Molecular Mechanisms for Changing Brain Connectivity in Mice and Humans.

The goal of this study was to examine commonalities in the molecular basis of learning in mice and humans. In previous work we have demonstrated that the anterior cingulate cortex (ACC) and hippocampus (HC) are involved in learning a two-choice visuospatial discrimination task. Here, we began by looking for candidate genes upregulated in mouse ACC and HC with learning. We then determined which of these were also upregulated in mouse blood. Finally, we used RT-PCR to compare candidate gene expression in mouse blood with that from humans following one of two forms of learning: a working memory task (network training) or meditation (a generalized training shown to change many networks). Two genes were upregulated in mice following learning: caspase recruitment domain-containing protein 6 (Card6) and inosine monophosphate dehydrogenase 2 (Impdh2). The Impdh2 gene product catalyzes the first committed step of guanine nucleotide synthesis and is tightly linked to cell proliferation. The Card6 gene product positively modulates signal transduction. In humans, Card6 was significantly upregulated, and Impdh2 trended toward upregulation with training. These genes have been shown to regulate pathways that influence nuclear factor kappa B (NF-κB), a factor previously found to be related to enhanced synaptic function and learning.

A genetically identified population of layer 4 neurons in auditory cortex that contributes to pre-pulse inhibition of the acoustic startle response.

A fundamental task faced by the auditory system is the detection of events that are signaled by fluctuations in sound. Spiking in auditory cortical neurons is critical for sound detection, but the causal roles of specific cell types and circuits are still mostly unknown. Here we tested the role of a genetically identified population of layer 4 auditory cortical neurons in sound detection. We measured sound detection using a common variant of pre-pulse inhibition of the acoustic startle response, in which a silent gap in background noise acts as a cue that attenuates startle. We used a Gpr26-Cre driver line, which we found expressed predominantly in layer 4 of auditory cortex. Photostimulation of these cells, which were responsive to gaps in noise, was sufficient to attenuate the startle reflex. Photosuppression of these cells reduced neural responses to gaps throughout cortex, and impaired behavioral gap detection. These data demonstrate that cortical Gpr26 neurons are both necessary and sufficient for top-down modulation of the acoustic startle reflex, and are thus likely to be involved in sound detection.

Amyloid Pathology in the Central Auditory Pathway of 5XFAD Mice Appears First in Auditory Cortex.

BACKGROUND: Effective treatment of Alzheimer's disease (AD) will hinge on early detection. This has led to the search for early biomarkers that use non-invasive testing. One possible early biomarker is auditory temporal processing deficits, which reflect central auditory pathway dysfunction and precede cognitive and memory declines in AD. Gap detection is a measure of auditory temporal processing, is impaired in human AD, and is also impaired in the 5XFAD mouse model of AD. Gap detection deficits appear as early as postnatal day 60 in 5XFAD mice, months before cognitive deficits or cell death, supporting gap detection as an early biomarker. However, it remains unclear how gap detection deficits relate to the progression of amyloid pathology in the auditory system. OBJECTIVE: To determine the progression of amyloid pathology throughout the central auditory system and across age in 5XFAD mice. METHODS: We quantified intracellular and extracellular antibody labelling of Aß42 in 6 regions of the central auditory system from p14 to p150. RESULTS: Pathology appeared first in primary auditory cortex (A1) as intracellular accumulation of Aß42 in layer 5 pyramidal neurons by age p21. Extracellular plaques appeared later, by age p90, in A1, medial geniculate body, and inferior colliculus. Auditory brainstem structures showed minimal amyloid pathology. We also observed pathology in the caudal pontine reticular nucleus, a brainstem structure that is outside of the central auditory pathway but which is involved in the acoustic startle reflex. CONCLUSION: These results suggest that Aß42 accumulation, but not plaques, may impair gap detection.

Decision Making as a Learned Skill in Mice and Humans.

Attention is a necessary component in many forms of human and animal learning. Numerous studies have described how attention and memory interact when confronted with a choice point during skill learning. In both animal and human studies, pathways have been found that connect the executive and orienting networks of attention to the hippocampus. The anterior cingulate cortex, part of the executive attention network, is linked to the hippocampus via the nucleus reuniens of the thalamus. The parietal cortex, part of the orienting attention network, accesses the hippocampus via the entorhinal cortex. These studies have led to specific predictions concerning the functional role of each pathway in connecting the cortex to the hippocampus. Here, we review some of the predictions arising from these studies. We then discuss potential methods for manipulating the two pathways and assessing the directionality of their functional connection using viral expression techniques in mice. New studies may allow testing of a behavioral model specifying how the two pathways work together during skill learning.

A Layer 3→5 Circuit in Auditory Cortex That Contributes to Pre-pulse Inhibition of the Acoustic Startle Response.

While connectivity within sensory cortical circuits has been studied extensively, how these connections contribute to perception and behavior is not well understood. Here we tested the role of a circuit between layers 3 and 5 of auditory cortex in sound detection. We measured sound detection using a common variant of pre-pulse inhibition of the acoustic startle response, in which a silent gap in background noise acts as a cue that attenuates startle. We used the Nr5a-Cre driver line, which we found drove expression in the auditory cortex restricted predominantly to layer 3. Photoactivation of these cells evoked short-latency, highly reliable spiking in downstream layer 5 neurons, and attenuated startle responses similarly to gaps in noise. Photosuppression of these cells did not affect behavioral gap detection. Our data provide the first demonstration that direct activation of auditory cortical neurons is sufficient to attenuate the acoustic startle response, similar to the detection of a sound.

5XFAD mice show early-onset gap encoding deficits in the auditory cortex.

Early detection will be crucial for effective treatment or prevention of Alzheimer's disease. The identification and validation of early, noninvasive biomarkers is therefore key to avoiding the most devastating aspects of Alzheimer's disease. Measures of central auditory processing such as gap detection have recently emerged as potential biomarkers in both human patients and the 5XFAD mouse model of Alzheimer's disease. Full validation of gap detection deficits as a biomarker will require detailed understanding of the underlying neuropathology, including which brain structures are involved and how the operation of neural circuits is affected. Here we show that 5XFAD mice exhibit gap detection deficits as early as 2 months of age, well before development of Alzheimer's disease-associated pathology. We then examined responses of neurons in the auditory cortex to gaps in white noise. Both gap responses and baseline firing rates were robustly and progressively degraded in 5XFAD mice compared to littermate controls. These impairments were first evident at 2-4 months of age in males, and 4-6 months in females. This demonstrates early-onset impairments to the central auditory system, which could be due to damage in the auditory cortex, upstream subcortical structures, or both.

A Cortico-Collicular Amplification Mechanism for Gap Detection.

Auditory cortex (AC) is necessary for the detection of brief gaps in ongoing sounds, but not for the detection of longer gaps or other stimuli such as tones or noise. It remains unclear why this is so, and what is special about brief gaps in particular. Here, we used both optogenetic suppression and conventional lesions to show that the cortical dependence of brief gap detection hinges specifically on gap termination. We then identified a cortico-collicular gap detection circuit that amplifies cortical gap termination responses before projecting to inferior colliculus (IC) to impact behavior. We found that gaps evoked off-responses and on-responses in cortical neurons, which temporally overlapped for brief gaps, but not long gaps. This overlap specifically enhanced cortical responses to brief gaps, whereas IC neurons preferred longer gaps. Optogenetic suppression of AC reduced collicular responses specifically to brief gaps, indicating that under normal conditions, the enhanced cortical representation of brief gaps amplifies collicular gap responses. Together these mechanisms explain how and why AC contributes to the behavioral detection of brief gaps, which are critical cues for speech perception, perceptual grouping, and auditory scene analysis.

Auditory Cortex Contributes to Discrimination of Pure Tones.

The auditory cortex is topographically organized for sound frequency and contains highly selective frequency-tuned neurons, yet the role of auditory cortex in the perception of sound frequency remains unclear. Lesion studies have shown that auditory cortex is not essential for frequency discrimination of pure tones. However, transient pharmacological inactivation has been reported to impair frequency discrimination. This suggests the possibility that successful tone discrimination after recovery from lesion surgery could arise from long-term reorganization or plasticity of compensatory pathways. Here, we compared the effects of lesions and optogenetic suppression of auditory cortex on frequency discrimination in mice. We found that transient bilateral optogenetic suppression partially but significantly impaired discrimination performance. In contrast, bilateral electrolytic lesions of auditory cortex had no effect on performance of the identical task, even when tested only 4 h after lesion. This suggests that when auditory cortex is destroyed, an alternative pathway is almost immediately adequate for mediating frequency discrimination. Yet this alternative pathway is insufficient for task performance when auditory cortex is intact but has its activity suppressed. These results indicate a fundamental difference between the effects of brain lesions and optogenetic suppression, and suggest the existence of a rapid compensatory process possibly induced by injury.

Differential Involvement of Three Brain Regions during Mouse Skill Learning.

Human skill learning is marked by a gradual decrease in reaction time (RT) and errors as the skill is acquired. To better understand the influence of brain areas thought to be involved in skill learning, we trained mice to associate visual-spatial cues with specific motor behaviors for a water reward. Task acquisition occurred over weeks and performance approximated a power function as often found with human skill learning. Using optogenetics we suppressed the primary visual cortex (V1), anterior cingulate cortex (ACC), or dorsal hippocampus (dHC) on 20% of trials at different stages of learning. Intermittent suppression of the V1 greatly reduced task performance on suppressed trials across multiple stages but did not change the overall rate of learning. In accord with some recent models of skill learning, ACC suppression produced higher error rates on suppressed trials throughout learning the skill, with effects intensifying in the later stages. This would suggest that cognitive influences mediated by the anterior cingulate continue throughout learning. Suppression of the hippocampus only modestly affected performance, with largely similar effects seen across stages. These results indicate different degrees of V1, ACC, and dHC involvement in acquisition and performance of this visual-spatial task and that the structures operate in parallel, and not in series, across learning stages.

5XFAD Mice Show Early Onset Gap Detection Deficits.

Alzheimer's patients show auditory temporal processing deficits very early in disease progression, before the onset of major cognitive impairments. In addition to potentially contributing to speech perception and communication deficits in patients, this also represents a potential early biomarker for Alzheimer's. For this reason, tests of temporal processing such as gap detection have been proposed as an early diagnosis tool. For a biomarker such as gap detection deficits to have maximum clinical value, it is important to understand what underlying neuropathology it reflects. For example, temporal processing deficits could arise from alterations at cortical, midbrain, or brainstem levels. Mouse models of Alzheimer's disease can provide the ability to reveal in detail the molecular and circuit pathology underlying disease symptoms. Here we tested whether 5XFAD mice, a leading Alzheimer's mouse model, exhibit impaired temporal processing. We found that 5XFAD mice showed robust gap detection deficits. Gap detection deficits were first detectable at about 2 months of age and became progressively worse, especially for males and for longer gap durations. We conclude that 5XFAD mice are well-suited to serve as a model for understanding the circuit mechanisms that contribute to Alzheimer's-related gap detection deficits.

Disruption of Early or Late Epochs of Auditory Cortical Activity Impairs Speech Discrimination in Mice.

Speech evokes robust activity in auditory cortex, which contains information over a wide range of spatial and temporal scales. It remains unclear which components of these neural representations are causally involved in the perception and processing of speech sounds. Here we compared the relative importance of early and late speech-evoked activity for consonant discrimination. We trained mice to discriminate the initial consonants in spoken words, and then tested the effect of optogenetically suppressing different temporal windows of speech-evoked activity in auditory cortex. We found that both early and late suppression disrupted performance equivalently. These results suggest that mice are impaired at recognizing either type of disrupted representation because it differs from those learned in training.

Changes in white matter in mice resulting from low-frequency brain stimulation.

Recent reports have begun to elucidate mechanisms by which learning and experience produce white matter changes in the brain. We previously reported changes in white matter surrounding the anterior cingulate cortex in humans after 2-4 weeks of meditation training. We further found that low-frequency optogenetic stimulation of the anterior cingulate in mice increased time spent in the light in a light/dark box paradigm, suggesting decreased anxiety similar to what is observed following meditation training. Here, we investigated the impact of this stimulation at the cellular level. We found that laser stimulation in the range of 1-8 Hz results in changes to subcortical white matter projection fibers in the corpus callosum. Specifically, stimulation resulted in increased oligodendrocyte proliferation, accompanied by a decrease in the g-ratio within the corpus callosum underlying the anterior cingulate cortex. These results suggest that low-frequency stimulation can result in activity-dependent remodeling of myelin, giving rise to enhanced connectivity and altered behavior.

Rapid Rebalancing of Excitation and Inhibition by Cortical Circuitry.

Excitation is balanced by inhibition to cortical neurons across a wide range of conditions. To understand how this relationship is maintained, we broadly suppressed the activity of parvalbumin-expressing (PV+) inhibitory neurons and asked how this affected the balance of excitation and inhibition throughout auditory cortex. Activating archaerhodopsin in PV+ neurons effectively suppressed them in layer 4. However, the resulting increase in excitation outweighed Arch suppression and produced a net increase in PV+ activity in downstream layers. Consequently, suppressing PV+ neurons did not reduce inhibition to principal neurons (PNs) but instead resulted in a tightly coordinated increase in both excitation and inhibition. The increase in inhibition constrained the magnitude of PN spiking responses to the increase in excitation and produced nonlinear changes in spike tuning. Excitatory-inhibitory rebalancing is mediated by strong PN-PV+ connectivity within and between layers and is likely engaged during normal cortical operation to ensure balance in downstream neurons.

A Novel Mechanism for the Grid-to-Place Cell Transformation Revealed by Transgenic Depolarization of Medial Entorhinal Cortex Layer II.

The spatial receptive fields of neurons in medial entorhinal cortex layer II (MECII) and in the hippocampus suggest general and environment-specific maps of space, respectively. However, the relationship between these receptive fields remains unclear. We reversibly manipulated the activity of MECII neurons via chemogenetic receptors and compared the changes in downstream hippocampal place cells to those of neurons in MEC. Depolarization of MECII impaired spatial memory and elicited drastic changes in CA1 place cells in a familiar environment, similar to those seen during remapping between distinct environments, while hyperpolarization did not. In contrast, both manipulations altered the firing rate of MEC neurons without changing their firing locations. Interestingly, only depolarization caused significant changes in the relative firing rates of individual grid fields, reconfiguring the spatial input from MEC. This suggests a novel mechanism of hippocampal remapping whereby rate changes in MEC neurons lead to locational changes of hippocampal place fields.

Rhythmic brain stimulation reduces anxiety-related behavior in a mouse model based on meditation training.

Meditation training induces changes at both the behavioral and neural levels. A month of meditation training can reduce self-reported anxiety and other dimensions of negative affect. It also can change white matter as measured by diffusion tensor imaging and increase resting-state midline frontal theta activity. The current study tests the hypothesis that imposing rhythms in the mouse anterior cingulate cortex (ACC), by using optogenetics to induce oscillations in activity, can produce behavioral changes. Mice were randomly assigned to groups and were given twenty 30-min sessions of light pulses delivered at 1, 8, or 40 Hz over 4 wk or were assigned to a no-laser control condition. Before and after the month all mice were administered a battery of behavioral tests. In the light/dark box, mice receiving cortical stimulation had more light-side entries, spent more time in the light, and made more vertical rears than mice receiving rhythmic cortical suppression or no manipulation. These effects on light/dark box exploratory behaviors are associated with reduced anxiety and were most pronounced following stimulation at 1 and 8 Hz. No effects were seen related to basic motor behavior or exploration during tests of novel object and location recognition. These data support a relationship between lower-frequency oscillations in the mouse ACC and the expression of anxiety-related behaviors, potentially analogous to effects seen with human practitioners of some forms of meditation.

White matter and reaction time: Reply to commentaries.

We appreciate the many comments we received on our discussion paper and believe that they reflect a recognition of the importance of this topic worldwide. We point out in this reply that there appears to be a confusion between the role of oscillations in creating white matter and other functions of oscillations in communicating between neural areas during task performance or at rest. We also discuss some mechanisms other than the enhancement of white matter that must influence reaction time. We recognize the limited understanding we have of transfer and outline some future directions designed to improve our understanding of this process.

How changes in white matter might underlie improved reaction time due to practice.

Why does training on a task reduce the reaction time for performing it? New research points to changes in white matter pathways as one likely mechanism. These pathways connect remote brain areas involved in performing the task. Genetic variations may be involved in individual differences in the extent of this improvement. If white matter change is involved in improved reaction time with training, it may point the way toward understanding where and how generalization occurs. We examine the hypothesis that brain pathways shared by different tasks may result in improved performance of cognitive tasks remote from the training.

Auditory cortex is required for fear potentiation of gap detection.

Auditory cortex is necessary for the perceptual detection of brief gaps in noise, but is not necessary for many other auditory tasks such as frequency discrimination, prepulse inhibition of startle responses, or fear conditioning with pure tones. It remains unclear why auditory cortex should be necessary for some auditory tasks but not others. One possibility is that auditory cortex is causally involved in gap detection and other forms of temporal processing in order to associate meaning with temporally structured sounds. This predicts that auditory cortex should be necessary for associating meaning with gaps. To test this prediction, we developed a fear conditioning paradigm for mice based on gap detection. We found that pairing a 10 or 100 ms gap with an aversive stimulus caused a robust enhancement of gap detection measured 6 h later, which we refer to as fear potentiation of gap detection. Optogenetic suppression of auditory cortex during pairing abolished this fear potentiation, indicating that auditory cortex is critically involved in associating temporally structured sounds with emotionally salient events.

Perceptual gap detection is mediated by gap termination responses in auditory cortex.

BACKGROUND: Understanding speech in the presence of background noise often becomes increasingly difficult with age. These age-related speech processing deficits reflect impairments in temporal acuity. Gap detection is a model for temporal acuity in speech processing in which a gap inserted in white noise acts as a cue that attenuates subsequent startle responses. Lesion studies have shown that auditory cortex is necessary for the detection of brief gaps, and auditory cortical neurons respond to the end of the gap with a characteristic burst of spikes called the gap termination response (GTR). However, it remains unknown whether and how the GTR plays a causal role in gap detection. We tested this by optogenetically suppressing the activity of somatostatin- or parvalbumin-expressing inhibitory interneurons, or CaMKII-expressing excitatory neurons, in auditory cortex of behaving mice during specific epochs of a gap detection protocol. RESULTS: Suppressing interneuron activity during the postgap interval enhanced gap detection. Suppressing excitatory cells during this interval attenuated gap detection. Suppressing activity preceding the gap had the opposite behavioral effects, whereas prolonged suppression across both intervals had no effect on gap detection. CONCLUSIONS: In addition to confirming cortical involvement, we demonstrate here for the first time a causal relationship between postgap neural activity and perceptual gap detection. Furthermore, our results suggest that gap detection involves an ongoing comparison of pre- and postgap spiking activity. Finally, we propose a simple yet biologically plausible neural circuit that reproduces each of these neural and behavioral results.

Role of self-generated odor cues in contextual representation.

As first demonstrated in the patient H.M., the hippocampus is critically involved in forming episodic memories, the recall of "what" happened "where" and "when." In rodents, the clearest functional correlate of hippocampal primary neurons is the place field: a cell fires predominantly when the animal is in a specific part of the environment, typically defined relative to the available visuospatial cues. However, rodents have relatively poor visual acuity. Furthermore, they are highly adept at navigating in total darkness. This raises the question of how other sensory modalities might contribute to a hippocampal representation of an environment. Rodents have a highly developed olfactory system, suggesting that cues such as odor trails may be important. To test this, we familiarized mice to a visually cued environment over a number of days while maintaining odor cues. During familiarization, self-generated odor cues unique to each animal were collected by re-using absorbent paperboard flooring from one session to the next. Visual and odor cues were then put in conflict by counter-rotating the recording arena and the flooring. Perhaps surprisingly, place fields seemed to follow the visual cue rotation exclusively, raising the question of whether olfactory cues have any influence at all on a hippocampal spatial representation. However, subsequent removal of the familiar, self-generated odor cues severely disrupted both long-term stability and rotation to visual cues in a novel environment. Our data suggest that odor cues, in the absence of additional rule learning, do not provide a discriminative spatial signal that anchors place fields. Such cues do, however, become integral to the context over time and exert a powerful influence on the stability of its hippocampal representation.