Ultrafast (400 Hz) network oscillations induced in mouse barrel cortex by optogenetic activation of thalamocortical axons

  1. Hang Hu
  2. Rachel E Hostetler
  3. Ariel Agmon

  • Curated by eLife

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    eLife assessment

    This manuscript provides a successful model to study the ultrafast brain oscillation-mediated brain circuitry and cellular mechanisms in sensory processing. Utilizing this model, the authors studied potential cellular mechanisms that generate ultrafast oscillations (250-600Hz) in the cortex. These oscillations correlate with sensory stimulation and might be relevant for perceiving relevant sensory inputs. The data reasonably support most of the claims by the authors in this manuscript.

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Abstract

Oscillations of extracellular voltage, reflecting synchronous, rhythmic activity in large populations of neurons, are a ubiquitous feature in the mammalian brain, and are thought to subserve important, if not fully understood roles in normal and abnormal brain function. Oscillations at different frequency bands are hallmarks of specific brain and behavioral states. At the higher end of the spectrum, 150-200 Hz ripples occur in the hippocampus during slow-wave sleep, and ultrafast (400-600 Hz) oscillations arise in the somatosensory cortices of humans and several other mammalian species in response to peripheral nerve stimulation or punctate sensory stimuli. Here we report that brief optogenetic activation of thalamocortical axons, in brain slices from mouse somatosensory (barrel) cortex, elicited in the thalamorecipient layer local field potential (LFP) oscillations which we dubbed “ripplets”. Ripplets originated in the postsynaptic cortical network and consisted of a precisely repeating sequence of 2‑5 negative transients, closely resembling hippocampal ripples but, at ~400 Hz, over twice as fast. Fast-spiking (FS) inhibitory interneurons fired highly synchronous 400 Hz spike bursts entrained to the LFP oscillation, while regular-spiking (RS), excitatory neurons typically fired only 1-2 spikes per ripplet, in antiphase to FS spikes, and received synchronous sequences of alternating excitatory and inhibitory inputs. We suggest that ripplets are an intrinsically generated cortical response to a strong, synchronous thalamocortical volley, and could provide increased bandwidth for encoding and transmitting sensory information. Importantly, optogenetically induced ripplets are a uniquely accessible model system for studying synaptic mechanisms of fast and ultrafast cortical and hippocampal oscillations.

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  1. Version published to 10.7554/elife.82412 on eLife
  2. eLife

    eLife assessment

    This manuscript provides a successful model to study the ultrafast brain oscillation-mediated brain circuitry and cellular mechanisms in sensory processing. Utilizing this model, the authors studied potential cellular mechanisms that generate ultrafast oscillations (250-600Hz) in the cortex. These oscillations correlate with sensory stimulation and might be relevant for perceiving relevant sensory inputs. The data reasonably support most of the claims by the authors in this manuscript.

  3. eLife

    Reviewer #1 (Public Review):

    Hu et al. present findings that extend the understanding of the cellular and synaptic basis of fast network oscillations in the sensory cortex. They developed the ex vivo model system to study synaptic mechanisms of ultrafast (>400Hz) network oscillation ("ripplets") elicited in layer 4 (L4) of the barrel cortex in the mouse brain slice by optogenetically activating thalamocortical axon terminals at L4, which mimic the thalamic transmission of somatosensory information to the cortex. This model allowed them to reproduce extracellular ripplet oscillations in the slice preparation and investigate the temporal relationship of cellular and synaptic response in fast-spiking (FS) inhibitory interneurons and regular spiking (RS) with extracellular ripplet oscillations to common excitatory inputs at these cells. FS cells show precisely timed firing of spike bursts at ripplet frequency, and these spikes are highly synchronized with neighboring FS cells. Moreover, the phase-locked temporal relationship between the ripplets and responses of FS and RS cells, although different phases, to thalamocortical activation are found to closely coincide with EPSCs in RS cells, which suggests that common excitatory inputs to FS and RS cells and their synaptic connectivity are essential to generate reverberating network activity as ripplet oscillations. Additionally, they show that spikes of FS cells in layer 5 (L5) reduced in the slice with a cut between L4 and L5, proposing that recurrent excitation from L4 excitatory cells induced by thalamocortical optogenetic stimulation is necessary to drive FS spike bursts in layer 5 (L5).

    Overall, this study helps extend our knowledge of the synaptic mechanisms of ultrafast oscillations in the sensory cortex. However, it would have been nice if the authors had utilized various methodologies and systems.

    Although the overall findings are interesting, the conclusion of the study could have been strengthened according to the following points:

    1. The authors investigate the temporal relationship between ripplets and FS and RS cells' response elicited by optogenetic activation of TC axon terminals, which is mainly supported by phase-locked responses of FS and RS cells with local ripplets oscillations to optogenetic activation. They also show highly synchronized FS-FS firing by eliminating electrical gap-junction and inhibitory synaptic connections to this synchrony. Based on these findings, the authors suggest that common excitatory inputs to FS and RS cells in L4 would be essential to generate these local ripplets. However, it interferes with the ability to follow the logical flow for biding other findings of phase-locking responses of FS and RS cells in ripplet oscillations in L4.

    2. The authors suggest that the optogenetic activation of TC axon terminal elicits local ripplet oscillations via synchronized spike burst of FS inhibitory interneurons and alternating EPSC-IPSC of RS cells in phase-locked with ripplets in L4 barrel cortex, which would be generated by following common excitatory inputs from the local circuits to these cells at the ripple frequency. Thus they intend to investigate the source of these excitatory inputs at this local network of L4 by suppressing the firing of L4 RS cells. However, they show FS spike bursts in L5B, instead of L4, due to the technical limitations of their experimental setup, as described in the manuscript. Although L5 FS spike bursts decrease after cutting the L4/L5 boundary, supposedly inhibiting excitatory input from L4 as depicted in Fig 6D in the author's manuscript, the interpretation of data seems overly extended because it does not necessarily represent cellular and synaptic activities which are phase-locked with the ripplets observed in L4.

    3. Authors suggested a circuit model. It would be recommended that the authors try to perform in silico analysis using the suggested model to explore the function of thalamocortical axons on the fast-spiking and regular-spiking neurons to support their circuit model.

  4. eLife

    Reviewer #2 (Public Review):

    This manuscript studied potential cellular mechanisms that generate ultrafast oscillations (250-600Hz) in the cortex. These oscillations correlate with sensory stimulation and might be relevant for the perception of relevant sensory inputs. The authors combined ex-vivo whole-cell patch-clamp recordings, local field potential (LFP) recordings, and optogenetic stimulation of thalamocortical afferents. In a technical tour de force, they recorded pairs of fast-spiking (FS)-FS and FS-regular-spiking (RS) neurons in the cortex and correlated their activity with the LFP signal.

    Optogenetic activation of thalamic afferents generated ripple-like extracellular waveforms in the cortex, which the authors referred to as ripplets. The timing of the peaks and troughs within these ripplets was consistent across slices and animals. Activation of thalamic inputs induced precisely timed FS spike bursts and RS spikes, which were phase-locked to the ripplet oscillation. The authors described the sequences of RS and FS neuron discharge and how they phase-locked to the ripplet, providing a model for the cellular mechanism generating the ripplet.

    The manuscript is well-written and guides the reader step by step into the detailed analysis of the timing of ripplets and cellular discharges. The authors appropriately cite the known literature about ultrafast oscillations and carefully compare the novel ripplets to the well-known hippocampal ripples. The methods used (ex-vivo patch-clamp and LFP) were appropriate to study the cellular mechanisms underlying the ripplets.

    Overall, this manuscript develops means for studying the role of cortical ultrafast oscillations and proposes a coherent model for the cellular mechanism underlying these cortical ultrafast oscillations.

  5. eLife

    Reviewer #3 (Public Review):

    In this study, Hu et al. aimed to identify the neuronal basis of ultrafast network oscillations in S1 layer 4 and 5 evoked by the optogenetic activation of thalamocortical afferents in vitro. Although earlier in vivo demonstration of this short-lived (~25 ms) oscillation is sparse and its significance in detecting salient stimuli is not known the available publications clearly show that the phenomenon is consistently present in the sensory systems of several species including humans.

    In this study using optogenetic activation of thalamocortical (TC) fibers as a proxy for a strong sensory stimulus the in vitro model accurately captures the in vivo phenomenon. The authors measure the features of oscillatory LFP signals together with the intracellular activity of fast-spiking (FS) interneurons in layer 4 and 5 as well as in layer 4 regular spiking (RS) cells. They accurately measure the coherence of intra- and extracellular activity and convincingly demonstrate the synchronous firing of FS cells and antiphase firing of RS and FS cells relative to the field oscillation.

    Major points:

    1. The authors conclude the FS cell network has a primary role in setting the frequency of the oscillation. While these data are highly plausible and entirely consistent with the literature only correlational not causal results are shown thus direct demonstration of the critical role of GABAergic mechanisms is missing.

    2. The authors put a strong emphasis on the role of RS-RS interactions in maintaining the oscillation once it was launched by a TC activity. Its direct demonstration, however, is not presented. The alternative scenario is that TC excitation provides a tonic excitatory background drive (or envelope) for interacting FS cells which then impose ultrafast, synchronized IPSPs on RS cells. Similar to the RS-RS drive in this scenario RS cells can also only fire in the "windows of opportunity" which explains their antiphase activity relative to FS cells, but RS cells themselves do not participate in the maintenance of oscillation. Distinguishing between these two scenarios is critical to assess the potential impact of ultrafast oscillation in sensory transmission. If TC inputs are critical the magnitude of thalamic activity will set the threshold for the oscillation if RS-RS interactions are important intracortical operation will build up the activity in a graded manner.

    Earlier theoretical studies (e.g Brunel and Wang, 2003; Geisler et al., 2005) strongly suggested that even in the case of the much slower hippocampal ripples (below 200 Hz) phasic activation of local excitatory cells cannot operate at these frequencies. Indeed, rise time, propagation, and integration of EPSPs can likely not take place in the millisecond (or submillisecond) range required for efficient RS-RS interactions. The alternative scenario (tonic excitatory background coupled with FS-FS interactions) on the other hand has been clearly demonstrated in the case of the CA3 ripples in the hippocampus (Schlingloff et al., 2014. J.Nsci).

    When the properties of the ultrafast oscillation were tested as various stimulation strengths (Figure 2) weaker stimulation resulted in less precise timing. If TC input is indeed required only to launch the oscillation not to maintain it, this is not expected since once a critical number of RS cells were involved to start the activity their rhythmicity should no longer depend on the magnitude of the initial input. On the other hand, if the entire transient oscillation depends on TC excitation weaker input would result in less precise firing.

    1. The experiments indicating the spread of phasic activity from L4 RS to L5 FS cells can not be accepted as fully conclusive. The horizontal cut not only severed the L4 RS to L5 FS connections but also many TC inputs to the L5 FS apical dendrites as well as the axons of L4 FS cells to L5 FS cells both of which can be pivotal in the translaminar spread.
  6. eLife

    Author Response:

    We thank the three reviewers for their thoughtful comments and constructive critique.

    Reviewer #1 (Public Review):

    Hu et al. present findings that extend the understanding of the cellular and synaptic basis of fast network oscillations in the sensory cortex. They developed the ex vivo model system to study synaptic mechanisms of ultrafast (>400Hz) network oscillation ("ripplets") elicited in layer 4 (L4) of the barrel cortex in the mouse brain slice by optogenetically activating thalamocortical axon terminals at L4, which mimic the thalamic transmission of somatosensory information to the cortex. This model allowed them to reproduce extracellular ripplet oscillations in the slice preparation and investigate the temporal relationship of cellular and synaptic response in fast-spiking (FS) inhibitory interneurons and regular spiking (RS) with extracellular ripplet oscillations to common excitatory inputs at these cells. FS cells show precisely timed firing of spike bursts at ripplet frequency, and these spikes are highly synchronized with neighboring FS cells. Moreover, the phase-locked temporal relationship between the ripplets and responses of FS and RS cells, although different phases, to thalamocortical activation are found to closely coincide with EPSCs in RS cells, which suggests that common excitatory inputs to FS and RS cells and their synaptic connectivity are essential to generate reverberating network activity as ripplet oscillations. Additionally, they show that spikes of FS cells in layer 5 (L5) reduced in the slice with a cut between L4 and L5, proposing that recurrent excitation from L4 excitatory cells induced by thalamocortical optogenetic stimulation is necessary to drive FS spike bursts in layer 5 (L5).

    Overall, this study helps extend our knowledge of the synaptic mechanisms of ultrafast oscillations in the sensory cortex. However, it would have been nice if the authors had utilized various methodologies and systems.

    Although the overall findings are interesting, the conclusion of the study could have been strengthened according to the following points:

    1. The authors investigate the temporal relationship between ripplets and FS and RS cells' response elicited by optogenetic activation of TC axon terminals, which is mainly supported by phase-locked responses of FS and RS cells with local ripplets oscillations to optogenetic activation. They also show highly synchronized FS-FS firing by eliminating electrical gap-junction and inhibitory synaptic connections to this synchrony. Based on these findings, the authors suggest that common excitatory inputs to FS and RS cells in L4 would be essential to generate these local ripplets. However, it interferes with the ability to follow the logical flow for biding other findings of phase-locking responses of FS and RS cells in ripplet oscillations in L4.

    We understand the reviewer’s issue with the logical flow of our argument. We will address this concern by textual changes and/or by rearranging the order of the presentation and figures.

    2. The authors suggest that the optogenetic activation of TC axon terminal elicits local ripplet oscillations via synchronized spike burst of FS inhibitory interneurons and alternating EPSC-IPSC of RS cells in phase-locked with ripplets in L4 barrel cortex, which would be generated by following common excitatory inputs from the local circuits to these cells at the ripple frequency. Thus they intend to investigate the source of these excitatory inputs at this local network of L4 by suppressing the firing of L4 RS cells. However, they show FS spike bursts in L5B, instead of L4, due to the technical limitations of their experimental setup, as described in the manuscript. Although L5 FS spike bursts decrease after cutting the L4/L5 boundary, supposedly inhibiting excitatory input from L4 as depicted in Fig 6D in the author's manuscript, the interpretation of data seems overly extended because it does not necessarily represent cellular and synaptic activities which are phase-locked with the ripplets observed in L4.

    We have not studied network oscillation in layer 5 at the same level of detail we have studied layer 4; however the oscillations in both layers are phase locked. We will show this as supplemental data in the revised manuscript.

    3. Authors suggested a circuit model. It would be recommended that the authors try to perform in silico analysis using the suggested model to explore the function of thalamocortical axons on the fast-spiking and regular-spiking neurons to support their circuit model.

    We agree that a computational model of the layer 4 network, demonstrating ripplets in silico, would enhance our understanding of this re-discovered ultrafast oscillation. Moreover, such a model would also help constrain the allowable parameter space of other, existing models of layer 4 or of the complete cortical column, as the ability of these existing models to recreate ripplets in response to strong, synchronous thalamocortical activation could now be used as a stringent test of the assumptions underlying these models. We hope to reproduce ripplets in silico, within an experimentally constrained parameter space, in a near future study.

    Reviewer #2 (Public Review):

    This manuscript studied potential cellular mechanisms that generate ultrafast oscillations (250-600Hz) in the cortex. These oscillations correlate with sensory stimulation and might be relevant for the perception of relevant sensory inputs. The authors combined ex-vivo whole-cell patch-clamp recordings, local field potential (LFP) recordings, and optogenetic stimulation of thalamocortical afferents. In a technical tour de force, they recorded pairs of fast-spiking (FS)-FS and FS-regular-spiking (RS) neurons in the cortex and correlated their activity with the LFP signal.

    Optogenetic activation of thalamic afferents generated ripple-like extracellular waveforms in the cortex, which the authors referred to as ripplets. The timing of the peaks and troughs within these ripplets was consistent across slices and animals. Activation of thalamic inputs induced precisely timed FS spike bursts and RS spikes, which were phase-locked to the ripplet oscillation. The authors described the sequences of RS and FS neuron discharge and how they phase-locked to the ripplet, providing a model for the cellular mechanism generating the ripplet.

    The manuscript is well-written and guides the reader step by step into the detailed analysis of the timing of ripplets and cellular discharges. The authors appropriately cite the known literature about ultrafast oscillations and carefully compare the novel ripplets to the well-known hippocampal ripples. The methods used (ex-vivo patch-clamp and LFP) were appropriate to study the cellular mechanisms underlying the ripplets.

    Overall, this manuscript develops means for studying the role of cortical ultrafast oscillations and proposes a coherent model for the cellular mechanism underlying these cortical ultrafast oscillations.

    We thank the reviewer for his supportive comments.

    Reviewer #3 (Public Review):

    In this study, Hu et al. aimed to identify the neuronal basis of ultrafast network oscillations in S1 layer 4 and 5 evoked by the optogenetic activation of thalamocortical afferents in vitro. Although earlier in vivo demonstration of this short-lived (~25 ms) oscillation is sparse and its significance in detecting salient stimuli is not known the available publications clearly show that the phenomenon is consistently present in the sensory systems of several species including humans.

    In this study using optogenetic activation of thalamocortical (TC) fibers as a proxy for a strong sensory stimulus the in vitro model accurately captures the in vivo phenomenon. The authors measure the features of oscillatory LFP signals together with the intracellular activity of fast-spiking (FS) interneurons in layer 4 and 5 as well as in layer 4 regular spiking (RS) cells. They accurately measure the coherence of intra- and extracellular activity and convincingly demonstrate the synchronous firing of FS cells and antiphase firing of RS and FS cells relative to the field oscillation.

    Major points:

    1. The authors conclude the FS cell network has a primary role in setting the frequency of the oscillation. While these data are highly plausible and entirely consistent with the literature only correlational not causal results are shown thus direct demonstration of the critical role of GABAergic mechanisms is missing.

    We find that blocking fast inhibition (by puffing a gabazine solution locally) converts ripplets into long-duration paroxysmal events with high-frequency firing of both RS and FS cells. While we do not think that this experiment is diagnostic in distinguishing between competing models (in all models fast inhibition is a necessary component), we will add these experiments as supplemental material.

    1. The authors put a strong emphasis on the role of RS-RS interactions in maintaining the oscillation once it was launched by a TC activity. Its direct demonstration, however, is not presented. The alternative scenario is that TC excitation provides a tonic excitatory background drive (or envelope) for interacting FS cells which then impose ultrafast, synchronized IPSPs on RS cells. Similar to the RS-RS drive in this scenario RS cells can also only fire in the "windows of opportunity" which explains their antiphase activity relative to FS cells, but RS cells themselves do not participate in the maintenance of oscillation. Distinguishing between these two scenarios is critical to assess the potential impact of ultrafast oscillation in sensory transmission. If TC inputs are critical the magnitude of thalamic activity will set the threshold for the oscillation if RS-RS interactions are important intracortical operation will build up the activity in a graded manner.

    Earlier theoretical studies (e.g Brunel and Wang, 2003; Geisler et al., 2005) strongly suggested that even in the case of the much slower hippocampal ripples (below 200 Hz) phasic activation of local excitatory cells cannot operate at these frequencies. Indeed, rise time, propagation, and integration of EPSPs can likely not take place in the millisecond (or submillisecond) range required for efficient RS-RS interactions. The alternative scenario (tonic excitatory background coupled with FS-FS interactions) on the other hand has been clearly demonstrated in the case of the CA3 ripples in the hippocampus (Schlingloff et al., 2014. J.Nsci).

    The Schlingloff et al. study is important, and we actually think that their results, and many of their conclusions, are consistent with our own. We agree with these authors that “…PV cells are essential for the initiation and maintenance of sharp waves and the generation of ripple oscillations”, that “…perisomatic inhibition enforces ripple synchrony by phase-locking firing during SWRs”, and also that “…neuronal coupling via gap junctions is not essential in ripple synchronization”. We also agree that “The tonic excitatory ‘envelope’ arising from the buildup of activity of PCs drives the firing of PV cells”, as far as initiation of ripples in CA3 is concerned. In our model system, thalamocortical excitation serves the same role, of initiating the oscillation. However I do not see how the data of Schlingloff et al. support the conclusion that (in the legend to their Fig. 11) “…there is no cycle-by-cycle reciprocal interaction between the PCs and the PV [interneurons]”, or the implication that FS cells function as independent pacemakers “…because of their reciprocal inhibition”, as their FINO model suggests. The Schlingloff et al. data clearly show cycle-by-cycle alternations of EPSCs and IPSCs (their Fig. 1C, D, as well as their Fig. 7B), as we show in our Fig. 5A. These phasic EPSCs, occurring at ripple frequency, by necessity originate from pyramidal cells synchronized (as a population) to the ripple oscillation, as indeed shown in their multi-unit recordings. This precise, phasic (and clearly not “tonic”) excitatory drive cannot be uncoupled from the ripple (or ripplet) oscillation, and therefore cannot be dismissed as a factor driving the oscillation.

    The strongest evidence the Schlingloff et al. study provides that FS cells synchronize independently of excitatory cells – and then impose this oscillation on the excitatory cells - is in their demonstration of ripples generated by prolonged direct optogenetic stimulation of PV cells, in the presence of glutamatergic blockers (their Fig. 6). However this manipulation worked only in some of their slices, and the oscillations only lasted as long as the light stimulus and therefore were exogenously driven rather than network driven. They do not show intracellular responses from either inhibitory or excitatory cells, nor multi-unit activity, during this manipulation, so it is difficult to know if excitatory cells were indeed entrained to the same frequency, as the FINO model posits. Nevertheless this is a very interesting experiment which we plan to attempt in our own model system in a future study.

    When the properties of the ultrafast oscillation were tested as various stimulation strengths (Figure 2) weaker stimulation resulted in less precise timing. If TC input is indeed required only to launch the oscillation not to maintain it, this is not expected since once a critical number of RS cells were involved to start the activity their rhythmicity should no longer depend on the magnitude of the initial input. On the other hand, if the entire transient oscillation depends on TC excitation weaker input would result in less precise firing.

    Our interpretation for the lesser spike precision with a weaker optogenetic stimulation is that fewer FS cells fired upon the initial thalamocortical volley, and therefore a weaker IPSP wavefront was propagated to RS cells allowing for a wider “window of opportunity” for RS firing, and this loss of synchrony then propagated from cycle to cycle. This interpretation will be added in the revised manuscript.

    1. The experiments indicating the spread of phasic activity from L4 RS to L5 FS cells can not be accepted as fully conclusive. The horizontal cut not only severed the L4 RS to L5 FS connections but also many TC inputs to the L5 FS apical dendrites as well as the axons of L4 FS cells to L5 FS cells both of which can be pivotal in the translaminar spread.

    FS cells do not have apical dendrites so we assume the reviewer meant to say “L5 RS apical dendrites”; however if the cut reduced the excitability of L5 RS cells, that only strengthens our conclusion that RS firing is required for maintaining the oscillation. While the cut could have also disrupted L4 FS to L5 FS connections, we are not aware of any evidence that such inter-laminar connections exist. On the other hand, the Pluta et al. 2015 study shows very robust excitatory connections between L4 RS and L5 FS cells.

    Having said that, we agree with the reviewer (indeed, with all three reviewers) that the L4/L5 cut experiments are not conclusive, and we will make this clear in our discussion in the revised manuscript. We plan to do a more conclusive test of our model by using a transgenic line to express inhibitory opsins specifically in L4. This will require expressing ChR2 in the thalamus by virus injection and a careful comparison of ripplets between the two models; we therefore reserve these experiments to a future study.

  7. Version published to 10.1101/2022.09.02.506344v1 on bioRxiv