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. Author manuscript; available in PMC: 2026 Jul 1.
Published in final edited form as: Exp Neurol. 2025 Apr 4;389:115233. doi: 10.1016/j.expneurol.2025.115233

Decreased locus coeruleus multiunit activity in a mouse model of temporal lobe seizures with impaired consciousness

Marcus Valcarce-Aspegren 1, Patrick Paszkowski 1, Shixin Liu 1, Qian Wu 1, Sarah McGill 1, Lim-Anna Sieu 1, Hal Blumenfeld 1,2,3
PMCID: PMC12083539  NIHMSID: NIHMS2073863  PMID: 40189126

Abstract

People with temporal lobe epilepsy often suffer debilitating loss of consciousness during seizures. Rodent models have previously implicated the inhibition of brainstem and basal forebrain cholinergic neurons in the cortical impairment during these periods of impaired consciousness. However, there are still other subcortical pathways that remain largely unexplored. Our goal was to record multiunit activity in the locus coeruleus (LC) in an awake mouse model to help elucidate its potential role in this pathophysiology. Recordings were performed using head-fixed mice running on a wheel with chronically implanted bipolar electrodes in the right orbitofrontal cortex and bilateral dorsal hippocampi. Focal limbic seizures were induced via the application of current pulses into the HC and multiunit recordings were simultaneously obtained from the LC. We observed a significant decrease in firing of LC neurons during ictal impairment of running wheel behavior. There was also a concurrent, significant increase in power in the 1-4 Hz band in the OFC. This provides evidence of a LC noradrenergic pathway contributing to depressed arousal in focal limbic seizures. Further elucidation of these, and other pathways, will contribute better mechanistic understanding of ictal unconsciousness and may lead to novel, improved treatments for people with epilepsy.

Keywords: locus coeruleus, norepinephrine, consciousness, epilepsy, focal limbic seizure

Introduction

Loss of consciousness during focal limbic seizures significantly contributes to reduced quality of life for people with epilepsy (Vickrey et al., 2000). Previous work has established the network inhibition hypothesis, which posits that focal limbic seizures impair arousal through inhibition of subcortical structures, causing sleep-like, low-frequency cortical oscillations and impaired consciousness (Blumenfeld, 2021). Inhibited brainstem and basal forebrain cholinergic, serotonergic as well as putative glutamatergic thalamic signaling have previously been implicated as contributing pathways for depressed arousal in rat focal limbic seizures (Englot et al., 2008; Feng et al., 2017; Motelow et al., 2015; Zhan et al., 2016). A recent novel, awake, head-fixed mouse model demonstrated impaired behavioral responsiveness and cortical slow waves resembling human temporal lobe seizures, along with reduced cortical cholinergic neurotransmission (Sieu et al., 2024). However, the role of norepinephrine (NE) and the locus coeruleus (LC) during ictal unconsciousness has not been studied in detail. The LC-NE system is a target of interest in this context as it has been shown to have bilateral projections both to the limbic system and the frontal cortex (Room et al., 1981), has long been associated with the regulation of arousal and plays a significant role in sleep-wake transitions. For example, in awake mice optogenetic inhibition of LC increased cortical slow-wave activity and induced slow wave sleep (Carter et al., 2010). A recent study of LC neurons in anesthetized rats showed variable changes in neuronal activity during hippocampal seizures related to the location of neurons in the LC (Larsen et al., 2023). Therefore, our goal was to investigate the potential role of the LC-NE system in decreased ictal arousal in an awake behaving mouse model of focal temporal lobe seizures, and to determine if activity in this arousal system is decreased, as would be predicted by the network inhibition hypothesis (Blumenfeld, 2021). To this end, electrophysiology recordings in an awake, behaving mouse model were employed (Sieu et al., 2024). Better understanding of these pathways is critical to identify potential targets for therapeutic intervention.

Materials & Methods

Animal Preparation and Surgery

All procedures were performed in accordance with approved protocols of Yale University’s Institutional Animal Care and Use Committee. Animals were adult male and female C57BL/6 mice age 3-6 months. All surgeries were performed under deep anesthesia with ketamine (90 mg/kg) and xylazine (9 mg/kg). The mice were fixed in a stereotaxic head frame and received either pre- and post-operative buprenorphine (0.05 mg/kg), or pre-operative, long-acting Buprenorphine Ethiqa XR (3.25 mg/kg) as analgesia. Lidocaine (0.03 mg/kg) was injected into the scalp before the incision was made. After the scalp was removed, an electrical drill (Microtorque II, Ram Products) was used to make burr holes at coordinates above targeted structures relative to Bregma(Paxinos and Franklin, 2012). Stereotaxic coordinates relative to Bregma of final targeted electrode tip positions for right lateral orbitofrontal cortex (OFC) were anterior-posterior (AP) +2.22 mm, medial-lateral (ML) ±1.25 mm, dorsal-ventral (DV): −2.75 mm; for left and right CA1 of dorsal hippocampi (HC) they were AP −1.46 to −2.30 mm, ML ±1.50 mm, DV: −1.40 to −1.75 mm. Bipolar Teflon coated stainless steel electrodes (outer diameter: 0.150 mm, 8IE36332TWLE, P1 Technologies, Roanoke Va.) were placed in the right OFC and in the bilateral dorsal HC. An additional burr hole was drilled in the left frontal bone for a grounding screw (diameter: 1.6 mm, 8IE3639616XE, P1 Technologies). Bilateral access chambers were also drilled over the LC using stereotaxic coordinates of AP −5.45 mm, ML ±0.85mm and sealed with silicone for later, unilateral acute recordings of either the left or right LC. The animals received Carprofen (5 mg/kg) subcutaneously before waking up as well as 24h later. They then recovered for five days postoperatively before being trained to run on the wheel for one additional day prior to the recording session. All recordings occurred in awake, head-fixed animals running freely on a wheel (Figure 1A). Once experiments were completed, animals were euthanized using intraperitoneal injection of Euthasol (>450mg/kg) combined with Ketamine-xylazine. Brains were then harvested for histological analysis.

Figure 1. Decreased multiunit activity in locus coeruleus during focal limbic seizures accompanied by depressed cortical arousal and impaired running wheel speed.

Figure 1.

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A. Experimental setup showing mouse head-fixed on running wheel with electrodes recording multiunit activity (MUA) from locus coeruleus (LC), as well as local field potentials from hippocampus (HC) and lateral orbital frontal cortex (OFC). Stimulation (Stim) is delivered to HC to induce focal limbic seizures.

B. Example of histology showing electrode trace marked with DiI in the locus coeruleus, with noradrenergic neurons marked with GFP-linked anti-tyrosine hydroxylase (Anti-TH).

C. Example of a focal limbic seizure in the awake, head-fixed mouse. Stimulus artifact from current injection (2s, 60Hz) initiating seizure is marked by “STIM.” Note that the animal stopped running (indicated by no change in wheel position in bottom trace), during the ictal period.

D. Zoomed in comparison of MUA and LFP tracings comparing pre-ictal baseline and ictal recordings. The ictal recording shows polyspike discharges in HC, a visibly appreciable decrease in LC MUA activity, and concurrent slow waves in the OFC.

Electrophysiology

Mice were head-fixed on a running wheel, and the silicone was gently removed. Sterile saline was used to clean the exposed brain surface and a high-impedance monopolar tungsten microelectrode (UEWMGGSEDNNM, FHC), was slowly lowered into one of the access chambers to reach the left or right LC (DV −3.75mm). The location of the LC electrode was marked by dipping it in fluorescent carbocyanine dye DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Thermo Fisher Scientific D3911)(Matsubayashi et al., 2008). LC signals from the monopolar microelectrode were acquired with, and amplified (x 1000) by, an A-M Systems amplifier (Model 1800) at 400 - 10,000 Hz to acquire multiunit activity (MUA). LFP were concurrently measured with the bilaterally implanted chronic bipolar electrodes in the dorsal HCs as well as from the right OFC using an A-M Systems Model 1800, with 1000x amplification and 0.1 to 100 Hz band pass filters for OFC; and with 1000x amplification and 1 to 500 Hz band pass filters for HC. Wheel position was measured with a MA3 Analogue 10-Bit Encoder (US Digital Corporation). All signals were digitized with a Power 1401 using Spike2 v8 software (CED). MUA were sampled at 20,000 Hz; LFP and wheel position at 1,000Hz.

Focal seizures were induced unilaterally in one hippocampus using a 2-second, 60-Hz train of square biphasic pulses, 1ms duration per phase (Model 2100, A-M Systems). Current was titrated to the smallest amplitude which induced a seizure of >15-second duration, with amplitudes ranging from 20 to 40 μA. Activity in the OFC was monitored for propagation of polyspike activity and seizures that were deemed to be generalizing in this way were subsequently excluded from analysis. Ultimately, there were n=6 animals and n=8 seizures analyzed. Three mice were male and three females, there were no statistically significant differences between the female and male animals. In five out of six animals, only one seizure was able to be induced per mouse without secondary generalization. However, in one of the animals we were able to trigger three separate focal seizures with no generalization. There was always a 20-minute interval between stimulations, with an observed return of electrophysiological baseline prior.

Immunohistochemistry and microscopy

Brains were fixed with 4% paraformaldehyde and cut in 60 μm slices for histology. Placement of chronically implanted electrodes was visualized with Cresyl violet staining. Noradrenergic neurons were stained with anti-tyrosine hydroxylase monoclonal antibodies with Alexa Fluor 488 conjugate (TH: normal donkey serum, 1:1000; Millipore Sigma MAB318-AF488)(Itoi et al., 2011). The electrode trace was visualized with DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Thermo Fisher Scientific D3911) (Matsubayashi et al., 2008) (Figure 1B). A Leica DM6B Microsystems microscope and the Leica LAS Microimage system were used to identify the electrode trace and relevant anatomic structures, identified using a standard anatomical atlas of the mouse brain(Paxinos and Franklin, 2012).

Data analysis

Spike-2 (v8, CED) was used to obtain LFP delta power (1–4 Hz fast Fourier transform) and MUA root-mean-square voltage (Vrms), both calculated in overlapping 1-second windows. Subsequent analysis was in MATLAB (v9.8.0.1380330 (R2020a) Update 2; MathWorks). LFP delta power and Vrms were further smoothed with a 2s running average to improve readability. Wheel position values divided by sampling interval was used to derive wheel speed. A 5Hz low-pass filter was applied to the speed values to remove artifacts created by the wheel position resetting after each full revolution. Wheel speed was smoothed to improve readability using a Gaussian-weighted moving average filter with a 2 s window length. Analysis epochs were as follows: pre-ictal (baseline) = 15 s preceding seizure; ictal = first 14 s of seizure (defined as HC polyspike discharges); post-ictal = 15 s following end of HC seizure. Values shown are mean across seizures ±SEM. Data between analysis epochs were compared statistically using two-sided Wilcoxon signed ranked tests, with Bonferroni-adjusted significance threshold p < 0.05.

Results

MUA decreases in LC during focal limbic seizures with increased cortical LFP delta power and impaired behavior

Like human temporal lobe seizures with impaired consciousness(Englot et al., 2010), focal limbic seizures in the mouse model exhibited polyspike discharges in the hippocampus, along with cortical slow wave activity in OFC and behavioral arrest demonstrated through decreased locomotion (Fig 1C, D). These changes in the mouse model were accompanied by decreased MUA firing in the LC during seizures (Fig 1C, D). The ictal decrease in LC MUA, increase in cortical slow waves, and decrease in running wheel speed all showed recovery during the post-ictal period (Fig 2).

Figure 2. Group data showing decreased locus coeruleus MUA, increased cortical slow waves and reduced running wheel speed across seizures.

Figure 2.

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A. Locus coeruleus (LC) multiunit activity (MUA) root mean squared voltage (Vrms) mean timecourses in the pre-ictal, ictal, and post-ictal periods. Stimulus (STIM) was 60 Hz, 2s train of pulses to hippocampus (Fig 1). B. Change in LC MUA Vrms for each seizure in the ictal and post-ictal periods compared to pre-ictal baseline period. C. Lateral orbital frontal cortical (OFC) delta frequency (1-4Hz) power mean timecourse. D. Changes in OFC delta power for each seizure compared to pre-ictal period. E. Running wheel speed timecourse. F. Changes in running wheel speed compared to pre-ictal period. Traces in A, C, E are mean ± SEM. Round symbols in B, D, F are mean percent change for the relevant analysis epoch in each seizure, with boxes indicating 25th percentile (lower line), median (middle line), and 75th percentile (upper line), and whiskers indicating minimum and maximum overall values. * p < 0.05 by Wilcoxon signed-rank test with Bonferroni correction. N=8 seizures in 6 animals.

To quantify the importance of LC modulation during ictal unconsciousness in focal limbic seizures, we calculated group statistics across seizures (Fig 2). The MUA Vrms in LC decreased on average by 16.5 ± 10.7 % (mean ± SEM) in the ictal period compared to baseline (P = 0.02; Figure 2A, B) and recovered with no significant difference from baseline during the post-ictal period (P = 0.55; Figure 2A, B). Simultaneously, LFP delta (1–4 Hz) power in OFC increased markedly by a mean of 1,762 ± 1,887% during the ictal period (P = .008; Fig 2C, D) and trended towards recovery in the post-ictal period but remaining significantly elevated at 553 ± 752% above baseline (P = .008; Fig 2C, D). There was a concurrent decrease in behavior quantified by running wheel speed, which showed decreases in the ictal epoch with a mean change of 79.1 ± 22.4% compared to baseline (P = .008; Fig 2E, F). During the post ictal period speeds remained somewhat decreased by a mean of 52.6 ± 41.4% compared to baseline (P = .02; Fig 2E, F). These results demonstrate a significant decrease in LC MUA activity during focal limbic seizures with concurrent OFC slow-waves and impaired behavior.

Discussion

There is a growing body of evidence for the idea that impaired consciousness in focal seizures involves complex, long-range inhibitory effects on cortical neurons by way of the subcortical arousal systems and their neurotransmitters(Blumenfeld, 2012; Blumenfeld et al., 2004; Englot et al., 2009; Kundishora et al., 2017; Zhao et al., 2020). This indirect mechanism by which seizures originating in the hippocampus interact with subcortical structures that project to the neocortex results in cortical slowing similar to what is observed in deep sleep or anesthesia (Yue et al., 2020). This similarity between sleep and ictal unconsciousness is helpful in selecting targets of interest. For instance, as mentioned previously, the LC has been shown to play significant causal role in sleep-wake transitions, as well as in generating increased cortical slow-wave activity and propensity to enter slow-wave sleep when inhibited optogenetically in normal, awake mice (Carter et al., 2010). It therefore is easy to imagine that pathological inhibition of the LC during focal limbic seizures could similarly play a role in generating cortical slow wave activity, and the related cognitive impairment.

In this study, using a previously validated acute seizure model for focal limbic seizures with impaired arousal (Sieu et al., 2024), we acquired results that support this notion by showing a statistically significant decrease in LC activity during ictal periods of cortical slowing with behavioral arrest. However, it is noteworthy that running behavior did not recover to pre-ictal levels and that there was a continued increase in OFC delta power in the immediate post-ictal interval despite the return of LC firing. This suggests that there are other mechanisms aside from NE that contribute to postictal impairment that deserve further study. More generally, we also showed that it is possible to obtain stable, multiunit recordings from nuclei deep in the brainstem of awake, moving animals.

Previous studies have shown that complete disconnection of the limbic system from subcortical structures prevented cortical slowing (Englot et al., 2009). In addition, stimulation of subcortical arousal areas showed decreased cortical slow-wave activity and reversal of seizure-related impairment of consciousness (Furman et al., 2015; Gummadavelli et al., 2015; Kundishora et al., 2017; Xu et al., 2020). The LC projects to both the limbic system (Room et al., 1981) and arousal regions of the intralaminar thalamus (Condés-Lara, 1998). For instance, In addition and as mentioned previously, the LC plays a critical role in normal sleep-wake transition, with optogenetic stimulation of the LC during sleep resulting in consistent and immediate transition to wakefulness. These findings, taken in conjunction with the results of our study, position the LC as an excellent target for optogenetic stimulation during focal limbic seizures in order to potentially restore arousal and to establish a causal role for the LC in the network inhibition hypothesis. In addition to optogenetic or electrical stimulation as possible future mechanistic and therapeutic avenues of investigation, a valuable approach would be pharmacological modulation of noradrenergic, cholinergic or other receptors to see if there are significant behavioral or electrophysiological effects during focal limbic seizures.

Another important area for future investigation is the role of other neurotransmitters interacting with modulation of NE to produce impaired arousal. Prior work suggests that both increased inhibitory GABAergic input and reduced excitatory input may contribute in parallel to decreased subcortical arousal in the cholinergic system during focal limbic seizures (Andrews et al., 2019; Zhao et al., 2020). Similar mechanisms may contribute to reduced subcortical arousal in noradrenergic systems in the ictal and postictal periods, and these mechanisms as well as other arousal systems such as dopamine or serotonin (Zhan et al., 2016) should be investigated further in future work. In addition, although the acute electrically-induced seizure model used here has been validated in prior studies of focal limbic seizures (Englot et al., 2008; Englot et al., 2009; Feng et al., 2017; Kundishora et al., 2017; Motelow et al., 2015; Sieu et al., 2024) and has the advantage of predictable seizure occurrence to facilitate data acquisition, these findings should ultimately be confirmed in a chronic spontaneous seizure model.

Conclusion

A novel awake mouse model of focal limbic seizures4 was used to record multiunit activity from behaving mice before, during and after focal limbic seizures. During periods of ictal behavioral arrest measured by running wheel speed there was a concurrent, significant increase in OFC slow-wave activity together with significantly decreased firing in the LC, which eventually returned to baseline in the postictal period. This suggests that modulation of the LC plays a role in loss of consciousness during focal limbic seizures. Better understanding of the subcortical pathways that contribute to impaired consciousness during focal seizures could establish novel therapeutic avenues aiming to restore arousal and improve function during and following seizures.

Highlights.

  • Stable brainstem recordings possible in awake mouse model of focal limbic seizures

  • Locus coeruleus activity decreases during focal seizures with decreased awareness

  • Locus coeruleus activity decreases during focal seizures with cortical slowing

Acknowledgements

This work was supported by NIH R01 NS066974, R37 NS100901, the Mark Loughridge and Michele Williams Foundation, the Betsy and Jonathan Blattmachr family (to HB), the Richard K. Gershon, MD Medical Student Research Fellowship and the James G. Hirsch, MD Endowed Medical Student Research Award (to MVA)

Footnotes

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Declaration of Interests

None of the authors has anything to declare.

Conflict of Interest/Ethical Publication Statement

We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. None of the authors has any conflict of interest to disclose.

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