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Abstract

Mild traumatic brain injury (mTBI) is the most prevalent form of brain injury, and persons sustaining an mTBI can experience a number of neurological and behavioral impairments that in some cases can linger for weeks to months. The hippocampus plays a prominent role in learning and memory and in novelty detection. The activity of hippocampal neurons is modulated by oscillations of the local field potential, especially theta oscillations. Theta oscillations are thought to coordinate and organize hippocampal neuronal activities to encode information essential for memory formation. Both clinical and experimental studies have indicated that hippocampal function is often impaired in persons with mTBI. However, the neural mechanisms underlying these impairments are not well understood. In the present study, we examined the activity of hippocampal neurons and their coupling to local theta oscillations in awake, freely moving animals with mTBI. Electrodes were implanted into the CA1 subfield of the hippocampus in sham and mTBI rats in order to simultaneously record local field potentials and the activity of CA1 neurons. Consistent with previous studies, we found that theta power is reduced after mTBI. This reduction was not associated with any significant loss of either cholinergic or parvalbumin-positive interneurons in the medial septum nucleus/diagonal band. Furthermore, an analysis of waveform properties of unit activity indicated that the coupling of both excitatory and inhibitory (wide and narrow) neurons to theta in rats with mild fluid percussion injury (mFPI) is severely compromised. The lack of hippocampal neuronal coupling to theta may contribute to the poor memory of animals with mFPI.

Introduction

Mild traumatic brain injury (mTBI) is the most prevalent form of TBI^1,2 and can result from a blow or jolt to the head. This sudden movement of the head causes rapid acceleration/deceleration of the brain, which stretches brain cells and alters their membrane properties and triggers intracellular cascades.^3–5 The symptoms of an mTBI may include neurological (e.g., headache and balance problems), cognitive (e.g., learning and memory impairments), and behavioral (e.g., anxiety and depression) problems that can last for days, weeks, or even longer.^6–15 As these symptoms can manifest in the absence of overt contusion, hemorrhage, or cell loss, neuronal dysfunction is likely an underlying mechanism.^16–21

The hippocampus is a key structure involved in learning and memory, and dysfunction to this structure has been consistently implicated in the cognitive dysfunction that occurs following TBI.^16,22,23 Hippocampal activity is coordinated, in part, by the coupling of hippocampal neurons to local field potentials (LFPs), including theta oscillations.^24–28 Hippocampal theta oscillations are generated by cholinergic and GABAergic input from within the medial septal nucleus/diagonal band (MSN/DB), and its coupling to hippocampal neurons is thought to coordinate and organize neuronal activity in order to encode information essential for memory formation.^25,29,30 For example, studies have demonstrated that disruption of theta oscillations is associated with poor performance in tasks that involve spatial navigation or recognizing changes within an environment.^31,32 In the context of TBI, a few studies have shown that experimental TBI can reduce theta power in rats and mice^33–35 and that the recovery of theta power correlates with improved cognitive function.^36,37 Furthermore, recent studies have demonstrated that electrical stimulation of the MSN/DB or optogenetic stimulation of CA1 neurons at theta frequency improves learning and memory.^38,39

Although mild fluid percussion injury (mFPI) has been reported to cause impairments in hippocampal tasks, it was not known if these impairments were associated with an inability of hippocampal neurons to couple to theta. Recently, it was observed that the ability of hippocampal neurons to couple to theta is unperturbed after FPI.⁴⁰ However, this study was carried out in anesthetized animals, and theta power under anesthesia was not impaired in brain-injured rats compared with controls. Thus, it remains to be determined if the ability of hippocampal neurons to couple to theta oscillations is impaired in non-anaesthetized animals. To address this knowledge gap, we simultaneously measured LFPs and the activity/coupling of neurons in the ipsilateral CA1 subfield of awake, freely moving rats with mFPI,^41–43 and compared these with uninjured control rats.

Methods

Materials

All experimental procedures were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the recommendations provided in the Guide for the Care and Use of Laboratory Animals. A total of 16 male Sprague–Dawley rats (Envigo, 300–350 g) were used in these studies. For immunohistochemistry, antibodies to NeuN were purchased from Millipore (Burlington, MA, USA), antibodies against parvalbumin were obtained from Abcam (Cambridge, UK), and antibodies that recognize somatostatin were purchased from Invitrogen (Waltham, MA, USA). Thirty-two channel electrode arrays were obtained from Bio-Signal (Dallas, TX, USA), which were plugged into 32-channel unity gain headstages obtained from Blackrock (Salt Lake City, UT, USA).

Lateral mFPI and electrode implantation surgeries

Lateral mFPI was delivered as described previously.^44–46 Anesthetized rats were mounted on a stereotaxic frame, and a midline 4.8 mm diameter craniotomy was made midway between bregma and lambda and 1.0 mm lateral to the midline on the right hemisphere. A hub (modified from a 20-gauge needle) was implanted into the burr hole and affixed using dental cement. Once the rat regained its toe pinch reflex, a single sterile saline pulse was delivered with a pressure of 1.5 atm over base room pressure (Custom Design & Fabrication, Richmond, VA, USA). Sham animals were anesthetized and received a scalp incision but not the hub implantation or injury. Following a 7-day recovery period, both sham (n = 4) and injured (n = 4) rats were implanted with 32-channel (BioSignal, Dallas, TX, USA) electrodes directed at the dorsal CA1 subfield of the hippocampus. The electrode array was positioned 3.8 mm posterior to bregma and 2.0 mm lateral from the midline, at a depth of 2.2 mm, which was adjusted during surgery to maximize the yield of these fixed arrays.

Electrophysiological recordings and spike analysis

Recordings were performed on days 17–19 post-mFPI while animals explored a 60 cm × 60 cm arena. LFPs were sampled at 10 kHz and digitally filtered (0.5–500 Hz). Spike data were sampled at 30 kHz, digitally filtered (250 Hz hi-pass filter) and thresholds set to 50–80 µV. Spikes were sorted using a manual clustering program (Blackrock Offline Spike Sorter) based on peak amplitude, spike width, valley amplitude, area, and principal components 1–4.

Cell type classification

The units isolated through spike sorting were classified based on three criteria, their firing rate, tau rise, and peak latency values.^45,47 Before the classification, a minimum firing rate threshold of 0.62 Hz was established that reliably estimates peak latency based on a multistep subsampling of a highly active reference neuron. Units with a firing rate below this threshold were excluded from the analysis. Tau rise values were used as a criterion for a subset of neurons and calculated by fitting an exponential function to their autocorrelograms (ACGs) using the fit_ACG function from the Cell Explorer library.⁴⁵ Neurons were classified as putative pyramidal neurons if they exhibited peak latencies >0.425 ms and mean firing rates below 5 Hz.^48,49 Cells with peak latencies < 0.425 ms and a firing rate above 5 Hz were classified as putative narrow interneurons, while cells with tau rise value above 6 ms, a mean firing rate above 5 Hz, and a peak latency above 0.425 ms were identified as wide interneurons.

Phase coupling analysis

To obtain the theta oscillation from LFP, a single recording site with the highest theta power was selected as the reference signal and was band-pass filtered between 6 and 12 Hz. A threshold was set so that periods where theta power was <10% of the total band power were excluded from this analysis. Theta cycles were identified in sliding 1-second windows with 0.5-second overlaps using the find oscillations function in NeuroExplorer. A phase angle (trough designated as 0 degrees) was assigned to each recorded neuronal spike. The phase distribution of each neuron was tested for nonuniformity using Rayleigh’s method.^50,51 This produced a Rayleigh statistic and p value based on the length of mean vector and number of spikes. No neurons with an obvious multimodal phase distribution was detected. Any cells with a Bonferoni adjusted p value <0.05 were considered theta coupled.

Immunohistochemistry and quantification

In order to avoid the potential damage caused by electrode implantation/explantation, separate groups of sham and mFPI rats (n = 4/group) were generated for histopathological analysis. At one month post-injury, animals were euthanized by an overdose of sodium pentobarbital (100 mg/kg) and then transcardially perfused with 4% paraformaldehyde. Thirty µm thick sections were prepared, and multiplex immunohistochemistry was carried out using antibodies directed against NeuN, choline acetyltransferase (ChAT), parvalbumin, and somatostain.^52,53 Images of brain sections were captured using a Zeiss Axio Scan.Z1 slide scanning microscope using acquisition parameters that were kept constant for each multiplex panel. Initial tile stitching and shading corrections were performed online using Zen Blue (version 2.6). Images containing the MSN/DB or CA1 subfield of the ipsilateral hippocampus were captured using Zen Lite (version 3.5), then converted to grayscale, and thresholded. Signals having circularity (0.50–1.00) and size (0–1 mm²) were counted using ImageJ 1.54b. Cell outlines were then independently verified by comparison to the original photomicrograph by a trained observer.

Statistical analysis

For immunohistochemistry, comparisons between groups were carried out using two-tailed t-tests. Analysis of firing rate properties for sham and mFPI groups used a nonparametric Mann–Whitney U test due to unequal sample sizes. Results are presented as mean ± standard error of mean, and a Bonferroni correction was applied separately for each comparison within the sham and mFPI groups to control for multiple testing errors. All data were tested for normality.

Results

1.5 Atm FPI does not cause overt brain damage or cell loss in the MSN/DB

An mFPI is typically defined at the pressure range of 0.9–1.5 atm^42,54,55 and does not cause gross damage or neuronal loss. Consistent with these previous reports, the representative images shown in Figure 1 show that 1.5 atm FPI did not cause any overt brain damage (Fig. 1A) or visible neuronal loss in either the injured cortex (*, site of injury) or the hippocampus (Fig. 1B). However, as we have observed that mFPI significantly reduces theta power, we questioned if this is associated with a loss of neurons in the MSN/DB. To address this possibility, we counted the number of ChAT- and parvalbumin-positive neurons, as these have been shown to be critical for the regulation of hippocampal theta oscillations (Fig. 1C). Representative ChAT-immunostained sections containing the MSN/DB from a sham and an mFPI animal are shown in Figure 1D. Unlike more severe forms of injury,⁵⁶ mFPI did not cause any significant change in the number of ChAT-positive neurons in the MSN/DB (t = 0.363, p = 0.729). The representative images and summary data in Figure 1E show that the number of parvalbumin-positive neurons within the MSN/DB was also unaffected by 1.5 atm mFPI (t = 0.105, p = 0.920). Within the hippocampus (Fig. 1F), the cholinergic and parvalbumin-positive inhibitory neurons of the MSN/DB project onto both excitatory and inhibitory neurons to regulate their firing. We have previously observed that mFPI reduces the number of parvalbumin-positive inhibitory neurons,⁴⁵ a finding replicated here (t = 5.569, p = 0.002, Fig. 1G). However, whether somatostatin-positive cells are lost as a result of mFPI had not been examined. Figure 1H shows representative montage images (and summary cell counts) of the ipsilateral CA1 subfield from a sham and a 1.5 atm FPI rat immunostained for somatostatin. Quantification of somatostatin-positive cells did not reveal any significant loss of these cells in the ipsilateral CA1 as a result of mFPI compared to sham controls (t = −0.291, p = 0.788).

FIG. 1.

mFPI does not cause overt damage or cell loss in the MSN/DB. (A) Representative pictures of brains taken from a sham and a 1.5 atm FPI rat demonstrating no visible damage. (B) Montage images of NeuN immunoreactivity in coronal tissue sections from a sham and an mFPI rat. No overt cell loss was seen in the injured cortex (yellow asterisk: site of injury) or the hippocampus. (C) Simplified schematic showing the key cell types in the MSN/DB for generating hippocampal theta rhythms. (D) Representative photomicrographs and cell count summary data for ChAT-positive cells in the medial septal nucleus. (E) Representative photomicrographs and cell count summary data for PV-positive cells in the MSN/DB. (F) Simplified schematic showing the key cell types in the hippocampus regulated by theta rhythms. (G) Representative photomicrographs and cell count summary data for PV-positive cells in the ipsilateral CA1 subfield of the hippocampus. (H) Representative photomicrographs and cell count summary data for SST-positive cells in the ipsilateral CA1 subfield of the hippocampus. Sham, n = 4; mFPI, n = 4. Data are presented as mean ± SEM. *, p < 0.05. ChAT, choline acetyltransferase; mFPI, mild fluid percussion injury; MSN/DB, medial septal nucleus/diagonal band; PV, parvalbumin; SEM, standard error of mean; SST, somatostatin.

mFPI impairs neuronal coupling to hippocampal theta oscillations in awake animals

Whether hippocampal neurons retain the ability to couple their firing to theta oscillations (i.e., firing at a specific phase angle of theta) in awake brain-injured animals has not been examined. We used single unit activity from the CA1 subfield and oscillations of LFPs to evaluate if neuronal spike-theta phase coupling is altered in mFPI rats.⁴⁵ Neurons were initially grouped, based on their waveform properties, as either excitatory or inhibitory.⁴⁰ Representative spike waveforms (black line = mean; gray = standard deviation) of a representative pyramidal neuron, a narrow interneuron, and a wide interneuron are shown in Figure 2A. Figure 2B shows the spike autocorrelograms for each of these neurons. An equation with triple exponential functions⁴⁷ was fitted to each autocorrelogram, yielding a tau rise value (red line) that can be used to help distinguish between pyramidal neurons (which have a long latency, and low tau rise time) and wide interneurons (having a long latency, but high tau rise time). Neurons that could not be assigned to any of these three groups remained as unclassified. Figure 2C (3D plots) and Supplementary Figure S1 (2D plots) illustrate the putative identity of all recorded neurons in sham and mFPI animals, plotted according to their peak latency (X-axis), tau rise time (Y-axis), and firing rate (Z-axis). Our analysis showed a dramatic decrease in the proportion of cells that could be classified as narrow or wide interneurons in mFPI animals (7% of neurons) compared with sham (15% of neurons). Although it has been reported that the firing rate of neurons is not significantly altered in anesthetized FPI rats,⁴⁰ we observed a significant decrease in overall firing rate comparing awake mFPI (median = 2.237 Hz) rats to sham (median = 3.974 Hz) controls (z = 4.084, p = 4.427 × 10⁻⁵, Fig. 2D). This decrease was not due to reductions in the firing rate of any specific neuronal subtype, as each class of identified neurons showed reduced activity, though these reductions did not reach statistical significance (Fig. 2E).

FIG. 2.

Classification of neuron types in sham and mFPI animals. (A) Representative spike waveforms of a pyramidal neuron, a narrow interneuron, and a wide interneuron, with peak latencies noted. (B) Representative autocorrelograms of the neurons presented in (A). An exponential function (red) is fitted to each autocorrelogram, yielding a tau rise value that is used to distinguish between pyramidal neurons and wide interneurons. (C) 3D plot illustrating all identified neurons with their putative cell types in sham (n = 202) and mFPI (n = 106) animals. Neurons that do not meet the criteria of any cell type are labeled as unclassified. Axes represent peak latency (X-axis), tau rise time (Y-axis), and firing rate (Z-axis). (D) Overall firing rates of recorded neurons in sham and mFPI rats. Median firing rates for sham and mFPI animals were 3.974 and 2.237 spikes/s, respectively. (E) Distribution of firing rates across all cell types in sham and mFPI animals. Data are presented as the median (white dots) with an interquartile range spanning from the first to the third (black bar). *, p < 0.05. mFPI, mild fluid percussion injury.

We next examined the degree of coupling to the local theta oscillation for each identified neuron. Figure 3A shows the raw LFP presented in light gray and filtered theta signal (6–12 Hz band-pass filter) in dark gray. Consistent with previous reports,^33,34,38,57 animals with mFPI exhibited overall lower theta power compared to sham controls. When the firing patterns of a representative pyramidal neuron recorded in a sham and an mFPI rat were overlaid on the local theta oscillation, it can be seen that while the pyramidal neuron in the sham tends to fire (blue dots) near the peak of a theta cycle, the pyramidal neuron from the rat with mFPI (red dots) appears to fire randomly with respect to theta. Polar histograms (Bin size = 14.4°) of these cells are shown in Figure 3B (blue bar = length of the mean vector adjusted to the maximum bin count). The polar plots indicate that while the representative pyramidal neuron from the sham operated animal fires close to the peak of theta (approximately at 190^°), the firing of the pyramidal neuron from the mFPI rat shows no preference for any phase of the local theta oscillation. Figure 3C shows the mean vector amplitude relative to the spike count for each identified neuron. Spike-phase coupling to the local theta oscillation was evaluated using the Rayleigh test.⁵¹ The dotted line represents the vector amplitude for each spike count above which the firing pattern is significantly different from a uniform distribution (after correction for multiple comparisons). The summary results shown in Figure 3C indicate that for sham animals, the firing of the majority of pyramidal neurons, as well as narrow and wide inhibitory neurons, is coupled (filled circles above dashed line) to the local theta oscillation. In contrast, the vast majority of neurons recorded from animals with mFPI show no coupling (open circles) to the local theta oscillation.

FIG. 3.

Reduced spike-theta coupling in mFPI animals. (A) Representative LFP signals from sham and mFPI animals. Raw LFP signals are presented in light gray and filtered signals (6–12 Hz band-pass filter) are presented in dark gray. mFPI animals exhibited overall lower theta power compared to sham controls. Colored dots (sham represented as blue; mFPI represented as red) indicate spike timings of a single putative pyramidal neuron in each animal relative to the local theta oscillation. Note that the majority of blue dots appear near the peak of the waveform (phase angle of 180°) in the sham animal, whereas the pyramidal neuron in the mFPI rat appears to fire at random in relation to theta. (B) Example polar histograms of the excitatory neurons shown in (A), where bins represent total spike phase counts relative to theta oscillations. The color bar indicates the length of mean vector of the polar histogram, adjusted to the maximum bin count. (C) The length of the mean vector are plotted against spike counts for each neuron in sham and mFPI animals. In each neuron, spike-phase coupling to theta cycle was tested using a Rayleigh test (p < 0.05, with a Bonferroni correction applied post hoc). Filled dots represent cells with significant spike-phase coupling, while unfilled dots indicate no coupling. Dashed lines indicate the threshold of Rayleigh test as a function of spike count. Sham animals demonstrated a significantly larger proportion of theta-coupled cells compared to mFPI animals. LFP, local field potential; mFPI, mild fluid percussion injury.

Discussion

The results presented in this article revealed the following key findings: (1) The decrease in theta power observed following mFPI was not associated with a decrease in the number of cholinergic or parvalbumin-positive interneurons within the MSN/DB but was concomitant with the selective loss of parvalbumin-positive neurons within the CA1 subfield of the hippocampus. This is consistent with the reduction in the number of neurons that could be classified as either narrow or wide inhibitory neurons. (2) In awake, freely moving rats, the overall firing rate of neurons resident to the ipsilateral CA1 subfield is significantly reduced in rats with mFPI. (3) While the majority of excitatory and inhibitory neurons within the CA1 subfield of sham animals are coupled to a preferred phase of the local theta rhythm, only a few neurons in the CA1 subfield of mFPI animals were coupled to theta. These findings may have implications in the learning and memory impairments observed following mTBI.

A large body of evidence supports specific roles for phase coupling in the timing of the firing of individual neurons. Although theta coupling has been reported to play a role in both spatial and nonspatial memory,^58,59 it has been primarily studied in the context of place cells and spatial navigation. When an animal enters a place cell’s field, the firing of the place cell becomes synchronized with a specific phase of the local theta oscillation (a process known as phase locking or coupling).^27,60 As the animal repeatedly explores its environment, the sequential activation of place cells causes them to become linked to each other, allowing them to be activated in sequence within a theta cycle.^28,61 This allows the animal to know where it has been and where it is going.⁶² During development, the ability to form theta sequenced firing patterns can be observed concurrent with the ability to form memories,⁶³ suggesting that theta locking is a critical component of spatial learning and memory. Consistent with this, phase locking has also been observed in sequence order memory formation in the hippocampus of humans.^64,65 Although a reduction in hippocampal theta power after brain injury had been previously reported,^33,34,38,57 whether this reduction was sufficient to alter neuronal coupling had not been investigated. Our results show that the coupling of both excitatory and inhibitory neurons within the CA1 subfield of awake, freely moving animals was significantly impaired after mFPI. These findings, coupled with our previous observations that place cell properties are perturbed after mFPI,⁴⁵ suggest that the inability of hippocampal neurons to couple to theta may contribute to learning and memory impairments.

Some limitations of this study need to be acknowledged. First, although we observed that the coupling of excitatory neurons to theta was impaired after mFPI, we did not specifically assess if the coupling of place cells was altered. However, given the robust nature of the lack of coupling we observed, and the estimation that 25–30% of recorded neurons have a place field,⁶⁶ it is likely that place cell coupling to theta was impaired. Second, although we have observed that hippocampal neurons fail to couple to local theta oscillations, whether reduced theta coupling contributes to learning and memory impairments in brain-injured animals has yet to be shown. We have previously demonstrated that optogenetic stimulation of hippocampal neurons at theta rescued learning and memory in brain-injured rats.³⁸ However, whether this improvement in learning and memory results in enhanced theta phase coupling has yet to be determined. Finally, another limitation is that only male rats were used. Additional studies will be required to determine if there are sex differences in the consequences of mFPI on theta coupling and if the stage of estrus plays a role in these effects.^67–69

Footnotes

Authors’ Contributions

All authors have read and approved the article prior to submission. B.N.: Formal analysis, investigation, writing—original draft, writing—review and editing, and visualization; R.H.: Investigation, writing—review and editing, and supervision; J.I.B.: Investigation and writing—review and editing; D.R.: Investigation and writing—review and editing; A.N.M.: Writing—original draft, writing—review and editing, visualization, and supervision; J.Z.: Investigation, P.K.D.: Conceptualization, writing—original draft, writing—review and editing, visualization, supervision, project administration, and funding acquisition.

Author Disclosure Statement

J.I.B. is now the Senior Neuroscientist for Williams, Actuated Medical, Inc. The other authors do not have any potential conflicts of interest to disclose.

Funding Information

This work was supported by the National Institutes of Health (NS118329) grant awarded to P.K.D. and funding from the TIRR Foundation awarded to J.I.B. and R.H.

Supplementary Material

Abbreviations Used

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Mild Traumatic Brain Injury Impairs Coupling of CA1 Neuronal Activity to Theta Oscillations

Abstract

Introduction

Methods

Materials

Lateral mFPI and electrode implantation surgeries

Electrophysiological recordings and spike analysis

Cell type classification

Phase coupling analysis

Immunohistochemistry and quantification

Statistical analysis

Results

1.5 Atm FPI does not cause overt brain damage or cell loss in the MSN/DB

mFPI impairs neuronal coupling to hippocampal theta oscillations in awake animals

Discussion

Footnotes

Authors’ Contributions

Author Disclosure Statement

Funding Information

Supplementary Material

Abbreviations Used

References

Supplementary Material