Microglia transform in response to changes in sensory or neural activity, such as sensory deprivation. However, little is known about how specific frequencies of neural activity, or brain rhythms, affect microglia and cytokine signaling. Using visual noninvasive flickering sensory stimulation (flicker) to induce electrical neural activity at 40 hertz, within the gamma band, and 20 hertz, within the beta band, we found that these brain rhythms differentially affect microglial morphology and cytokine expression in healthy animals. Flicker induced expression of certain cytokines independently of microglia, including interleukin-10 and macrophage colony-stimulating factor. We hypothesized that nuclear factor κB (NF-κB) plays a causal role in frequency-specific cytokine and microglial responses because this pathway is activated by synaptic activity and regulates cytokines. After flicker, phospho–NF-κB colabeled with neurons more than microglia. Inhibition of NF-κB signaling down-regulated flicker-induced cytokine expression and attenuated flicker-induced changes in microglial morphology. These results reveal a mechanism through which brain rhythms affect brain function by altering microglial morphology and cytokines via NF-κB.

Repeated sequences of neural activity are a pervasive feature of neural networks in vivo and in vitro. In the hippocampus, sequential firing of many neurons over periods of 100-300 ms reoccurs during behavior and during periods of quiescence. However, it is not known whether the hippocampus produces longer sequences of activity or whether such sequences are restricted to specific network states. Furthermore, whether long repeated patterns of activity are transmitted to single cells downstream is unclear. To answer these questions, we recorded intracellularly from hippocampal CA1 of awake, behaving male mice to examine both subthreshold activity and spiking output in single neurons. In eight of nine recordings, we discovered long (900 ms) reoccurring subthreshold fluctuations or “repeats.” Repeats generally were high-amplitude, nonoscillatory events reoccurring with 10msprecision. Using statistical controls, we determined that repeats occurred more often than would be expected from unstructured network activity (e.g., by chance). Most spikes occurred during a repeat, and when a repeat contained a spike, the spike reoccurred with precision on the order of ≤ 20 ms, showing that long repeated patterns of subthreshold activity are strongly connected to spike output. Unexpectedly, we found that repeats occurred independently of classic hippocampal network states like theta oscillations or sharp-wave ripples. Together, these results reveal surprisingly long patterns of repeated activity in the hippocampal network that occur nonstochastically, are transmitted to single downstream neurons, and strongly shape their output. This suggests that the timescale of information transmission in the hippocampal network is much longer than previously thought.

We previously reported that inducing gamma oscillations with a non-invasive light flicker (gamma entrainment using sensory stimulus or GENUS) impacted pathology in the visual cortex of Alzheimer's disease mouse models. Here, we designed auditory tone stimulation that drove gamma frequency neural activity in auditory cortex (AC) and hippocampal CA1. Seven days of auditory GENUS improved spatial and recognition memory and reduced amyloid in AC and hippocampus of 5XFAD mice. Changes in activation responses were evident in microglia, astrocytes, and vasculature. Auditory GENUS also reduced phosphorylated tau in the P301S tauopathy model. Furthermore, combined auditory and visual GENUS, but not either alone, produced microglial-clustering responses, and decreased amyloid in medial prefrontal cortex. Whole brain analysis using SHIELD revealed widespread reduction of amyloid plaques throughout neocortex after multi-sensory GENUS. Thus, GENUS can be achieved through multiple sensory modalities with wide-ranging effects across multiple brain areas to improve cognitive function. Auditory stimulation combined with light-induced gamma oscillations in the hippocampus CA1 and auditory cortex regions of the brain reduces amyloid levels and improves memory in animal models of Alzheimer's disease.

Modulating brain oscillations has strong therapeutic potential. However, commonly used non-invasive interventions such as transcranial magnetic or direct current stimulation have limited effects on deeper cortical structures like the medial temporal lobe. Repetitive audio-visual stimulation, or sensory flicker, modulates such structures in mice but little is known about its effects in humans. Using high spatiotemporal resolution, we mapped and quantified the neurophysiological effects of sensory flicker in human subjects undergoing presurgical intracranial seizure monitoring. We found that flicker modulates both local field potential and single neurons in higher cognitive regions, including the medial temporal lobe and prefrontal cortex, and that local field potential modulation is likely mediated via resonance of involved circuits. We then assessed how flicker affects pathological neural activity, specifically interictal epileptiform discharges, a biomarker of epilepsy also implicated in Alzheimer’s and other diseases. In our patient population with focal seizure onsets, sensory flicker decreased the rate interictal epileptiform discharges. Our findings support the use of sensory flicker to modulate deeper cortical structures and mitigate pathological activity in humans.

Rhythmic neural activity, which coordinates brain regions and neurons to achieve multiple brain functions, is impaired in many diseases. Despite the therapeutic potential of driving brain rhythms, methods to noninvasively target deep brain regions are limited. Accordingly, we recently introduced a noninvasive stimulation approach using flickering lights and sounds (“flicker”). Flicker drives rhythmic activity in deep and superficial brain regions. Gamma flicker spurs immune function, clears pathogens, and rescues memory performance in mice with amyloid pathology. Here, we present substantial improvements to this approach that is flexible, user-friendly, and generalizable across multiple experimental settings and species. We present novel opensource methods for flicker stimulation across rodents and humans. We demonstrate rapid, cross-species induction of rhythmic activity without behavioral confounds in multiple settings from electrophysiology to neuroimaging. This flicker approach provides an exceptional opportunity to discover the therapeutic effects of brain rhythms across scales and species.

Many neurodegenerative and neurological diseases are rooted in dysfunction of the neuroimmune system; therefore, manipulating this system has strong therapeutic potential. Prior work has shown that exposing mice to flickering lights at 40 Hz drives gamma frequency (~40 Hz) neural activity and recruits microglia, the primary immune cells of the brain, revealing a novel method to manipulate the neuroimmune system. However, the biochemical signaling mechanisms between 40 Hz neural activity and immune recruitment remain unknown. Here, we exposed wild-type male mice to 5–60 min of 40 Hz or control flicker and assessed cytokine and phosphoprotein networks known to play a role in immune function. We found that 40 Hz flicker leads to increases in the expression of cytokines which promote microglial phagocytic states, such as IL-6 and IL-4, and increased expression of microglial chemokines, such as macrophage-colony-stimulating factor and monokine induced by interferon-γ. Interestingly, cytokine effects differed as a function of stimulation frequency, revealing a range of neuroimmune effects of stimulation. To identify possible mechanisms underlying cytokine expression, we quantified the effect of the flicker on intracellular signaling pathways known to regulate cytokine levels. We found that a 40 Hz flicker upregulates phospho-signaling within the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. While cytokine expression increased after 1 h of 40 Hz flicker stimulation, protein phosphorylation in the NF-κB pathway was upregulated within minutes. Importantly, the cytokine expression profile induced by 40 Hz flicker was different from cytokine changes in response to acute neuroinflammation induced by lipopolysaccharides. These results are the first, to our knowledge, to show how visual stimulation rapidly induces critical neuroimmune signaling in healthy animals.

Synapse loss and altered synaptic strength are thought to underlie cognitive impairment in Alzheimer's disease (AD) by disrupting neural activity essential for memory. While synaptic dysfunction in AD has been well characterized in anesthetized animals and in vitro, it remains unknown how synaptic transmission is altered during behavior. By measuring synaptic efficacy as mice navigate in a virtual reality task, we find deficits in interneuron connection strength onto pyramidal cells in hippocampal CA1 in the 5XFAD mouse model of AD. These inhibitory synaptic deficits are most pronounced during sharp-wave ripples, network oscillations important for memory that require inhibition. Indeed, 5XFAD mice exhibit fewer and shorter sharp-wave ripples with impaired place cell reactivation. By showing inhibitory synaptic dysfunction in 5XFAD mice during spatial navigation behavior and suggesting a synaptic mechanism underlying deficits in network activity essential for memory, this work bridges the gap between synaptic and neural activity deficits in AD.

Introduction: We and collaborators discovered that flickering lights and sound at gamma frequency (40 Hz) reduce Alzheimer's disease (AD) pathology and alter immune cells and signaling in mice. To determine the feasibility of this intervention in humans we tested the safety, tolerability, and daily adherence to extended audiovisual gamma flicker stimulation. Methods: Ten patients with mild cognitive impairment due to underlying AD received 1-hour daily gamma flicker using audiovisual stimulation for 4 or 8 weeks at home with a delayed start design. Results: Gamma flicker was safe, tolerable, and adherable. Participants’ neural activity entrained to stimulation. Magnetic resonance imaging and cerebral spinal fluid proteomics show preliminary evidence that prolonged flicker affects neural networks and immune factors in the nervous system. Discussion: These findings show that prolonged gamma sensory flicker is safe, tolerable, and feasible with preliminary indications of immune and network effects, supporting further study of gamma stimulation in AD.

Memory-guided navigation relies on hippocampal neurons, like place cells, that encode features of the environment. However, little is known about hippocampal place codes when spatial cues provide ambiguous information about finding goals. Nonplace cells, pyramidal cells that fire without strong spatial modulation in an environment, may be well-suited to carry task-relevant information when spatial information is ambiguous. We find that when spatial cues and goal information are conflicting, nonplace cell firing distinguishes between ambiguous spatial cues. On correct trials nonplace cells had higher firing rates and altered gamma-phase modulation at task-relevant cues than on incorrect trials, while place cells showed no such differences. Finally, this goal discrimination in nonplace cells is absent in a mouse model of Alzheimer's disease that has memory impairment. Our findings show that nonplace cells differentiate ambiguous goal information that place cells do not, revealing a special contribution to coding by these nonplace cells.

Whole-cell patch clamping in vivo is an important neuroscience technique that uniquely provides access to both suprathreshold spiking and subthreshold synaptic events of single neurons in the brain. This article describes how to set up and use the autopatcher, which is a robot for automatically obtaining high-yield and high-quality whole-cell patch clamp recordings in vivo. By following this protocol, a functional experimental rig for automated whole-cell patch clamping can be set up in 1 week. High-quality surgical preparation of mice takes ∼1 h, and each autopatching experiment can be carried out over periods lasting several hours. Autopatching should enable in vivo intracellular investigations to be accessible by a substantial number of neuroscience laboratories, and it enables labs that are already doing in vivo patch clamping to scale up their efforts by reducing training time for new lab members and increasing experimental durations by handling mentally intensive tasks automatically.