Electrophysiology publications

The hearing abilities of mammals are impacted by factors such as social cues, habitat, and physical characteristics. Despite being used commonly to study social behaviors, hearing of the monogamous prairie vole (Microtus ochrogaster) has never been characterized. In this study, anatomical features are measured and auditory brainstem responses (ABRs) are used to measure auditory capabilities of prairie voles, characterizing monaural and binaural hearing and hearing range. Sexually naive male and female voles were measured to characterize differences due to sex. It was found that prairie voles show a hearing range with greatest sensitivity between 8 and 32 kHz, binaural hearing across interaural time difference ranges appropriate for their head sizes. No differences are shown between the sexes in binaural hearing or hearing range (except at 1 kHz), however, female voles have increased amplitude of peripheral ABR waves I and II and longer latency of waves III and IV compared to males. The results confirm that prairie voles have a broad hearing range, binaural hearing consistent with rodents of similar size, and differences in amplitudes and thresholds of monaural physiological measures between the sexes. These data further highlight the necessity to understand sex-specific differences in neural processing that may underly variability in responses between sexes.

Multielectrode arrays for interfacing with neurons are of great interest for a wide range of medical applications. However, current electrodes cause damage over time. Ultra small carbon fibers help to address issues but controlling the electrode site geometry is difficult. Here we propose a methodology to create small, pointed fiber electrodes (SPFe). We compare the SPFe to previously made blowtorched fibers in characterization. The SPFe result in small site sizes [Formula: see text] with consistently sharp points (20.8 ± 7.64°). Additionally, these electrodes were able to record and/or stimulate neurons multiple animal models including rat cortex, mouse retina, Aplysia ganglia and octopus axial cord. In rat cortex, these electrodes recorded significantly higher peak amplitudes than the traditional blowtorched fibers. These SPFe may be applicable to a wide range of applications requiring a highly specific interface with individual neurons.

Sudden and surprising sensory events trigger neural processes that swiftly adjust behavior. To study the phylogenesis and the mechanism of this phenomenon, we trained two male rhesus monkeys to keep a cursor inside a visual target by exerting force on an isometric joystick. We examined the effect of surprising auditory stimuli on exerted force, scalp electroencephalographic (EEG) activity, and local field potentials (LFPs) recorded from the dorsolateral prefrontal cortex. Auditory stimuli elicited (1) a biphasic modulation of isometric force, a transient decrease followed by a corrective tonic increase, and (2) EEG and LFP deflections dominated by two large negativepositive waves (N70 and P130). The EEG potential was symmetrical and maximal at the scalp vertex, highly reminiscent of the human vertex potential. Electrocortical potentials and force were tightly coupled: the P130 amplitude predicted the magnitude of the corrective force increase, particularly in the LFPs recorded from deep rather than superficial cortical layers. These results disclose a phylogenetically preserved corticomotor mechanism supporting adaptive behavior in response to salient sensory events. Significance Statement Survival in the natural world depends on an animals capacity to adapt ongoing behavior to abrupt unexpected events. To study the neural mechanisms underlying this capacity, we trained monkeys to apply constant force on a joystick while we recorded their brain activity from the scalp and the prefrontal cortex contralateral to the hand holding the joystick. Unexpected auditory stimuli elicited a biphasic force modulation: a transient reduction followed by a corrective adjustment. The same stimuli also elicited EEG and LFP responses, dominated by a biphasic wave that predicted the magnitude of the behavioral adjustment. These results disclose a phylogenetically preserved corticomotor mechanism supporting adaptive behavior in response to unexpected events.

The auditory steady-state response (ASSR) is a cortical oscillation induced by trains of 40 Hz acoustic stimuli. While the ASSR has been widely used in clinic measurement, the underlying neural mechanism remains poorly understood. In this study, we investigated the contribution of different stages of auditory thalamocortical pathwaymedial geniculate body (MGB), thalamic reticular nucleus (TRN), and auditory cortex (AC)to the generation and regulation of 40 Hz ASSR in C57BL/6 mice of both sexes. We found that the neural response synchronizing to 40 Hz sound stimuli was most prominent in the GABAergic neurons in the granular layer of AC and the ventral division of MGB (MGBv), which were regulated by optogenetic manipulation of TRN neurons. Behavioral experiments confirmed that disrupting TRN activity has a detrimental effect on the ability of mice to discriminate 40 Hz sounds. These findings revealed a thalamocortical mechanism helpful to interpret the results of clinical ASSR examinations. Significance Statement Our study contributes to clarifying the thalamocortical mechanisms underlying the generation and regulation of the auditory steady-state response (ASSR), which is commonly used in both clinical and neuroscience research to assess the integrity of auditory function. Combining a series of electrophysiological and optogenetic experiments, we demonstrate that the generation of cortical ASSR is dependent on the lemniscal thalamocortical projections originating from the ventral division of medial geniculate body to the GABAergic interneurons in the granule layer of the auditory cortex. Furthermore, the thalamocortical process for ASSR is strictly regulated by the activity of thalamic reticular nucleus (TRN) neurons. Behavioral experiments confirmed that dysfunction of TRN would cause a disruption of mice's behavioral performance in the auditory discrimination task.

Intracortical microelectrode arrays (MEAs) can be used in a range of applications, from basic neuroscience research to providing an intimate interface with the brain as part of a brain-computer interface (BCI) system aimed at restoring function for people living with neurological disorders or injuries. Unfortunately, MEAs tend to fail prematurely, leading to a loss in functionality for many applications. An important contributing factor in MEA failure is oxidative stress resulting from chronically inflammatory-activated microglia and macrophages releasing reactive oxygen species (ROS) around the implant site. Antioxidants offer a means for mitigating oxidative stress and improving tissue health and MEA performance. Here, we investigate using the clinically available antioxidant dimethyl fumarate (DMF) to reduce the neuroinflammatory response and improve MEA performance in a rat MEA model. Daily treatment of DMF for 16 weeks resulted in a significant improvement in the recording capabilities of MEA devices during the sub-chronic (Weeks 5–11) phase (42% active electrode yield vs. 35% for control). However, these sub-chronic improvements were lost in the chronic implantation phase, as a more exacerbated neuroinflammatory response occurs in DMF-treated animals by 16 weeks post-implantation. Yet, neuroinflammation was indiscriminate between treatment and control groups during the sub-chronic phase. Although worse for chronic use, a temporary improvement (<12 weeks) in MEA performance is meaningful. Providing short-term improvement to MEA devices using DMF can allow for improved use for limited-duration studies. Further efforts should be taken to explore the mechanism behind a worsened neuroinflammatory response at the 16-week time point for DMF-treated animals and assess its usefulness for specific applications.

Prediction provides key advantages for survival, and cognitive studies have demonstrated that the brain computes multilevel predictions. Evidence for predictions remains elusive at the neuronal level because of the complexity of separating neural activity into predictions and stimulus responses. We overcome this challenge by recording from single neurons from cortical and subcortical auditory regions in anesthetized and awake preparations, during unexpected stimulus omissions interspersed in a regular sequence of tones. We find a subset of neurons that responds reliably to omitted tones. In awake animals, omission responses are similar to anesthetized animals, but larger and more frequent, indicating that the arousal and attentional state levels affect the degree to which predictions are neuronally represented. Omission-sensitive neurons also responded to frequency deviants, with their omission responses getting emphasized in the awake state. Because omission responses occur in the absence of sensory input, they provide solid and empirical evidence for the implementation of a predictive process.

Objective. Characterizing the relationship between neuron spiking and the signals that electrodes record is vital to defining the neural circuits driving brain function and informing clinical brain-machine interface design. However, high electrode biocompatibility and precisely localizing neurons around the electrodes are critical to defining this relationship. Approach. Here, we demonstrate consistent localization of the recording site tips of subcellular-scale (6.8 µ m diameter) carbon fiber electrodes and the positions of surrounding neurons. We implanted male rats with carbon fiber electrode arrays for 6 or 12+ weeks targeting layer V motor cortex. After explanting the arrays, we immunostained the implant site and localized putative recording site tips with subcellular-cellular resolution. We then 3D segmented neuron somata within a 50 µ m radius from implanted tips to measure neuron positions and health and compare to healthy cortex with symmetric stereotaxic coordinates. Main results. Immunostaining of astrocyte, microglia, and neuron markers confirmed that overall tissue health was indicative of high biocompatibility near the tips. While neurons near implanted carbon fibers were stretched, their number and distribution were similar to hypothetical fibers placed in healthy contralateral brain. Such similar neuron distributions suggest that these minimally invasive electrodes demonstrate the potential to sample naturalistic neural populations. This motivated the prediction of spikes produced by nearby neurons using a simple point source model fit using recorded electrophysiology and the mean positions of the nearest neurons observed in histology. Comparing spike amplitudes suggests that the radius at which single units can be distinguished from others is near the fourth closest neuron (30.7 ± 4.6 µ m, X ˉ ± S) in layer V motor cortex. Significance. Collectively, these data and simulations provide the first direct evidence that neuron placement in the immediate vicinity of the recording site influences how many spike clusters can be reliably identified by spike sorting.

The ability to localize a sound source in complex environments is essential for communication and navigation. Spatial hearing relies predominantly on the comparison of differences in the arrival time of sound between the two ears, the interaural time differences (ITDs). Hearing impairments are highly detrimental to sound localization. While cochlear implants (CIs) have been successful in restoring many crucial hearing capabilities, sound localization via ITD detection with bilateral CIs remains poor. The underlying reasons are not well understood. Neuronally, ITD sensitivity is generated by coincidence detection between excitatory and inhibitory inputs from the two ears performed by specialized brainstem neurons. Due to the lack of electrophysiological brainstem recordings during CI stimulation, it is unclear to what extent the apparent deficits are caused by the binaural comparator neurons or arise already on the input level. Here, we use a bottom-up approach to compare response features between electric and acoustic stimulation in an animal model of CI hearing. Conducting extracellular single neuron recordings in gerbils, we find severe hyper-precision and moderate hyper-entrainment of both the excitatory and inhibitory brainstem inputs to the binaural comparator neurons during electrical pulse-train stimulation. This finding establishes conclusively that the binaural processing stage must cope with highly altered input statistics during CI stimulation. To estimate the consequences of these effects on ITD sensitivity, we used a computational model of the auditory brainstem. After tuning the model parameters to match its response properties to our physiological data during either stimulation type, the model predicted that ITD sensitivity to electrical pulses is maintained even for the hyper-precise inputs. However, the model exhibits severely altered spatial sensitivity during electrical stimulation compared to acoustic: while resolution of ITDs near midline was increased, more lateralized adjacent source locations became inseparable. These results directly resemble recent findings in rodent and human CI listeners. Notably, decreasing the phase-locking precision of inputs during electrical stimulation recovered a wider range of separable ITDs. Together, our findings suggest that a central problem underlying the diminished ITD sensitivity in CI users might be the temporal hyper-precision of inputs to the binaural comparator stage induced by electrical stimulation.

Sensory hypersensitivity, especially in the auditory system, is a common symptom in Fragile X syndrome (FXS), the most common monogenic form of intellectual disability. However, linking phenotypes across genetic background strains of mouse models has been a challenge and could underly some of the issues with translatability of drug studies to the human condition. This study is the first to characterize the auditory brain stem response (ABR), a minimally invasive physiological readout of early auditory processing that is also used in humans, in a commonly used mouse background strain model of FXS, C57BL/6J. We measured morphological features of pinna and head and used ABR to measure the hearing range, and monaural and binaural auditory responses in hemizygous males, homozygous females, and heterozygous females compared with those in wild-type mice. Consistent with previous study, we showed no difference in morphological parameters across genotypes or sexes. There was no significant difference in hearing range between the sexes or genotypes, however there was a trend towards high frequency hearing loss in male FXS mice. In contrast, female mice with homozygous FXS had a decreased amplitude of wave IV of the monaural ABR, while there was no difference in males for amplitudes and no change in latency of ABR waveforms across sexes and genotypes. Finally, males with FXS had an increased latency of the binaural interaction component (BIC) at 0 interaural timing difference compared with that in wild-type males. These findings further clarify auditory brain stem processing in FXS by adding more information across genetic background strains allowing for a better understanding of shared phenotypes.

Dominant neuroanatomical models hold that humans regulate their movements via loop-like cortico-subcortical networks, which include the subthalamic nucleus (STN), motor thalamus, and sensorimotor cortex (SMC). Inhibitory commands across these networks are purportedly sent via transient, burst-like signals in the β frequency (15–29 Hz). However, since human depth-recording studies are typically limited to one recording site, direct evidence for this proposition is hitherto lacking. Here, we present simultaneous multi-site recordings from SMC and either STN or motor thalamus in humans performing the stop-signal task. In line with their purported function as inhibitory signals, subcortical β-bursts were increased on successful stop-trials. STN bursts in particular were followed within 50 ms by increased β-bursting over SMC. Moreover, between-site comparisons (including in a patient with simultaneous recordings from SMC, thalamus, and STN) confirmed that β-bursts in STN temporally precede thalamic β-bursts. This highly unique set of recordings provides empirical evidence for the role of β-bursts in conveying inhibitory commands along long-proposed cortico-subcortical networks underlying movement regulation in humans.

Peripheral nerves carry sensory (afferent) and motor (efferent) signals between the central nervous system and other parts of the body. The peripheral nervous system (PNS) is therefore rich in targets for therapeutic neuromodulation, bioelectronic medicine, and neuroprosthetics. Peripheral nerve interfaces (PNIs) generally suffer from a tradeoff between selectivity and invasiveness. This work describes the fabrication, evaluation, and chronic implantation in zebra finches of a novel PNI that breaks this tradeoff by interfacing with small nerves. This PNI integrates a soft, stretchable microelectrode array with a 2‐photon 3D printed microclip (μcPNI). The advantages of this μcPNI compared to other designs are: a) increased spatial resolution due to bi‐layer wiring of the electrode leads, b) reduced mismatch in biomechanical properties with the nerve, c) reduced disturbance to the host tissue due to the small size, d) elimination of sutures or adhesives, e) high circumferential contact with small nerves, f) functionality under considerable strain, and g) graded neuromodulation in a low‐threshold stimulation regime. Results demonstrate that the μcPNIs are electromechanically robust, and are capable of reliably recording and stimulating neural activity in vivo in small nerves. The μcPNI may also inform the development of new optical, thermal, ultrasonic, or chemical PNIs as well.

Chronically implanted neural probes are powerful tools to decode brain activity however, recording population and spiking activity over long periods remains a major challenge. Here, we designed and fabricated flexible intracortical Michigan-style arrays with a shank cross-section per electrode of 250 μm $$^2$$ 2 utilizing the polymer paryleneC with the goal to improve the immune acceptance. As flexible neural probes are unable to penetrate the brain due to the low buckling force threshold, a tissue-friendly insertion system was developed by reducing the effective shank length. The insertion strategy enabled the implantation of the four, bare, flexible shanks up to 2 mm into the mouse brain without increasing the implantation footprint and therefore, minimizing the acute trauma. In acute recordings from the mouse somatosensory cortex and the olfactory bulb, we demonstrated that the flexible probes were able to simultaneously detect local field potentials as well as single and multi-unit activity. Additionally, the flexible arrays outperformed stiff probes with respect to yield of single unit activity. Following the successful in vivo validation, we further improved the microfabrication towards a double-metal-layer process, and were able to double the number of electrodes per shank by keeping the shank width resulting in a cross-section per electrode of 118 μm $$^2$$ 2 .

Bioelectric medicine treatments target disorders of the nervous system unresponsive to pharmacological methods. While current stimulation paradigms effectively treat many disorders, the underlying mechanisms are relatively unknown, and current neuroscience recording electrodes are often limited in their specificity to gross averages across many neurons or axons. Here, we develop a novel, durable carbon fiber electrode array adaptable to many neural structures for precise neural recording. Carbon fibers ( [Formula: see text] diameter) were sharpened using a reproducible blowtorchmethod that uses the reflection of fibers against the surface of a water bath. The arrays were developed by partially embedding carbon fibers in medical-grade silicone to improve durability. We recorded acute spontaneous electrophysiology from the rat cervical vagus nerve (CVN), feline dorsal root ganglia (DRG), and rat brain. Blowtorching resulted in fibers of 72.3 ± 33.5-degree tip angle with [Formula: see text] exposed carbon. Observable neural clusters were recorded using sharpened carbon fiber electrodes fromrat CVN ( [Formula: see text]), feline DRG ( [Formula: see text]), and rat brain ( [Formula: see text]). Recordings from the feline DRG included physiologically relevant signals from increased bladder pressure and cutaneous brushing. These results suggest that this carbon fiber array is a uniquely durable and adaptable neural recordingdevice. In the future, this device may be useful as a bioelectric medicine tool for diagnosis and closed-loop neural control of therapeutic treatments and monitoring systems.

The temporal frequency of sensory stimulation is a decisive factor in the plasticity of perceptual detection thresholds. However, surprisingly little is known about how distinct temporal parameters of sensory input differentially recruit activity of neuronal circuits in sensory cortices. Here we demonstrate that brief repetitive visual stimulation induces long-term plasticity of visual responses revealed 24 h after stimulation and that the location and generalization of visual response plasticity is determined by the temporal frequency of the visual stimulation. Brief repetitive low-frequency stimulation (2 Hz) is sufficient to induce a visual response potentiation that is expressed exclusively in visual cortex layer 4 and in response to a familiar stimulus. In contrast, brief, repetitive high-frequency stimulation (HFS, 20 Hz) is sufficient to induce a visual response potentiation that is expressed in all cortical layers and transfers to novel stimuli. HFS induces a long-term suppression of the activity of fast-spiking interneurons and primes ongoing gamma oscillatory rhythms for phase reset by subsequent visual stimulation. This novel form of generalized visual response enhancement induced by HFS is paralleled by an increase in visual acuity, measured as improved performance in a visual detection task.

Polychlorinated biphenyls (PCBs) are enduring environmental toxicants and exposure is associated with neurodevelopmental deficits. The auditory system appears particularly sensitive, as previous work has shown that developmental PCB exposure causes both hearing loss and gross disruptions in the organization of the rat auditory cortex. However, the mechanisms underlying PCB-induced changes are not known, nor is it known whether the central effects of PCBs are a consequence of peripheral hearing loss. Here, we study changes in both peripheral and central auditory function in rats with developmental PCB exposure using a combination of optical and electrophysiological approaches. Female rats were exposed to an environmental PCB mixture in utero and until weaning. At adulthood, auditory brainstem responses (ABRs) were measured, and synaptic currents were recorded in slices from auditory cortex layer 2/3 neurons. Spontaneous IPSCs (sIPSCs) and miniature IPSCs (mIPSCs) were more frequent in PCB-exposed rats compared with controls and the normal relationship between IPSC parameters and peripheral hearing was eliminated in PCB-exposed rats. No changes in spontaneous EPSCs were found. Conversely, when synaptic currents were evoked by laser photostimulation of caged-glutamate, PCB exposure did not affect evoked inhibitory transmission, but increased the total excitatory charge, the number and distance of sites that evoke a significant response. Together, these findings indicate that early developmental exposure to PCBs causes long-lasting changes in both inhibitory and excitatory neurotransmission in the auditory cortex that are independent of peripheral hearing changes, suggesting the effects are because of the direct impact of PCBs on the developing auditory cortex.

The mismatch negativity (MMN) is a key biomarker of automatic deviance detection thought to emerge from 2 cortical sources. First, the auditory cortex (AC) encodes spectral regularities and reports frequency-specific deviances. Then, more abstract representations in the prefrontal cortex (PFC) allow to detect contextual changes of potential behavioral relevance. However, the precise location and time asynchronies between neuronal correlates underlying this frontotemporal network remain unclear and elusive. Our study presented auditory oddball paradigms along with “no-repetition” controls to record mismatch responses in neuronal spiking activity and local field potentials at the rat medial PFC. Whereas mismatch responses in the auditory system are mainly induced by stimulus-dependent effects, we found that auditory responsiveness in the PFC was driven by unpredictability, yielding context-dependent, comparatively delayed, more robust and longer-lasting mismatch responses mostly comprised of prediction error signaling activity. This characteristically different composition discarded that mismatch responses in the PFC could be simply inherited or amplified downstream from the auditory system. Conversely, it is more plausible for the PFC to exert top-down influences on the AC, since the PFC exhibited flexible and potent predictive processing, capable of suppressing redundant input more efficiently than the AC. Remarkably, the time course of the mismatch responses we observed in the spiking activity and local field potentials of the AC and the PFC combined coincided with the time course of the large-scale MMN-like signals reported in the rat brain, thereby linking the microscopic, mesoscopic, and macroscopic levels of automatic deviance detection.

The nascent field of bioelectronic medicine seeks to decode and modulate peripheral nervous system signals to obtain therapeutic control of targeted end organs and effectors. Current approaches rely heavily on electrode-based devices, but size scalability, material and microfabrication challenges, limited surgical accessibility, and the biomechanically dynamic implantation environment are significant impediments to developing and deploying peripheral interfacing technologies. Here, we present a microscale implantable device – the nanoclip – for chronic interfacing with fine peripheral nerves in small animal models that begins to meet these constraints. We demonstrate the capability to make stable, high signal-to-noise ratio recordings of behaviorally-linked nerve activity over multi-week timescales. In addition, we show that multi-channel, current-steering-based stimulation within the confines of the small device can achieve multi-dimensional control of a small nerve. These results highlight the potential of new microscale design and fabrication techniques for realizing viable devices for long-term peripheral interfacing.

Selective enhancement of GABAergic inhibition is thought to impact many vital brain functions and interferes with the genesis and/or progression of numerous brain disorders. Here, we show that selectively increasing NMDA receptor activity in inhibitory neurons using an NMDAR positive allosteric modulator (PAM) elevates spiking activity of inhibitory neurons in vitro and in vivo. In vivo infusion of PAM increases spontaneous and sound-evoked spiking in inhibitory and decreases spiking in excitatory neurons, and increases signal-to-noise ratio in the primary auditory cortex. In addition, PAM infusion prior to noise trauma prevents the occurrence of tinnitus and reduction in GABAergic inhibition. These results reveal that selectively enhancing endogenous NMDAR activity on the GABAergic neurons can effectively enhance inhibitory activity and alter excitatory–inhibitory balance, and may be useful for preventing diseases that involve reduced inhibition as the major cause.

Dopamine guides behavior and learning through pleasure, according to classic understanding. Dopaminergic neurons are traditionally thought to signal positive or negative prediction errors (PEs) when reward expectations are, respectively, exceeded or not matched. These signed PEs are quite different from the unsigned PEs, which report surprise during sensory processing. But mounting theoretical accounts from the predictive processing framework postulate that dopamine, as a neuromodulator, could potentially regulate the postsynaptic gain of sensory neurons, thereby scaling unsigned PEs according to their expected precision or confidence. Despite ample modeling work, the physiological effects of dopamine on the processing of surprising sensory information are yet to be addressed experimentally. In this study, we tested how dopamine modulates midbrain processing of unexpected tones. We recorded extracellular responses from the rat inferior colliculus to oddball and cascade sequences, before, during, and after the microiontophoretic application of dopamine or eticlopride (a D2-like receptor antagonist). Results demonstrate that dopamine reduces the net neuronal responsiveness exclusively to unexpected sensory input without significantly altering the processing of expected input. We conclude that dopaminergic projections from the thalamic subparafascicular nucleus to the inferior colliculus could encode the expected precision of unsigned PEs, attenuating via D2-like receptors the postsynaptic gain of sensory inputs forwarded by the auditory midbrain neurons. This direct dopaminergic modulation of sensory PE signaling has profound implications for both the predictive coding framework and the understanding of dopamine function.

Visual cortex (VC) over-activation analysed by evoked responses has been demonstrated in congenital deafness and after long-term acquired hearing loss in humans. However, permanent hearing deprivation has not yet been explored in animal models. Thus, the present study aimed to examine functional and molecular changes underlying the visual and auditory cross-modal reaction. For such purpose, we analysed cortical visual evoked potentials (VEPs) and the gene expression (RT-qPCR) of a set of markers for neuronal activation (c-Fos) and activity-dependent homeostatic compensation (Arc/Arg3.1). To determine the state of excitation and inhibition, we performed RT-qPCR and quantitative immunocytochemistry for excitatory (receptor subunits GluA2/3) and inhibitory (GABAA-α1, GABAB-R2, GAD65/67 and parvalbumin-PV) markers. VC over-activation was demonstrated by a significant increase in VEPs wave N1 and by up-regulation of the activity-dependent early genes c-Fos and Arc/Arg3.1 (thus confirming, by RT-qPCR, our previously published immunocytochemical results). GluA2 gene and protein expression were significantly increased in the auditory cortex (AC), particularly in layers 2/3 pyramidal neurons, but inhibitory markers (GAD65/67 and PV-GABA interneurons) were also significantly upregulated in the AC, indicating a concurrent increase in inhibition. Therefore, after permanent hearing loss in the rat, the VC is not only over-activated but also potentially balanced by homeostatic regulation, while excitatory and inhibitory markers remain imbalanced in the AC, most likely resulting from changes in horizontal intermodal regulation.