Disrupted Hippocampal Theta-Gamma Coupling and Spike-Field Coherence Following Experimental Traumatic Brain Injury

Reviewer #1 (Public review):

Summary:

This study investigated how traumatic brain injury affects oscillatory and single-unit hippocampal activity in awake-behaving rats.

Strengths:

The use of high-density laminar electrodes enabled precise localization of recording sites. To ensure an unbiased, rigorous approach, single-unit analysis was performed by a reviewer who was blind to experimental conditions. A proof of concept study was undertaken to characterize the pathology that resulted from the specific TBI model used in the main study. There was an effort to link abnormalities in hippocampal activity to memory disruption by running a cohort of rats on the Morris Water Maze task.

Weaknesses:

The paper is written as if the experiment was exploratory and not hypothesis-driven despite the fact that there is a wealth of experimental evidence about this TBI model that could have informed very specific predictions to test a hypothesis that is only hinted at in the discussion. The number of rats used for the spatial working memory experiment is not reported. Some of the statistics are not completely reported. It is also unclear what the rationale was for recording single units in a novel and familiar environment. Furthermore, this analysis comparing single-unit activity between familiar and novel environments is quite rudimentary. There are much more rigorous analyses to answer the question of how hippocampal single-unit firing patterns differ across changes in environments. There are details lacking about the number of units recorded per session and per rat, all of which are usually reported in studies that record single units. Spatial working memory assessment is delegated to a single panel of a supplementary figure. More importantly, there is no effort to dissociate between spatial working memory deficits and other motor, motivational, or sensory deficits that could have been driving the lower "memory score" in the experimental group.

Reviewer #2 (Public review):

Summary:

The authors investigate changes in theta-gamma phase amplitude coupling, and action potential entrainment to theta following traumatic brain injury (TBI). Both phenomena are widely hypothesized to be important for cognition, and the authors report deficits in both after TBI. The manuscript is well-written, the figures are well-constructed, and the author's use of high-level analysis methods for TBI EEG data collected from awake, behaving animals is welcome.

Major Comments:

- The animal n's are small (4 sham and 5 injured). In Figure 3, for instance, one wonders if panels D and E might have shown significant differences if more animals had been recorded.

- The text focuses on deficits in the theta and gamma bands, but the reduction in power appears to be broadband (see Figure 1F, especially Pyramidal cell layer panel). Therefore, the overall decrease in broadband (in the injured population) must be normalized between sham and injured animals before a selective comparison between sham and injured animals can be conducted. That is the only way that selective narrow bands i.e., theta and low gamma can be compared between the two cohorts. A brief discussion of the significance of a broadband decrease would be appreciated.

Reviewer #3 (Public review):

Summary:

In this study, the authors studied the effects of traumatic brain injury created by LFPI procedure on the CA1 at the network level. The major findings in this study seem to be that the TBI reduces theta and gamma powers in CA1, reduces phase-amplitude coupling in between theta and gamma bands as well as disrupts the gamma entrainment of interneurons. I think the authors have made some important discoveries that could help advance the understanding of TBI effects at the physiological level, however, more investigations into deciphering the relationship of the behavioral and brain states to the observed effects would help clarify the interpretations for the readers.

Strengths:

The authors in this study were able to combine behavioral verification of the TBI model with the laminar electrophysiological recordings of the CA1 region to bring forward network-level anomalies such as the temporal coordination of network-level oscillations as well as in the firing of the interneurons. Indeed, it seems that the findings may serve future studies to functionally better understand and/or refine the therapies for the TBI.

Weaknesses:

Discoveries made in the paper and their broad interpretations can be helped with further characterization and comparison among the brain and behavioral states both during immobility and movement. The impact of brain injury in several parts of the brain can alter brain-wide LFP and/or behavior. The altered behavior and/or LFP patterns might then lead to reduced spiking and unreliable LFP oscillations in the hippocampus. Hence, claims made in the abstract such as "These results reveal deficits in information encoding and retrieval schemes essential to cognition that likely underlie TBI-associated learning and memory impairments, and elucidate potential targets for future neuromodulation therapies" do not have enough evidence to test whether the disruptions were information encoding and retrieval related or due to sensory-motor and/or behavioral deficits that could also occur during TBI.

Movement velocity is already known to be correlated to the entrainment of spikes with the theta rhythm and also in some cases with the gamma oscillations. So, it is important to disentangle the differences in behavioral variables and the observed effects. As an example, the author's claims of disrupted temporal coding (as shown in the graphical abstract) might have suffered from these confounds. The observed results of reduced entrainment might, on one hand, be due to the decreased LFP power (induced by injury in different brain areas) resulting in altered behavior and/or the unreliable oscillations of the LFP bands such as theta and gamma, rather than memory encoding and retrieval related disruption of spikes synchrony to the rhythms, while on the other hand, they may simply be due to reduced excitability in the neurons particularly in the behavioral and brain state in which the effects were observed, rather than disrupted temporal code. Hence, further investigations into dissociating these factors could help readers mechanistically understand the interesting results observed by the authors.

Author response:

We would like to thank the editors and reviewers for their constructive feedback, and we look forward to addressing their comments in the revised manuscript. We also appreciate the acknowledgment that the use of laminar electrodes in awake-behaving animals is an important advancement for the TBI community, and that our results provide a potential physiological link between coalescing TBI pathologies and cognitive deficits. We believe that integrating the reviewer comments will help to make our analyses even more rigorous and will improve the overall manuscript. Please find comments related to specific concerns raised in the public review below:

The paper is written as if the experiment was exploratory and not hypothesis-driven despite the fact that there is a wealth of experimental evidence about this TBI model that could have informed very specific predictions to test a hypothesis that is only hinted at in the discussion… It is also unclear what the rationale was for recording single units in a novel and familiar environment. Furthermore, this analysis comparing single-unit activity between familiar and novel environments is quite rudimentary. There are much more rigorous analyses to answer the question of how hippocampal single-unit firing patterns differ across changes in environments.

Previous mechanistic and physiological studies suggested interneuronal dysfunction following TBI that we hypothesized would disrupt oscillatory dynamics underlying temporal coding (single unit entrainment to theta/gamma, phase precession, and phase-amplitude coupling). These are known to support hippocampal-dependent learning and memory tasks such as the Morris Water Maze. While we did not record during a goal-directed behavioral task, the goal of recording in a familiar and novel environment was to assess remapping across these environments. Unfortunately, occupancy in the two environments was not high enough to rigorously characterize place cell specificity and phase precession or and investigate remapping, although putative place cells were identified. Despite this shortcoming, we were still able to confirm that the spike timing of interneurons relative to hippocampal oscillations was disrupted which we believe underlies the massive reduction in theta-gamma phase amplitude coupling reported. This opens the door to more strongly hypothesis-driven, mechanistic studies (i.e. closed loop stimulation) to alter the spike timing of interneurons relative to theta phase and potentially rescue these effects on phase amplitude coupling and behavior.

The number of rats used for the spatial working memory experiment is not reported. Some of the statistics are not completely reported… There are details lacking about the number of units recorded per session and per rat, all of which are usually reported in studies that record single units.

The number of rats used for the spatial working memory task was reported in the text and Figure legend where the statistics were reported, but we will ensure that the statistics are more completely reported by including relevant statistical results and parameters outside of the test used and p-value. Additionally, we will report the number of units recorded per animal.

Spatial working memory assessment is delegated to a single panel of a supplementary figure. More importantly, there is no effort to dissociate between spatial working memory deficits and other motor, motivational, or sensory deficits that could have been driving the lower "memory score" in the experimental group

The spatial working memory deficit that we report in the Morris Water Maze is not a novel finding and has been demonstrated numerous times in this TBI model. Our goal in including this was to increase the rigor of the study by verifying this deficit in our hands at the injury level used for these physiology experiments. The dissociation between spatial working memory deficits and other motor, motivational, or sensory deficits from TBI in the Morris Water Maze (e.g. swim speed and escape latency with visible platforms) has been well characterized in this TBI model at many injury levels including more severe injuries than those used in this study. We will address this in the Discussion as it is an important point.

The text focuses on deficits in the theta and gamma bands, but the reduction in power appears to be broadband (see Figure 1F, especially Pyramidal cell layer panel). Therefore, the overall decrease in broadband (in the injured population) must be normalized between sham and injured animals before a selective comparison between sham and injured animals can be conducted. That is the only way that selective narrow bands i.e., theta and low gamma can be compared between the two cohorts. A brief discussion of the significance of a broadband decrease would be appreciated.

We agree that there is a broadband downward shift in power following TBI especially in the pyramidal cell layer. We will include a normalization of the power spectra in order to specifically compare the theta and gamma bands between sham and injured rats and include discussion about the broadband decrease.

Discoveries made in the paper and their broad interpretations can be helped with further characterization and comparison among the brain and behavioral states both during immobility and movement. The impact of brain injury in several parts of the brain can alter brain-wide LFP and/or behavior. The altered behavior and/or LFP patterns might then lead to reduced spiking and unreliable LFP oscillations in the hippocampus. Hence, claims made in the abstract such as "These results reveal deficits in information encoding and retrieval schemes essential to cognition that likely underlie TBI-associated learning and memory impairments, and elucidate potential targets for future neuromodulation therapies" do not have enough evidence to test whether the disruptions were information encoding and retrieval related or due to sensory-motor and/or behavioral deficits that could also occur during TBI.

Movement velocity is already known to be correlated to the entrainment of spikes with the theta rhythm and also in some cases with the gamma oscillations. So, it is important to disentangle the differences in behavioral variables and the observed effects. As an example, the author's claims of disrupted temporal coding (as shown in the graphical abstract) might have suffered from these confounds. The observed results of reduced entrainment might, on one hand, be due to the decreased LFP power (induced by injury in different brain areas) resulting in altered behavior and/or the unreliable oscillations of the LFP bands such as theta and gamma, rather than memory encoding and retrieval related disruption of spikes synchrony to the rhythms, while on the other hand, they may simply be due to reduced excitability in the neurons particularly in the behavioral and brain state in which the effects were observed, rather than disrupted temporal code. Hence, further investigations into dissociating these factors could help readers mechanistically understand the interesting results observed by the authors.

We agree that changes in hippocampal physiology that we report could arise due to disrupted inputs from TBI, and this study is inherently limited due to recording exclusively from CA1. We chose to record from the hippocampus due to its importance for learning and memory, and its vulnerability in TBI. Future studies will investigate how hippocampal afferents are affected by injury, and we hope that the layer-specific changes we report will help to inform which inputs may be preferentially disrupted. Importantly, these inputs along with local processing within the hippocampus change drastically depending on the behavior of the animal. We will more rigorously assess movement and the behavioral state of the rats when comparing physiological properties, especially the firing rates reported in Figure 3.