the goal of this study is to show that excitability changes alone can promote ensemble integration).
Major comments:
Line 79 f: It is not clear from this paragraph, which of the cited papers provide experimental details and which one presents the 'alternative hypothesis' (Titley et al., 2017; see above). This should be described more accurately to share the precise status quo of this research field with the audience.
Figure 2: This is the critical point, and my experimental comments will focus on this: the authors show in this figure that both the optical stimulation as well as the electrical stimulation trigger synaptic plasticity, consisting of an immediate depression, followed after a pause by a potentiation. This potentiation is - in the case of electrical stimulation - significantly different from the baseline values (not significant for the opto group, but the number of recordings is quite low). It thus is conceivable that this effect contributes to the enhanced correlation of activity in the network that is shown in Figure 1.
This is a physiological observation, but attempts should be made to block/prevent the synaptic change and to assess whether enhanced correlation can persist with only excitability changes being available as a cellular mechanism. One way to do this is to perform recordings with physiological calcium and magnesium concentrations in the ACSF. As stated in the methods, the authors used 2mM Calcium and 1mM Magnesium. The physiological concentrations are about 1.2 mM Calcium and 1 mM Magnesium, thus creating an ionic milieu that is likely to be less permissive for LTP. The authors should try whether under these conditions the biphasic synaptic change is gone/reduced and the excitability change persists. If so, they can then test whether the enhanced activity correlation is still seen. Also, these recordings should be performed at physiological temperature.
If this is not successful (or as an alternative to start with), the authors might try pharmacological or genetic LTP blockade (e.g. targeting NMDA receptors or CaMKII) or use weaker stimulation protocols (intrinsic plasticity has a lower induction threshold than LTP).
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