Functional diversities within neurons and astrocytes in the adult rat auditory cortex revealed by single-nucleus RNA sequencing.

The mammalian cerebral cortex is composed of a rich diversity of cell types. Cortical cells are organized into networks that rely on their functional diversity to ultimately carry out a variety of sophisticated cognitive functions. To investigate the breadth of transcriptional diverse cell types in the sensory cortex, we have used single-nucleus RNA sequencing (snRNA-seq) in the auditory cortex of the adult rat. A variety of unique excitatory and inhibitory neuron types were identified. In addition, we report for the first time a diversity of astrocytes in the auditory cortex that may represent functionally unique subtypes. Together, these results pave the way for building models of how neurons in the sensory cortex work in concert with astrocytes at synapses to fulfill high-cognitive functions like learning and memory.

Epigenetic mechanisms regulate cue memory underlying discriminative behavior.

The burgeoning field of neuroepigenetics has introduced chromatin modification as an important interface between experience and brain function. For example, epigenetic mechanisms like histone acetylation and DNA methylation operate throughout a lifetime to powerfully regulate gene expression in the brain that is required for experiences to be transformed into long-term memories. This review highlights emerging evidence from sensory models of memory that converge on the premise that epigenetic regulation of activity-dependent transcription in the sensory brain facilitates highly precise memory recall. Chromatin modifications may be key for neurophysiological responses to transient sensory cue features experienced in the "here and now" to be recapitulated over the long term. We conclude that the function of epigenetic control of sensory system neuroplasticity is to regulate the amount and type of sensory information retained in long-term memories by regulating neural representations of behaviorally relevant cues that guide behavior. This is of broad importance in the neuroscience field because there are few circumstances in which behavioral acts are devoid of an initiating sensory experience.

Sex differences in auditory processing vary across estrous cycle.

In humans, females process a sound's harmonics more robustly than males. As estrogen regulates auditory plasticity in a sex-specific manner in seasonally breeding animals, estrogen signaling is one hypothesized mechanism for this difference in humans. To investigate whether sex differences in harmonic encoding vary similarly across the reproductive cycle of mammals, we recorded frequency-following responses (FFRs) to a complex sound in male and female rats. Female FFRs were collected during both low and high levels of circulating estrogen during the estrous cycle. Overall, female rodents had larger harmonic encoding than male rodents, and greater harmonic strength was seen during periods of greater estrogen production in the females. These results argue that hormonal differences, specifically estrogen, underlie sex differences in harmonic encoding in rodents and suggest that a similar mechanism may underlie differences seen in humans.

Memory Specific to Temporal Features of Sound Is Formed by Cue-Selective Enhancements in Temporal Coding Enabled by Inhibition of an Epigenetic Regulator.

Recent investigation of memory-related functions in the auditory system have capitalized on the use of memory-modulating molecules to probe the relationship between memory and substrates of memory in auditory system coding. For example, epigenetic mechanisms, which regulate gene expression necessary for memory consolidation, are powerful modulators of learning-induced neuroplasticity and long-term memory (LTM) formation. Inhibition of the epigenetic regulator histone deacetylase 3 (HDAC3) promotes LTM, which is highly specific for spectral features of sound. The present work demonstrates for the first time that HDAC3 inhibition also enables memory for temporal features of sound. Adult male rats trained in an amplitude modulation (AM) rate discrimination task and treated with a selective inhibitor of HDAC3 formed memory that was highly specific to the AM rate paired with reward. Sound-specific memory revealed behaviorally was associated with a signal-specific enhancement in temporal coding in the auditory system; stronger phase locking that was specific to the rewarded AM rate was revealed in both the surface-recorded frequency following response and auditory cortical multiunit activity in rats treated with the HDAC3 inhibitor. Furthermore, HDAC3 inhibition increased trial-to-trial cortical response consistency (relative to naive and trained vehicle-treated rats), which generalized across different AM rates. Stronger signal-specific phase locking correlated with individual behavioral differences in memory specificity for the AM signal. These findings support that epigenetic mechanisms regulate activity-dependent processes that enhance discriminability of sensory cues encoded into LTM in both spectral and temporal domains, which may be important for remembering spectrotemporal features of sounds, for example, as in human voices and speech.SIGNIFICANCE STATEMENT Epigenetic mechanisms have recently been implicated in memory and information processing. Here, we use a pharmacological inhibitor of HDAC3 in a sensory model of learning to reveal the ability of HDAC3 to enable precise memory for amplitude-modulated sound cues. In so doing, we uncover neural substrates for memory's specificity for temporal sound cues. Memory specificity was supported by auditory cortical changes in temporal coding, including greater response consistency and stronger phase locking. HDAC3 appears to regulate effects across domains that determine specific cue saliency for behavior. Thus, epigenetic players may gate how sensory information is stored in long-term memory and can be leveraged to reveal the neural substrates of sensory details stored in memory.

Sensory cortical and subcortical auditory neurophysiological changes predict cue-specific extinction behavior enabled by the pharmacological inhibition of an epigenetic regulator during memory formation.

Extinction learning and memory have been broadly investigated at both behavioral and neural levels, but sensory system contributions to extinction processes have been less explored. Using a sound-reward extinction paradigm in male rats, we reveal both cortical and subcortical forms of plasticity associated with the cue-specificity of behavioral extinction memory. In the auditory cortex, frequency tuning narrowed by up to two-thirds of an octave around the remembered extinguished sound cue. Subcortical signals revealed in the auditory brainstem response (ABR) in the same animals developed smaller amplitudes of some (but not all) ABR peaks evoked by the extinguished sound frequency. Interestingly, treatment with an inhibitor of histone deacetylase 3 (HDAC3-i) facilitated both auditory cortical tuning bandwidth changes and changes in subcortical peak amplitude evoked only by the extinguished sound frequency. These neurophysiological changes were correlated to each other, and to the highly precise extinction behavior enabled by HDAC3-i (compared to vehicle controls). Thus, we show for the first time that HDAC3 regulates the specificity of sensory features consolidated in extinction memory. Further, the sensory cortical changes in tuning bandwidth recapitulate known effects of blocking HDAC3 to enhance cue specificity in other behavioral tasks. Therefore, the findings demonstrate how some forms of sensory neuroplasticity may encode specific sensory features of learning experiences in order to enable cue-specific behaviors.

Precise memory for pure tones is predicted by measures of learning-induced sensory system neurophysiological plasticity at cortical and subcortical levels.

Despite identical learning experiences, individuals differ in the memory formed of those experiences. Molecular mechanisms that control the neurophysiological bases of long-term memory formation might control how precisely the memory formed reflects the actually perceived experience. Memory formed with sensory specificity determines its utility for selectively cueing subsequent behavior, even in novel situations. Here, a rodent model of auditory learning capitalized on individual differences in learning-induced auditory neuroplasticity to identify and characterize neural substrates for sound-specific (vs. general) memory of the training signal's acoustic frequency. Animals that behaviorally revealed a naturally induced signal-"specific" memory exhibited long-lasting signal-specific neurophysiological plasticity in auditory cortical and subcortical evoked responses. Animals with "general" memories did not exhibit learning-induced changes in these same measures. Manipulating a histone deacetylase during memory consolidation biased animals to have more signal-specific memory. Individual differences validated this brain-behavior relationship in both natural and manipulated memory formation, such that the degree of change in sensory cortical and subcortical neurophysiological responses could be used to predict the behavioral precision of memory.

Inhibition of histone deacetylase 3 via RGFP966 facilitates cortical plasticity underlying unusually accurate auditory associative cue memory for excitatory and inhibitory cue-reward associations.

Epigenetic mechanisms are key for regulating long-term memory (LTM) and are known to exert control on memory formation in multiple systems of the adult brain, including the sensory cortex. One epigenetic mechanism is chromatin modification by histone acetylation. Blocking the action of histone de-acetylases (HDACs) that normally negatively regulate LTM by repressing transcription has been shown to enable memory formation. Indeed, HDAC inhibition appears to facilitate memory by altering the dynamics of gene expression events important for memory consolidation. However, less understood are the ways in which molecular-level consolidation processes alter subsequent memory to enhance storage or facilitate retrieval. Here we used a sensory perspective to investigate whether the characteristics of memory formed with HDAC inhibitors are different from naturally-formed memory. One possibility is that HDAC inhibition enables memory to form with greater sensory detail than normal. Because the auditory system undergoes learning-induced remodeling that provides substrates for sound-specific LTM, we aimed to identify behavioral effects of HDAC inhibition on memory for specific sound features using a standard model of auditory associative cue-reward learning, memory, and cortical plasticity. We found that three systemic post-training treatments of an HDAC3-inhibitor (RGPF966, Abcam Inc.) in rats in the early phase of training facilitated auditory discriminative learning, changed auditory cortical tuning, and increased the specificity for acoustic frequency formed in memory of both excitatory (S+) and inhibitory (S-) associations for at least 2 weeks. The findings support that epigenetic mechanisms act on neural and behavioral sensory acuity to increase the precision of associative cue memory, which can be revealed by studying the sensory characteristics of long-term associative memory formation with HDAC inhibitors.

HDAC3 Inhibitor RGFP966 Modulates Neuronal Memory for Vocal Communication Signals in a Songbird Model.

Epigenetic mechanisms that modify chromatin conformation have recently been under investigation for their contributions to learning and the formation of memory. For example, the role of enzymes involved in histone acetylation are studied in the formation of long-lasting memories because memory consolidation requires gene expression events that are facilitated by an open state of chromatin. We recently proposed that epigenetic events may control the entry of specific sensory features into long-term memory by enabling transcription-mediated neuronal plasticity in sensory brain areas. Histone deacetylases, like HDAC3, may thereby regulate the specific sensory information that is captured for entry into long-term memory stores (Phan and Bieszczad, 2016). To test this hypothesis, we used an HDAC3-selective inhibitor (RGFP966) to determine whether its application after an experience with a sound stimulus with unique acoustic features could contribute to the formation of a memory that would assist in mediating its later recognition. We gave adult male zebra finches limited exposure to unique conspecific songs (20 repetitions each, well below the normal threshold to form long-term memory), followed by treatment with RGFP966 or vehicle. In different groups, we either made multi-electrode recordings in the higher auditory area NCM (caudal medial nidopallidum), or determined expression of an immediate early gene, zenk (also identified as zif268, egr-1, ngfi-a and krox24), known to participate in neuronal memory in this system. We found that birds treated with RGFP966 showed neuronal memory after only limited exposure, while birds treated with vehicle did not. Strikingly, evidence of neuronal memory in NCM induced by HDAC3-inhibition was lateralized to the left-hemisphere, consistent with our finding that RGFP966-treatment also elevated zenk expression only in the left hemisphere. The present findings show feasibility for epigenetic mechanisms to control neural plasticity underlying the formation of specific memories for conspecific communication sounds. This is the first evidence in zebra finches that epigenetic mechanisms may contribute to gene expression events for memory of acoustically-rich sensory cues.

Sensory Cortical Plasticity Participates in the Epigenetic Regulation of Robust Memory Formation.

Neuroplasticity remodels sensory cortex across the lifespan. A function of adult sensory cortical plasticity may be capturing available information during perception for memory formation. The degree of experience-dependent remodeling in sensory cortex appears to determine memory strength and specificity for important sensory signals. A key open question is how plasticity is engaged to induce different degrees of sensory cortical remodeling. Neural plasticity for long-term memory requires the expression of genes underlying stable changes in neuronal function, structure, connectivity, and, ultimately, behavior. Lasting changes in transcriptional activity may depend on epigenetic mechanisms; some of the best studied in behavioral neuroscience are DNA methylation and histone acetylation and deacetylation, which, respectively, promote and repress gene expression. One purpose of this review is to propose epigenetic regulation of sensory cortical remodeling as a mechanism enabling the transformation of significant information from experiences into content-rich memories of those experiences. Recent evidence suggests how epigenetic mechanisms regulate highly specific reorganization of sensory cortical representations that establish a widespread network for memory. Thus, epigenetic mechanisms could initiate events to establish exceptionally persistent and robust memories at a systems-wide level by engaging sensory cortical plasticity for gating what and how much information becomes encoded.

Learning strategy refinement reverses early sensory cortical map expansion but not behavior: Support for a theory of directed cortical substrates of learning and memory.

Primary sensory cortical fields develop highly specific associative representational plasticity, notably enlarged area of representation of reinforced signal stimuli within their topographic maps. However, overtraining subjects after they have solved an instrumental task can reduce or eliminate the expansion while the successful behavior remains. As the development of this plasticity depends on the learning strategy used to solve a task, we asked whether the loss of expansion is due to the strategy used during overtraining. Adult male rats were trained in a three-tone auditory discrimination task to bar-press to the CS+ for water reward and refrain from doing so during the CS- tones and silent intertrial intervals; errors were punished by a flashing light and time-out penalty. Groups acquired this task to a criterion within seven training sessions by relying on a strategy that was "bar-press from tone-onset-to-error signal" ("TOTE"). Three groups then received different levels of overtraining: Group ST, none; Group RT, one week; Group OT, three weeks. Post-training mapping of their primary auditory fields (A1) showed that Groups ST and RT had developed significantly expanded representational areas, specifically restricted to the frequency band of the CS+ tone. In contrast, the A1 of Group OT was no different from naïve controls. Analysis of learning strategy revealed this group had shifted strategy to a refinement of TOTE in which they self-terminated bar-presses before making an error ("iTOTE"). Across all animals, the greater the use of iTOTE, the smaller was the representation of the CS+ in A1. Thus, the loss of cortical expansion is attributable to a shift or refinement in strategy. This reversal of expansion was considered in light of a novel theoretical framework (CONCERTO) highlighting four basic principles of brain function that resolve anomalous findings and explaining why even a minor change in strategy would involve concomitant shifts of involved brain sites, including reversal of cortical expansion.

Histone Deacetylase Inhibition via RGFP966 Releases the Brakes on Sensory Cortical Plasticity and the Specificity of Memory Formation.

Research over the past decade indicates a novel role for epigenetic mechanisms in memory formation. Of particular interest is chromatin modification by histone deacetylases (HDACs), which, in general, negatively regulate transcription. HDAC deletion or inhibition facilitates transcription during memory consolidation and enhances long-lasting forms of synaptic plasticity and long-term memory. A key open question remains: How does blocking HDAC activity lead to memory enhancements? To address this question, we tested whether a normal function of HDACs is to gate information processing during memory formation. We used a class I HDAC inhibitor, RGFP966 (C21H19FN4O), to test the role of HDAC inhibition for information processing in an auditory memory model of learning-induced cortical plasticity. HDAC inhibition may act beyond memory enhancement per se to instead regulate information in ways that lead to encoding more vivid sensory details into memory. Indeed, we found that RGFP966 controls memory induction for acoustic details of sound-to-reward learning. Rats treated with RGFP966 while learning to associate sound with reward had stronger memory and additional information encoded into memory for highly specific features of sounds associated with reward. Moreover, behavioral effects occurred with unusually specific plasticity in primary auditory cortex (A1). Class I HDAC inhibition appears to engage A1 plasticity that enables additional acoustic features to become encoded in memory. Thus, epigenetic mechanisms act to regulate sensory cortical plasticity, which offers an information processing mechanism for gating what and how much is encoded to produce exceptionally persistent and vivid memories. Significance statement: Here we provide evidence of an epigenetic mechanism for information processing. The study reveals that a class I HDAC inhibitor (Malvaez et al., 2013; Rumbaugh et al., 2015; RGFP966, chemical formula C21H19FN4O) alters the formation of auditory memory by enabling more acoustic information to become encoded into memory. Moreover, RGFP966 appears to affect cortical plasticity: the primary auditory cortex reorganized in a manner that was unusually "tuned-in" to the specific sound cues and acoustic features that were related to reward and subsequently remembered. We propose that HDACs control "informational capture" at a systems level for what and how much information is encoded by gating sensory cortical plasticity that underlies the sensory richness of newly formed memories.

Gamma band plasticity in sensory cortex is a signature of the strongest memory rather than memory of the training stimulus.

Gamma oscillations (∼30-120Hz) are considered to be a reflection of coordinated neuronal activity, linked to processes underlying synaptic integration and plasticity. Increases in gamma power within the cerebral cortex have been found during many cognitive processes such as attention, learning, memory and problem solving in both humans and animals. However, the specificity of gamma to the detailed contents of memory remains largely unknown. We investigated the relationship between learning-induced increased gamma power in the primary auditory cortex (A1) and the strength of memory for acoustic frequency. Adult male rats (n=16) received three days (200 trials each) of pairing a tone (3.66 kHz) with stimulation of the nucleus basalis, which implanted a memory for acoustic frequency as assessed by associatively-induced disruption of ongoing behavior, viz., respiration. Post-training frequency generalization gradients (FGGs) revealed peaks at non-CS frequencies in 11/16 cases, likely reflecting normal variation in pre-training acoustic experiences. A stronger relationship was found between increased gamma power and the frequency with the strongest memory (peak of the difference between individual post- and pre-training FGGs) vs. behavioral responses to the CS training frequency. No such relationship was found for the theta/alpha band (4-15 Hz). These findings indicate that the strength of specific increased neuronal synchronization within primary sensory cortical fields can determine the specific contents of memory.

Extinction reveals that primary sensory cortex predicts reinforcement outcome.

Primary sensory cortices are traditionally regarded as stimulus analysers. However, studies of associative learning-induced plasticity in the primary auditory cortex (A1) indicate involvement in learning, memory and other cognitive processes. For example, the area of representation of a tone becomes larger for stronger auditory memories and the magnitude of area gain is proportional to the degree that a tone becomes behaviorally important. Here, we used extinction to investigate whether 'behavioral importance' specifically reflects a sound's ability to predict reinforcement (reward or punishment) vs. to predict any significant change in the meaning of a sound. If the former, then extinction should reverse area gains as the signal no longer predicts reinforcement. Rats (n = 11) were trained to bar-press to a signal tone (5.0 kHz) for water-rewards, to induce signal-specific area gains in A1. After subsequent withdrawal of reward, A1 was mapped to determine representational areas. Signal-specific area gains, estimated from a previously established brain-behavior quantitative function, were reversed, supporting the 'reinforcement prediction' hypothesis. Area loss was specific to the signal tone vs. test tones, further indicating that withdrawal of reinforcement, rather than unreinforced tone presentation per se, was responsible for area loss. Importantly, the amount of area loss was correlated with the amount of extinction (r = 0.82, P < 0.01). These findings show that primary sensory cortical representation can encode behavioral importance as a signal's value to predict reinforcement, and that the number of cells tuned to a stimulus can dictate its ability to command behavior.

Nicotinic acetylcholine receptors in rat forebrain that bind ¹8F-nifene: relating PET imaging, autoradiography, and behavior.

Nicotinic acetylcholine receptors (nAChRs) in the brain are important for cognitive function; however, their specific role in relevant brain regions remains unclear. In this study, we used the novel compound ¹8F-nifene to examine the distribution of nAChRs in the rat forebrain, and for individual animals related the results to behavioral performance on an auditory-cognitive task. We first show negligible binding of ¹8F-nifene in mice lacking the ß2 nAChR subunit, consistent with previous findings that ¹8F-nifene binds to α4ß2* nAChRs. We then examined the distribution of ¹8F-nifene in rat using three methods: in vivo PET, ex vivo PET and autoradiography. Generally, ¹8F-nifene labeled forebrain regions known to contain nAChRs, and the three methods produced similar relative binding among regions. Importantly, ¹8F-nifene also labeled some white matter (myelinated axon) tracts, most prominently in the temporal subcortical region that contains the auditory thalamocortical pathway. Finally, we related ¹8F-nifene binding in several forebrain regions to each animal's performance on an auditory-cued, active avoidance task. The strongest correlations with performance after 14 days training were found for ¹8F-nifene binding in the temporal subcortical white matter, subiculum, and medial frontal cortex (correlation coefficients, r > 0.8); there was no correlation with binding in the auditory thalamus or auditory cortex. These findings suggest that individual performance is linked to nicotinic functions in specific brain regions, and further support a role for nAChRs in sensory-cognitive function.

Remodeling the cortex in memory: Increased use of a learning strategy increases the representational area of relevant acoustic cues.

Associative learning induces plasticity in the representation of sensory information in sensory cortices. Such high-order associative representational plasticity (HARP) in the primary auditory cortex (A1) is a likely substrate of auditory memory: it is specific, rapidly acquired, long-lasting and consolidates. Because HARP is likely to support the detailed content of memory, it is important to identify the necessary behavioral factors that dictate its induction. Learning strategy is a critical factor for the induction of plasticity (Bieszczad & Weinberger, 2010b). Specifically, use of a strategy that relies on tone onsets induces HARP in A1 in the form of signal-specific decreased threshold and bandwidth. The present study tested the hypothesis that the form and degree of HARP in A1 reflects the amount of use of an "onset strategy". Adult male rats (n=7) were trained in a protocol that increased the use of this strategy from approximately 20% in prior studies to approximately 80%. They developed signal-specific gains in representational area, transcending plasticity in the form of local changes in threshold and bandwidth. Furthermore, the degree of area gain was proportional to the amount of use of the onset strategy. A second complementary experiment demonstrated that use of a learning strategy that specifically did not rely on tone onsets did not produce gains in representational area; but rather produced area loss. Together, the findings indicate that the amount of strategy use is a dominant factor for the induction of learning-induced cortical plasticity along a continuum of both form and degree.

Representational gain in cortical area underlies increase of memory strength.

Neuronal plasticity that develops in the cortex during learning is assumed to represent memory content, but the functions of such plasticity are actually unknown. The shift in spectral tuning in primary auditory cortex (A1) to the frequency of a tone signal is a compelling candidate for a substrate of memory because it has all of the cardinal attributes of associative memory: associativity, specificity, rapid induction, consolidation, and long-term retention. Tuning shifts increase the representational area of the signal in A1, as an increasing function of performance level, suggesting that area encodes the magnitude of acquired stimulus significance. The present study addresses the question of the specific function of learning-induced associative representational plasticity. We tested the hypothesis that specific increases in A1 representational area for an auditory signal serve the mnemonic function of enhancing memory strength for that signal. Rats were trained to bar-press for reward contingent on the presence of a signal tone (5.0 kHz), and assessed for memory strength during extinction. The amount of representational area gain for the signal frequency band was significantly positively correlated with resistance to extinction to the signal frequency in two studies that spanned the range of task difficulty. These findings indicate that specific gain in cortical representational area underlies the strength of the behaviorally-relevant contents of memory. Thus, mnemonic functions of cortical plasticity are determinable.

Learning strategy trumps motivational level in determining learning-induced auditory cortical plasticity.

Associative memory for auditory-cued events involves specific plasticity in the primary auditory cortex (A1) that facilitates responses to tones which gain behavioral significance, by modifying representational parameters of sensory coding. Learning strategy, rather than the amount or content of learning, can determine this learning-induced cortical (high order) associative representational plasticity (HARP). Thus, tone-contingent learning with signaled errors can be accomplished either by (1) responding only during tone duration ("tone-duration" strategy, T-Dur), or (2) responding from tone onset until receiving an error signal for responses made immediately after tone offset ("tone-onset-to-error", TOTE). While rats using both strategies achieve the same high level of performance, only those using the TOTE strategy develop HARP, viz., frequency-specific decreased threshold (increased sensitivity) and decreased bandwidth (increased selectivity) (Berlau & Weinberger, 2008). The present study challenged the generality of learning strategy by determining if high motivation dominates in the formation of HARP. Two groups of adult male rats were trained to bar-press during a 5.0kHz (10s, 70dB) tone for a water reward under either high (HiMot) or moderate (ModMot) levels of motivation. The HiMot group achieved a higher level of correct performance. However, terminal mapping of A1 showed that only the ModMot group developed HARP, i.e., increased sensitivity and selectivity in the signal-frequency band. Behavioral analysis revealed that the ModMot group used the TOTE strategy while HiMot subjects used the T-Dur strategy. Thus, type of learning strategy, not level of learning or motivation, is dominant for the formation of cortical plasticity.