Selective Deletion of Methyl CpG Binding Protein 2 from Parvalbumin Interneurons in the Auditory Cortex Delays the Onset of Maternal Retrieval in Mice.

Mutations in MECP2 cause the neurodevelopmental disorder Rett syndrome. MECP2 codes for methyl CpG binding protein 2 (MECP2), a transcriptional regulator that activates genetic programs for experience-dependent plasticity. Many neural and behavioral symptoms of Rett syndrome may result from dysregulated timing and thresholds for plasticity. As a model of adult plasticity, we examine changes to auditory cortex inhibitory circuits in female mice when they are first exposed to pups; this plasticity facilitates behavioral responses to pups emitting distress calls. Brainwide deletion of Mecp2 alters expression of markers associated with GABAergic parvalbumin interneurons (PVins) and impairs the emergence of pup retrieval. We hypothesized that loss of Mecp2 in PVins disproportionately contributes to the phenotype. Here, we find that deletion of Mecp2 from PVins delayed the onset of maternal retrieval behavior and recapitulated the major molecular and neurophysiological features of brainwide deletion of Mecp2 We observed that when PVin-selective mutants were exposed to pups, auditory cortical expression of PVin markers increased relative to that in wild-type littermates. PVin-specific mutants also failed to show the inhibitory auditory cortex plasticity seen in wild-type mice on exposure to pups and their vocalizations. Finally, using an intersectional viral genetic strategy, we demonstrate that postdevelopmental loss of Mecp2 in PVins of the auditory cortex is sufficient to delay onset of maternal retrieval. Our results support a model in which PVins play a central role in adult cortical plasticity and may be particularly impaired by loss of Mecp2 SIGNIFICANCE STATEMENT Rett syndrome is a neurodevelopmental disorder that includes deficits in both communication and the ability to update brain connections and activity during learning (plasticity). This condition is caused by mutations in the gene MECP2 We use a maternal behavioral test in mice requiring both vocal perception and neural plasticity to probe the role of Mecp2 in social and sensory learning. Mecp2 is normally active in all brain cells, but here we remove it from a specific population (parvalbumin neurons). We find that this is sufficient to delay learned behavioral responses to pups and recreates many deficits seen in whole-brain Mecp2 deletion. Our findings suggest that parvalbumin neurons specifically are central to the consequences of loss of Mecp2 activity and yield clues as to possible mechanisms by which Rett syndrome impairs brain function.

A circuit from the locus coeruleus to the anterior cingulate cortex modulates offspring interactions in mice.

Social sensitivity to other individuals in distress is crucial for survival. The anterior cingulate cortex (ACC) is a structure involved in making behavioral choices and is influenced by observed pain or distress. Nevertheless, our understanding of the neural circuitry underlying this sensitivity is incomplete. Here, we reveal unexpected sex-dependent activation of ACC when parental mice respond to distressed pups by returning them to the nest ("pup retrieval"). We observe sex differences in the interactions between excitatory and inhibitory ACC neurons during parental care, and inactivation of ACC excitatory neurons increased pup neglect. Locus coeruleus (LC) releases noradrenaline in ACC during pup retrieval, and inactivation of the LC-ACC pathway disrupts parental care. We conclude that ACC maintains sex-dependent sensitivity to pup distress under LC modulation. We propose that ACC's involvement in parenting presents an opportunity to identify neural circuits that support sensitivity to the emotional distress of others.

Selective deletion of Methyl CpG binding protein 2 from parvalbumin interneurons in the auditory cortex delays the onset of maternal retrieval in mice.

Mutations in MECP2 cause the neurodevelopmental disorder Rett syndrome. MECP2 codes for methyl CpG binding protein 2 (MECP2), a transcriptional regulator that activates genetic programs for experience-dependent plasticity. Many neural and behavioral symptoms of Rett syndrome may result from dysregulated timing and threshold for plasticity. As a model of adult plasticity, we examine changes to auditory cortex inhibitory circuits in female mice when they are first exposed to pups; this plasticity facilitates behavioral responses to pups emitting distress calls. Brain-wide deletion of Mecp2 alters expression of markers associated with GABAergic parvalbumin interneurons (PVin) and impairs the emergence of pup retrieval. We hypothesized that loss of Mecp2 in PVin disproportionately contributes to the phenotype. Here we find that deletion of Mecp2 from PVin delayed the onset of maternal retrieval behavior and recapitulated the major molecular and neurophysiological features of brain-wide deletion of Mecp2 . We observed that when PVin-selective mutants were exposed to pups, auditory cortical expression of PVin markers increased relative to that in wild type littermates. PVin-specific mutants also failed to show the inhibitory auditory cortex plasticity seen in wild type mice upon exposure to pups and their vocalizations. Finally, using an intersectional viral genetic strategy, we demonstrate that post-developmental loss of Mecp2 in PVin of the auditory cortex is sufficient to delay onset of maternal retrieval. Our results support a model in which PVin play a central role in adult cortical plasticity and may be particularly impaired by loss of Mecp2 . SIGNIFICANCE STATEMENT: Rett syndrome is a neurodevelopmental disorder that includes deficits in both communication and the ability to update brain connections and activity during learning ('plasticity'). This condition is caused by mutations in the gene MECP2 . We use a maternal behavioral test in mice requiring both vocal perception and neural plasticity to probe Mecp2' s role in social and sensory learning. Mecp2 is normally active in all brain cells, but here we remove it from a specific population ('parvalbumin neurons'). We find that this is sufficient to delay learned behavioral responses to pups and recreates many deficits seen in whole brain Mecp2 deletion. Our findings suggest that parvalbumin neurons specifically are central to the consequences of loss of Mecp2 activity and yield clues as to possible mechanisms by which Rett syndrome impairs brain function.

A dopaminergic reward prediction error signal shapes maternal behavior in mice.

How social contact is perceived as rewarding and subsequently modifies interactions is unclear. Dopamine (DA) from the ventral tegmental area (VTA) regulates sociality, but the ongoing, unstructured nature of free behavior makes it difficult to ascertain how. Here, we tracked the emergence of a repetitive stereotyped parental retrieval behavior and conclude that VTA DA neurons incrementally refine it by reinforcement learning (RL). Trial-by-trial performance was correlated with the history of DA neuron activity, but DA signals were inconsistent with VTA directly influencing the current trial. We manipulated the subject's expectation of imminent pup contact and show that DA signals convey reward prediction error, a fundamental component of RL. Finally, closed-loop optogenetic inactivation of DA neurons at the onset of pup contact dramatically slowed emergence of parental care. We conclude that this component of maternal behavior is shaped by an RL mechanism in which social contact itself is the primary reward.

The Bruce effect: Representational stability and memory formation in the accessory olfactory bulb of the female mouse.

In the Bruce effect, a mated female mouse becomes resistant to the pregnancy-blocking effect of the stud. Various lines of evidence suggest that this form of behavioral imprinting results from reduced sensitivity of the female's accessory olfactory bulb (AOB) to the stud's chemosignals. However, the AOB's combinatorial code implies that diminishing responses to one individual will distort representations of other stimuli. Here, we record extracellular responses of AOB neurons in mated and unmated female mice while presenting urine stimuli from the stud and from other sources. We find that, while initial sensory responses in the AOB (within a timescale required to guide social interactions) remain stable, responses to extended stimulation (as required for eliciting the pregnancy block) display selective attenuation of stud-responsive neurons. Such temporal disassociation could allow attenuation of slow-acting endocrine processes in a stimulus-specific manner without compromising ongoing representations that guide behavior.

Parvalbumin-Positive Interneurons Regulate Cortical Sensory Plasticity in Adulthood and Development Through Shared Mechanisms.

Parvalbumin-positive neurons are the largest class of GABAergic, inhibitory neurons in the central nervous system. In the cortex, these fast-spiking cells provide feedforward and feedback synaptic inhibition onto a diverse set of cell types, including pyramidal cells, other inhibitory interneurons, and themselves. Cortical inhibitory networks broadly, and cortical parvalbumin-expressing interneurons (cPVins) specifically, are crucial for regulating sensory plasticity during both development and adulthood. Here we review the functional properties of cPVins that enable plasticity in the cortex of adult mammals and the influence of cPVins on sensory activity at four spatiotemporal scales. First, cPVins regulate developmental critical periods and adult plasticity through molecular and structural interactions with the extracellular matrix. Second, they activate in precise sequence following feedforward excitation to enforce strict temporal limits in response to the presentation of sensory stimuli. Third, they implement gain control to normalize sensory inputs and compress the dynamic range of output. Fourth, they synchronize broad network activity patterns in response to behavioral events and state changes. Much of the evidence for the contribution of cPVins to plasticity comes from classic models that rely on sensory deprivation methods to probe experience-dependent changes in the brain. We support investigating naturally occurring, adaptive cortical plasticity to study cPVin circuits in an ethologically relevant framework, and discuss recent insights from our work on maternal experience-induced auditory cortical plasticity.

Precise and Pervasive Phasic Bursting in Locus Coeruleus during Maternal Behavior in Mice.

The noradrenergic locus coeruleus (LC) mediates key aspects of arousal, memory, and cognition in structured tasks, but its contribution to naturalistic behavior remains unclear. LC activity is thought to multiplex distinct signals by superimposing sustained ("tonic") firing patterns reflecting global brain states, such as arousal and anxiety, and rapidly fluctuating ("phasic") bursts signaling discrete behaviorally significant events. Manipulations of the LC noradrenergic system broadly impair social behavior, but the temporal structure of LC firing and its relationship to social interaction is unknown. One possibility is that tonic firing may increase in the presence of social partners; it is also possible that phasic bursts may accompany specific social events. We used chronic in vivo electrophysiology and fiber photometry to measure single-unit and population neural activity in LC of freely behaving mice during their interactions with pups. We find that pup retrieval elicits remarkably precise phasic activity in LC that cannot be attributed merely to sensory stimuli, motor activity, or reward. Correlation of LC activity with retrieval events shows that phasic events are most closely related to specific subsequent behaviors. The reliability and magnitude of phasic responses strongly suggest that these events are coordinated across LC and broadcast noradrenaline (NA) release throughout the brain. We also observed slow changes in tonic firing when females performed distinct maternal behaviors such as nest building and pup grooming. We therefore propose that LC signals state changes during sustained interactions and contributes to goal-directed action selection during social behavior with globally broadcast NA release.SIGNIFICANCE STATEMENT Locus coeruleus (LC) releases noradrenaline (NA) brain wide, influencing many cognitive, emotional, and physiological processes. Multifunctionality of LC is maintained by multiplexing NA signaling via brief "phasic" patterns of bursting and slowly changing "tonic" firing. Manipulations of NA impair social behavior, yet the structure of LC activity with respect to specific social events is unknown. We measured LC activity in mice freely interacting with pups. We find that pup retrieval elicits precisely timed and pervasive phasic activation of LC that anticipates specific behaviors. We also found that LC neurons exhibited slow fluctuations in firing during sustained behaviors. We propose that LC simultaneously contributes to goal-directed social action selection with globally broadcast NA release and signals social state changes with increased tonic firing.

Maternal Experience-Dependent Cortical Plasticity in Mice Is Circuit- and Stimulus-Specific and Requires MECP2.

The neurodevelopmental disorder Rett syndrome is caused by mutations in the gene Mecp2 Misexpression of the protein MECP2 is thought to contribute to neuropathology by causing dysregulation of plasticity. Female heterozygous Mecp2 mutants (Mecp2het ) failed to acquire a learned maternal retrieval behavior when exposed to pups, an effect linked to disruption of parvalbumin-expressing inhibitory interneurons (PV) in the auditory cortex. Nevertheless, how dysregulated PV networks affect the neural activity dynamics that underlie auditory cortical plasticity during early maternal experience is unknown. Here we show that maternal experience in WT adult female mice (WT) triggers suppression of PV auditory responses. We also observe concomitant disinhibition of auditory responses in deep-layer pyramidal neurons that is selective for behaviorally relevant pup vocalizations. These neurons further exhibit sharpened tuning for pup vocalizations following maternal experience. All of these neuronal changes are abolished in Mecp2het , suggesting that they are an essential component of maternal learning. This is further supported by our finding that genetic manipulation of GABAergic networks that restores accurate retrieval behavior in Mecp2het also restores maternal experience-dependent plasticity of PV. Our data are consistent with a growing body of evidence that cortical networks are particularly vulnerable to mutations of Mecp2 in PV neurons. Moreover, our work links, for the first time, impaired in vivo cortical plasticity in awake Mecp2 mutant animals to a natural, ethologically relevant behavior.SIGNIFICANCE STATEMENT Rett syndrome is a genetic disorder that includes language communication problems. Nearly all Rett syndrome is caused by mutations in the gene that produces the protein MECP2, which is important for changes in brain connectivity believed to underlie learning. We previously showed that female Mecp2 mutants fail to learn a simple maternal care behavior performed in response to their pups' distress cries. This impairment appeared to critically involve inhibitory neurons in the auditory cortex called parvalbumin neurons. Here we record from these neurons before and after maternal experience, and we show that they adapt their response to pup calls during maternal learning in nonmutants, but not in mutants. This adaptation is partially restored by a manipulation that improves learning.

MECP2 regulates cortical plasticity underlying a learned behaviour in adult female mice.

Neurodevelopmental disorders are marked by inappropriate synaptic connectivity early in life, but how disruption of experience-dependent plasticity contributes to cognitive and behavioural decline in adulthood is unclear. Here we show that pup gathering behaviour and associated auditory cortical plasticity are impaired in female Mecp2het mice, a model of Rett syndrome. In response to learned maternal experience, Mecp2het females exhibited transient changes to cortical inhibitory networks typically associated with limited plasticity. Averting these changes in Mecp2het through genetic or pharmacological manipulations targeting the GABAergic network restored gathering behaviour. We propose that pup gathering learning triggers a transient epoch of inhibitory plasticity in auditory cortex that is dysregulated in Mecp2het. In this window of heightened sensitivity to sensory and social cues, Mecp2 mutations suppress adult plasticity independently from their effects on early development.

Sexually divergent expression of active and passive conditioned fear responses in rats.

Traditional rodent models of Pavlovian fear conditioning assess the strength of learning by quantifying freezing responses. However, sole reliance on this measure includes the de facto assumption that any locomotor activity reflects an absence of fear. Consequently, alternative expressions of associative learning are rarely considered. Here we identify a novel, active fear response ('darting') that occurs primarily in female rats. In females, darting exhibits the characteristics of a learned fear behavior, appearing during the CS period as conditioning proceeds and disappearing from the CS period during extinction. This finding motivates a reinterpretation of rodent fear conditioning studies, particularly in females, and it suggests that conditioned fear behavior is more diverse than previously appreciated. Moreover, rats that darted during initial fear conditioning exhibited lower freezing during the second day of extinction testing, suggesting that females employ distinct and adaptive fear response strategies that improve long-term outcomes.

PTP1B inhibition suggests a therapeutic strategy for Rett syndrome.

The X-linked neurological disorder Rett syndrome (RTT) presents with autistic features and is caused primarily by mutations in a transcriptional regulator, methyl CpG-binding protein 2 (MECP2). Current treatment options for RTT are limited to alleviating some neurological symptoms; hence, more effective therapeutic strategies are needed. We identified the protein tyrosine phosphatase PTP1B as a therapeutic candidate for treatment of RTT. We demonstrated that the PTPN1 gene, which encodes PTP1B, was a target of MECP2 and that disruption of MECP2 function was associated with increased levels of PTP1B in RTT models. Pharmacological inhibition of PTP1B ameliorated the effects of MECP2 disruption in mouse models of RTT, including improved survival in young male (Mecp2-/y) mice and improved behavior in female heterozygous (Mecp2-/+) mice. We demonstrated that PTP1B was a negative regulator of tyrosine phosphorylation of the tyrosine kinase TRKB, the receptor for brain-derived neurotrophic factor (BDNF). Therefore, the elevated PTP1B that accompanies disruption of MECP2 function in RTT represents a barrier to BDNF signaling. Inhibition of PTP1B led to increased tyrosine phosphorylation of TRKB in the brain, which would augment BDNF signaling. This study presents PTP1B as a mechanism-based therapeutic target for RTT, validating a unique strategy for treating the disease by modifying signal transduction pathways with small-molecule drugs.

Noradrenergic plasticity of olfactory sensory neuron inputs to the main olfactory bulb.

Sensory responses are modulated by internal factors including attention, experience, and brain state. This is partly due to fluctuations in neuromodulatory input from regions such as the noradrenergic locus ceruleus (LC) in the brainstem. LC activity changes with arousal and modulates sensory processing, cognition, and memory. The main olfactory bulb (MOB) is richly targeted by LC fibers and noradrenaline profoundly influences MOB circuitry and odor-guided behavior. Noradrenaline-dependent plasticity affects the output of the MOB; however. it is unclear whether noradrenergic plasticity also affects the input to the MOB from olfactory sensory neurons (OSNs) in the glomerular layer. Noradrenergic terminals are found in the glomerular layer, but noradrenaline receptors do not seem to acutely modulate OSN terminals in vitro. We investigated whether noradrenaline induces plasticity at the glomerulus. We used wide-field optical imaging to measure changes in odor responses following electrical stimulation of LC in anesthetized mice. Surprisingly, odor-evoked intrinsic optical signals at the glomerulus were persistently weakened after LC activation. Calcium imaging selectively from OSNs confirmed that this effect was due to suppression of presynaptic input and was prevented by noradrenergic antagonists. Finally, suppression of responses to an odor did not require precise coincidence of the odor with LC activation. However, suppression was intensified by LC activation in the absence of odors. We conclude that noradrenaline release from LC has persistent effects on odor processing already at the first synapse of the main olfactory system. This mechanism could contribute to arousal-dependent memories.

Broadly tuned and respiration-independent inhibition in the olfactory bulb of awake mice.

Olfactory representations are shaped by brain state and respiration. The interaction and circuit substrates of these influences are unclear. Granule cells (GCs) in the main olfactory bulb (MOB) are presumed to sculpt activity reaching the cortex via inhibition of mitral/tufted cells (MTs). GCs potentially make ensemble activity more sparse by facilitating lateral inhibition among MTs and/or enforce temporally precise activity locked to breathing. Yet the selectivity and temporal structure of wakeful GC activity are unknown. We recorded GCs in the MOB of anesthetized and awake mice and identified state-dependent features of odor coding and temporal patterning. Under anesthesia, GCs were sparsely active and strongly and synchronously coupled to respiration. Upon waking, GCs desynchronized, broadened their tuning and largely fired independently from respiration. Thus, during wakefulness, GCs exhibited stronger odor responses with less temporal structure. We propose that during wakefulness GCs may shape MT odor responses through broadened lateral interactions rather than respiratory synchronization.

Behavioral state-dependent reconfiguration of song-related network activity and cholinergic systems.

The song system of oscine songbirds mediates multiple complex perceptive and productive behaviors. These discrete behaviors are modulated according to external variables such as social context, directed attention and other forms of experience. In addition, sleep has been implicated in song learning and song maintenance. Changes in behavioral state are associated with complex changes in auditory responsiveness and tonic/bursting properties of song system neurons. Cholinergic input, principally from the basal forebrain has been implicated in some of these state-dependent properties. Cholinergic modulation may affect numerous song system nuclei, with in vivo and in vitro studies indicating that a major target of cholinergic input is the forebrain nucleus HVC. Within HVC, a muscarinic cholinergic system has strong regulatory effects on most neurons, and may serve to couple and uncouple circuitry within HVC projecting along the premotor pathway with circuitry within HVC projecting along the cortico-basal ganglia pathway. These observations begin to describe how neuromodulatory regulation in the song system may contribute to learning phenomena.

Neuron-specific cholinergic modulation of a forebrain song control nucleus.

Cholinergic activation profoundly affects vertebrate forebrain networks, but pathway, cell type, and modality specificity remain poorly understood. Here we investigated cell-specific cholinergic modulation of neurons in the zebra finch forebrain song control nucleus HVC using in vitro whole cell recordings. The HVC contains projection neurons that exclusively project to either another song motor nucleus RA (robust nucleus of the arcopallium) (HVC-RAn) or the basal ganglia Area X (HVC-Xn) and these populations are synaptically coupled by a network of GABAergic interneurons. Among HVC-RAn, we observed two physiologically distinct classes that fire either phasically or tonically to injected current. Muscarine excited phasic HVC-RAn and most HVC-Xn. Effects were observed under conditions of blockade of fast synaptic transmission and were reversed by atropine. In contrast, unlike what is commonly observed in mammalian systems, HVC interneurons were inhibited by muscarine and these effects were reversed by atropine. Thus cholinergic modulation reconfigures the HVC network in a more complex fashion than that implied by monolithic "gating." The two projection pathways are decoupled through suppression of the inhibitory network that links them, whereas each is simultaneously predominantly excited. We speculate that fluctuating cholinergic tone in HVC could modulate the interaction of song motor commands with basal ganglia circuitry associated with song perception and modification. Furthermore, if the in vitro distinction between RA-projecting neurons that we observed is also present in vivo, then the song system motor pathway exhibits greater physiological diversity than has been commonly assumed.

Noradrenergic induction of odor-specific neural habituation and olfactory memories.

For many mammals, individual recognition of conspecifics relies on olfactory cues. Certain individual recognition memories are thought to be stored when conspecific odor cues coincide with surges of noradrenaline (NA) triggered by intensely arousing social events. Such familiar stimuli elicit reduced behavioral responses, a change likely related to NA-dependent plasticity in the olfactory bulb (OB). In addition to its role in these ethological memories, NA signaling in the OB appears to be relevant for the discrimination of more arbitrary odorants as well. Nonetheless, no NA-gated mechanism of long-term plasticity in the OB has ever been directly observed in vivo. Here, we report that NA release from locus ceruleus (LC), when coupled to odor presentation, acts locally in the main OB to cause a specific long-lasting suppression of responses to paired odors. These effects were observed for both food odors and urine, an important social recognition cue. Moreover, in subsequent behavioral tests, mice exhibited habituation to paired urine stimuli, suggesting that this LC-mediated olfactory neural plasticity, induced under anesthesia, can store an individual recognition memory that is observable after recovery.

Representation of natural stimuli in the rodent main olfactory bulb.

Natural odorants are complex mixtures of diverse chemical compounds. Monomolecular odorants are represented in the main olfactory bulb by distinct spatial patterns of activated glomeruli. However, it remains unclear how individual compounds contribute to population representations of natural stimuli, which appear to be unexpectedly sparse. We combined gas chromatography and intrinsic signal imaging to visualize glomerular responses to natural stimuli and their fractionated components. While whole stimuli activated up to 20 visible glomeruli, each fractionated component activated only one or few glomeruli, and most glomeruli were activated by only one component. Thus, responses to complex mixtures reflected activation by multiple components, with each contributing only a small part of the overall representation. We conclude that the population response to a complex stimulus is largely the sum of the responses to its individual components, and activation of an individual glomerulus independently signals the presence of a specific component.

Basal forebrain cholinergic modulation of auditory activity in the zebra finch song system.

The cholinergic basis of auditory "gating" in the sensorimotor nucleus HVc and its efferent target robustus archistriatalis (RA) was investigated in anesthetized zebra finches. Injections of cholinergic agonists carbachol or muscarine into HVc strongly affected discharge rates and diminished auditory responsiveness in both HVc and its target RA, changes toward an awake-like condition. HVc nicotine injections produced similar strong effects in HVc, but weaker and inconsistent effects in RA. Stimulation of basal forebrain (BF) produced an initial transient network shutdown followed by diminished auditory responsiveness in HVc and RA. All stimulation effects were blocked when preceded by HVc injections of nicotinic or muscarinic antagonists. Thus, BF cholinergic modulation of song system auditory activity acting via functionally distinct HVc circuits can contribute to auditory gating. We hypothesize that wakeful BF activity levels block sensory input to motor systems and adaptively change during behavior to allow sensorimotor feedback such as auditory feedback during singing.

State and neuronal class-dependent reconfiguration in the avian song system.

Sensory systems may adapt to behavioral requirements through state-dependent changes. In the forebrain song-system nucleus HVc of zebra finches, state-dependent auditory responses have been described in multiunit recordings. Here we report on behavioral state-dependent changes in the activity of distinct HVc neuronal classes. HVc projection neurons were identified by electrically stimulating HVc's target nuclei, the robust nucleus of the archistriatum and Area X, in anesthetized zebra finches. Projection neurons and two classes of putative interneurons could be distinguished on the basis of extracellular spike waveforms, with the first two factors of a principal components analysis accounting for 81% of the variance in spike morphometric values. Spike width was the best single variable for distinguishing among the neuronal classes. Putative interneurons had much higher firing rates spontaneously and in response to song than did projection neurons, which had extremely low spontaneous rates and phasic responses to song. Recordings from HVc in behaving animals were dominated by the two classes of putative interneurons. Both classes showed strong, selective, and temporally similar auditory responses during sleep, but only one class of interneurons reliably maintained auditory responses on waking. These responses were weaker and less selective than those seen during sleep. The observation that HVc auditory responsiveness in awake zebra finches is restricted to some classes of neurons may help explain prior multiunit results that suggested nearly complete suppression of HVc auditory responses in awake birds. We propose that the heterogeneous effects of behavioral state on distinct subpopulations of HVc neurons allow HVc to participate in multiple roles during song production, conspecific song recognition, and possibly memory consolidation during sleep.