Memory formation and retrieval of neuronal silencing in the auditory cortex

Sensory stimuli not only activate specific populations of cortical neurons but can also silence other populations. However, it remains unclear whether neuronal silencing per se leads to memory formation and behavioral expression. Here we show that mice can report optogenetic inactivation of auditory neuron ensembles by exhibiting fear responses or seeking a reward. Mice receiving pairings of footshock and silencing of a neuronal ensemble exhibited a fear response selectively to the subsequent silencing of the same ensemble. The valence of the neuronal silencing was preserved for at least 30 d and was susceptible to extinction training. When we silenced an ensemble in one side of auditory cortex for conditioning, silencing of an ensemble in another side induced no fear response. We also found that mice can find a reward based on the presence or absence of the silencing. Neuronal silencing was stored as working memory. Taken together, we propose that neuronal silencing without explicit activation in the cerebral cortex is enough to elicit a cognitive behavior.Cortical neurons exhibit spontaneous activity without explicit external stimuli (1–3), which may not only increase, but also be suppressed, by sensory stimuli (4, 5). For example, auditory stimuli suppress a subset of auditory cortical neurons in a frequency-dependent manner (5). Synaptic inhibition in the cerebral cortex is fundamental for neuronal modulation (6), including gain control (7), response selectivity (8, 9), and synchronized activities (10, 11). Inhibition-based modulations may contribute to stimulus-driven behaviors and associative memories of sensory stimuli (12); however, it remains unclear whether neuronal silencing (i.e., a transient reduction in firing rates from their spontaneous level) by itself can serve as a memory trace and bring about behavioral expressions. In this study, we tested this possibility by optogenetically silencing auditory cortical neurons.     

Extinction during reconsolidation of threat memory diminishes prefrontal cortex involvement

Controlling learned defensive responses through extinction does not alter the threat memory itself, but rather regulates its expression via inhibitory influence of the prefrontal cortex (PFC) over amygdala. Individual differences in amygdala–PFC circuitry function have been linked to trait anxiety and posttraumatic stress disorder. This finding suggests that exposure-based techniques may actually be least effective in those who suffer from anxiety disorders. A theoretical advantage of techniques influencing reconsolidation of threat memories is that the threat representation is altered, potentially diminishing reliance on this PFC circuitry, resulting in a more persistent reduction of defensive reactions. We hypothesized that timing extinction to coincide with threat memory reconsolidation would prevent the return of defensive reactions and diminish PFC involvement. Two conditioned stimuli (CS) were paired with shock and the third was not. A day later, one stimulus (reminded CS+) but not the other (nonreminded CS+) was presented 10 min before extinction to reactivate the threat memory, followed by extinction training for all CSs. The recovery of the threat memory was tested 24 h later. Extinction of the nonreminded CS+ (i.e., standard extinction) engaged the PFC, as previously shown, but extinction of the reminded CS+ (i.e., extinction during reconsolidation) did not. Moreover, only the nonreminded CS+ memory recovered on day 3. These results suggest that extinction during reconsolidation prevents the return of defensive reactions and diminishes PFC involvement. Reducing the necessity of the PFC–amygdala circuitry to control defensive reactions may help overcome a primary obstacle in the long-term efficacy of current treatments for anxiety disorders.Efforts to control maladaptive defensive reactions through extinction or exposure therapy are sometimes short-lived because these techniques do not significantly alter the threat memory itself, but rather regulate its expression via the prefrontal cortex’s (PFC) inhibition of the amygdala (1, 2). Individual variation in the integrity of this amygdala–prefrontal circuitry has been linked to trait anxiety and posttraumatic stress disorder, suggesting that exposure-based techniques may be least effective in those who suffer from anxiety disorders (3–9).Recently, it has been shown in mice (10, 11), rats (12), and humans (13–16) that precisely timing behavioral extinction to coincide with memory reconsolidation can persistently inhibit the return of defensive reactions (but see refs. 17–19 for a discussion of boundary conditions). Reconsolidation is the state to which memories enter upon retrieval, which makes them prone to interference (20–22). Behavioral interference of reconsolidation using extinction has been linked to alterations in glutamate receptor function in the amygdala, which plays a critical role in memory plasticity (10, 12). These findings are consistent with the suggestion that, in contrast to standard extinction training, extinction during reconsolidation may lead to long-lasting changes in the original threat memory (13, 16, 23).To date, the impact of extinction occurring during threat memory reconsolidation on PFC involvement is unknown in humans and other species. Animal studies of standard extinction training (i.e., repeated presentations of a conditioned stimulus without the aversive outcome) show that extinction learning and recall are mediated via the infralimbic (IL) region of the medial PFC and its connections with the amygdala; IL projections activate inhibitory cells within the amygdala that block the generation of the defense response (24, 25). Functional MRI (fMRI) studies of extinction in humans typically show a decrease in blood-oxygenation level-dependent (BOLD) signal in the ventral medial PFC (vmPFC; the human homolog of IL) in acquisition and early extinction, and a gradual increase in BOLD activity with the progression of extinction training (26, 27). If extinction occurs during reconsolidation, how might the vmPFC’s role change? One possibility is that processes occurring during reconsolidation alter the extinction circuitry, diminishing vmPFC involvement. To test this hypothesis, we used fMRI to examine the vmPFC during behavioral interference of reconsolidation in humans.BOLD responses were assessed during a 3-d protocol previously shown to interfere with reconsolidation (16). On day 1, two conditioned stimuli (CS+) were paired with a mild wrist shock (US, unconditioned stimulus); the third was not (CS−). On day 2, one CS+ (reminded CS+) but not the other (nonreminded CS+) was presented 10 min before extinction to reactivate the threat memory, followed by extinction training for all CSs. In this protocol, the reminded CS+ is presumably undergoing extinction during reconsolidation and the nonreminded CS+ is undergoing standard extinction training. On day 3, the threat memory was reinstated using four unsignaled shocks, followed by a test of recovery of the threat memory and another extinction session.We found that timing extinction training to coincide with threat memory reconsolidation prevents the return of defensive reactions, and indeed significantly diminishes PFC involvement. During extinction, only the nonreminded CS+ engaged the vmPFC, but not the reminded CS+ or the CS−. The vmPFC, moreover, showed enhanced functional connectivity with the amygdala only during extinction of the nonreminded, but not the reminded CS+. This altered connectivity during extinction of the reminded CS+ may play a role in enabling extinction learning training to more persistently modify the original threat-memory trace within the amygdala, thus preventing the return of defensive reactions on subsequent recovery tests. Reducing the necessity of the prefrontal–amygdala circuitry to control learned defensive reactions may help overcome a primary obstacle in the long-term efficacy of current treatments for anxiety disorders.     

Selective memory retrieval of auditory what and auditory where involves the ventrolateral prefrontal cortex

There is evidence from the visual, verbal, and tactile memory domains that the midventrolateral prefrontal cortex plays a critical role in the top–down modulation of activity within posterior cortical areas for the selective retrieval of specific aspects of a memorized experience, a functional process often referred to as active controlled retrieval. In the present functional neuroimaging study, we explore the neural bases of active retrieval for auditory nonverbal information, about which almost nothing is known. Human participants were scanned with functional magnetic resonance imaging (fMRI) in a task in which they were presented with short melodies from different locations in a simulated virtual acoustic environment within the scanner and were then instructed to retrieve selectively either the particular melody presented or its location. There were significant activity increases specifically within the midventrolateral prefrontal region during the selective retrieval of nonverbal auditory information. During the selective retrieval of information from auditory memory, the right midventrolateral prefrontal region increased its interaction with the auditory temporal region and the inferior parietal lobule in the right hemisphere. These findings provide evidence that the midventrolateral prefrontal cortical region interacts with specific posterior cortical areas in the human cerebral cortex for the selective retrieval of object and location features of an auditory memory experience.Functional neuroimaging studies have established a relationship between the ventrolateral prefrontal cortex and certain aspects of memory retrieval. For instance, there is evidence for the involvement of the left ventrolateral prefrontal region in selective verbal retrieval, such as the free recall of words that appear within particular contexts (lists) (1), verbal fluency (a form of selective verbal retrieval from semantic memory) (2, 3), and various other forms of verbal semantic retrieval (4–6). More precisely, it has been proposed that the two midventrolateral prefrontal cortical areas 45 and 47/12 are critical for the active selection and retrieval of information from memory when stimuli are related to other stimuli/contexts in multiple and more-or-less equiprobable ways, so that memory retrieval cannot be a matter of mere recognition of the stimuli or supported by strong and unique stimulus-to-stimulus or stimulus-to-context associations (7). In previous functional neuroimaging studies, we were able to show activity increases that were specific to the midventrolateral prefrontal cortex for the active retrieval of visual and tactile stimuli (8, 9). A more recent study has shown that patients with lesions to the ventrolateral prefrontal region, but not those with lesions involving the dorsolateral prefrontal region, show impairments in the active controlled retrieval of the visual contexts within which words had appeared (10). This impairment was not accompanied by general memory loss of the type observed after limbic medial temporal lobe lesions (11–13). Patients with ventrolateral prefrontal lesions performed as well as normal control subjects on recognition memory of the presented stimuli, but were impaired when they were asked to retrieve selectively specific aspects of the memorized information in a task in which words and their context entered into multiple relations with each other across trials and, therefore, retrieval could not be supported by simple recognition memory (10). Taken together, these results suggest that the midventrolateral prefrontal cortex plays a critical role in the top–down modulation of activity for the retrieval of specific features of mnemonic information when simple familiarity and/or unambiguous stimulus-to-stimulus relations are not sufficient for memory retrieval.Although there is considerable evidence of direct anatomical connections between auditory cortical regions in the temporal lobe and the ventrolateral prefrontal cortex (14–20) and the presence of auditory responsive neurons for complex sounds in the ventrolateral prefrontal cortex (21, 22), there is no study examining the potential involvement of the ventrolateral prefrontal region in the controlled selective retrieval of auditory nonverbal information in the human brain. Most studies investigating the role of the prefrontal cortex in the auditory domain have focused on verbal phonological and semantic memory (23–25). The aim of the present study was to examine the hypothesis that the midventrolateral prefrontal cortex is involved in the active controlled retrieval of nonverbal auditory information from memory, in a manner analogous to its previously demonstrated role in the retrieval of verbal and nonverbal visual and tactile information (8, 9, 26).In the present functional magnetic resonance imaging (fMRI) study, on each trial, one of four different melodies was presented from one of four locations in a virtual acoustic environment (Fig. 1A) and, after a short delay, the subjects were instructed via a cue to retrieve either the specific melody (regardless of its location) or the location (regardless of the melody that was presented there) (Fig. 1B). The subjects were thus required to isolate the instructed component of the previously memorized auditory stimulus complex, namely the melody or the location. The experimental design prevented the establishment of strong and unique relations between the melodies and the locations because, across trials, all four melodies were presented from each one of the four locations with equal probability. Intermixed with these “active retrieval” trials requiring the isolation of a specific feature of the stimulus complex were “recognition” control trials that were identical in terms of the initial stimulus presentation period but, following the delay, the subjects were instructed simply to recognize (based on familiarity) the previously encoded stimulus complex of the melody and its location. No isolation of a specific aspect of the memory (melody or location) was required in these recognition control trials. There were also “baseline” control trials in which no auditory stimulus was presented during the retrieval period. The hypothesis to be tested predicted selective activity increases in the midventrolateral prefrontal cortex during the active retrieval of specific aspects of the encoded auditory stimuli in comparison with the simple recognition of that stimulus, that is, during the test period of the active retrieval trials versus the same period in the recognition control trials.Open in a separate windowFig. 1.Schematic diagram of the experimental paradigm and scanning protocol. (A) Locations used to present the melodies. Gray indicates the four locations that were used only during the test period of the recognition control trials. (B) Illustration of the testing procedure. All trials started with the presentation of a cross in the middle of the screen followed by the encoding period, during which the auditory stimulus was presented. After a variable delay, a visual instructional cue was presented and the test period followed after the disappearance of the visual cue. A second auditory stimulus was presented during the test period for the experimental and recognition control trials but not during the baseline control trials. The subjects’ responses were recorded during the test period. The duration of the various periods was the same for the experimental and control trials. (C) Illustration of the sparse-sampling fMRI scanning used in this experiment.     

Selective pharmacogenetic inhibition of mammalian target of Rapamycin complex I (mTORC1) blocks long-term synaptic plasticity and memory storage

Both the formation of long-term memory (LTM) and late-long-term potentiation (L-LTP), which is thought to represent the cellular model of learning and memory, require de novo protein synthesis. The mammalian target of Rapamycin (mTOR) complex I (mTORC1) integrates information from various synaptic inputs and its best characterized function is the regulation of translation. Although initial studies have shown that rapamycin reduces L-LTP and partially blocks LTM, recent genetic and pharmacological evidence indicating that mTORC1 promotes L-LTP and LTM is controversial. Thus, the role of mTORC1 in L-LTP and LTM is unclear. To selectively inhibit mTORC1 activity in the adult brain, we used a "pharmacogenetic" approach that relies on the synergistic action of a drug (rapamycin) and a genetic manipulation (mTOR heterozygotes, mTOR(+/-) mice) on the same target (mTORC1). Although L-LTP and LTM are normal in mTOR(+/-) mice, application of a low concentration of rapamycin-one that is subthreshold for WT mice-prevented L-LTP and LTM only in mTOR(+/-) mice. Furthermore, we found that mTORC1-mediated translational control is required for memory reconsolidation. We provide here direct genetic evidence supporting the role of mTORC1 in L-LTP and behavioral memory.     

Spinophilin regulates the formation and function of dendritic spines

Spinophilin, a protein that interacts with actin and protein phosphatase-1, is highly enriched in dendritic spines. Here, through the use of spinophilin knockout mice, we provide evidence that spinophilin modulates both glutamatergic synaptic transmission and dendritic morphology. The ability of protein phosphatase-1 to regulate the activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors was reduced in spinophilin knockout mice. Consistent with altered glutamatergic transmission, spinophilin-deficient mice showed reduced long-term depression and exhibited resistance to kainate-induced seizures and neuronal apoptosis. In addition, deletion of the spinophilin gene caused a marked increase in spine density during development in vivo as well as altered filopodial formation in cultured neurons. In conclusion, spinophilin appears to be required for the regulation of the properties of dendritic spines.     

Sleep promotes the formation of dendritic filopodia and spines near learning-inactive existing spines

Changes in synaptic connections are believed to underlie long-term memory storage. Previous studies have suggested that sleep is important for synapse formation after learning, but how sleep is involved in the process of synapse formation remains unclear. To address this question, we used transcranial two-photon microscopy to investigate the effect of postlearning sleep on the location of newly formed dendritic filopodia and spines of layer 5 pyramidal neurons in the primary motor cortex of adolescent mice. We found that newly formed filopodia and spines were partially clustered with existing spines along individual dendritic segments 24 h after motor training. Notably, posttraining sleep was critical for promoting the formation of dendritic filopodia and spines clustered with existing spines within 8 h. A fraction of these filopodia was converted into new spines and contributed to clustered spine formation 24 h after motor training. This sleep-dependent spine formation via filopodia was different from retraining-induced new spine formation, which emerged from dendritic shafts without prior presence of filopodia. Furthermore, sleep-dependent new filopodia and spines tended to be formed away from existing spines that were active at the time of motor training. Taken together, these findings reveal a role of postlearning sleep in regulating the number and location of new synapses via promoting filopodial formation.

Learning and memory consolidation are associated with the rewiring of neuronal network connectivity (1–3). Previous studies have shown that motor training leads to the formation and elimination of postsynaptic dendritic spines of pyramidal neurons in the primary motor cortex (M1) (4–8). Learning-induced new spines stabilize and persist over long periods of time (4). The extent of spine remodeling correlates with behavioral improvement after learning (4, 9), and the disruption of spine remodeling impairs learned motor behavior (10–12). These studies suggest that learning-induced new synapses contribute to changes in neuronal circuits that are likely important for the retention of learned behaviors (13, 14).Cumulative evidence suggests that sleep affects synaptic structural plasticity in many brain regions (15–17). For example, sleep has been shown to promote spine formation and elimination in developing somatosensory and visual cortices (18, 19). In the motor cortex, sleep promotes branch-specific formation of new dendritic spines following motor learning and selectively stabilizes learning-induced new synaptic connections (11, 12). Sleep has also been shown to regulate dendritic spine numbers in hippocampal CA1 area (20–22). In addition, many lines of evidence have revealed the function of sleep in increasing, decreasing, or stabilizing synaptic strength and neuronal firing in various brain regions (23–31). Together, these studies strongly suggest that sleep has an important role in promoting synaptic structural plasticity in neuronal circuits during development and after learning.While sleep promotes the formation of new spines after learning (12), it remains unknown how postlearning sleep regulates new synapse formation along dendritic branches. Synapse formation is a prolonged process often involving the generation of dendritic filopodia, thin and long protrusions without bulbous heads (32–35). These highly dynamic filopodia have been shown to initiate the contact with presynaptic axonal terminals and transform into new spines (36, 37). It is not known whether sleep promotes new spine formation via filopodia formation and subsequent transformation. Furthermore, it is also unclear whether sleep-dependent formation of new dendritic protrusions (filopodia and spines) is distributed on dendritic branches in a random or nonrandom manner. On the one hand, new synapses may be formed in clusters with synapses of similar functions to allow nonlinear summation of inputs important for increasing memory storage capacity (9, 38–43). On the other hand, new connections may be formed preferentially near less active/strong synapses to avoid competition for limited synaptic resources (44–47).In this study, we found that dendritic filopodia and spines formed after motor training were partially clustered with existing spines on apical tuft dendrites of layer 5 (L5) pyramidal neurons in the mouse primary motor cortex. Posttraining sleep was critical for the clustered formation of new filopodia, some of which were transformed into new spines. In addition, the clustered new filopodia and spines tended to be formed near existing spines that were inactive at the time of motor training. These findings reveal a role for sleep in neuronal circuit plasticity by promoting clustered spine formation via dendritic filopodia near learning-inactive existing spines.     

Regional synapse gain and loss accompany memory formation in larval zebrafish

Defining the structural and functional changes in the nervous system underlying learning and memory represents a major challenge for modern neuroscience. Although changes in neuronal activity following memory formation have been studied [B. F. Grewe et al., Nature 543, 670–675 (2017); M. T. Rogan, U. V. Stäubli, J. E. LeDoux, Nature 390, 604–607 (1997)], the underlying structural changes at the synapse level remain poorly understood. Here, we capture synaptic changes in the midlarval zebrafish brain that occur during associative memory formation by imaging excitatory synapses labeled with recombinant probes using selective plane illumination microscopy. Imaging the same subjects before and after classical conditioning at single-synapse resolution provides an unbiased mapping of synaptic changes accompanying memory formation. In control animals and animals that failed to learn the task, there were no significant changes in the spatial patterns of synapses in the pallium, which contains the equivalent of the mammalian amygdala and is essential for associative learning in teleost fish [M. Portavella, J. P. Vargas, B. Torres, C. Salas, Brain Res. Bull. 57, 397–399 (2002)]. In zebrafish that formed memories, we saw a dramatic increase in the number of synapses in the ventrolateral pallium, which contains neurons active during memory formation and retrieval. Concurrently, synapse loss predominated in the dorsomedial pallium. Surprisingly, we did not observe significant changes in the intensity of synaptic labeling, a proxy for synaptic strength, with memory formation in any region of the pallium. Our results suggest that memory formation due to classical conditioning is associated with reciprocal changes in synapse numbers in the pallium.

It is widely believed that memories are formed as a result of alterations in synaptic connections between axons and dendrites, an idea first proposed by Ramon y Cajal (1–4). Although synapse changes have been extensively studied in brain slices in the context of long-term potentiation (5, 6), less is known about how synapses in a living vertebrate are modified when a memory is formed.Memory formation has been widely studied using classical conditioning (CC), a robust and straightforward form of learning in which an animal is exposed to a neutral stimulus (conditioned stimulus, CS) paired with an appetitive or aversive stimulus (unconditioned stimulus, US) that evokes a specific behavioral response (UR, unconditioned response) (7, 8). As a result of the pairing, animals learn to associate the CS with the US, causing them to respond to the CS with a conditioned response (CR) identical to the UR, signifying memory retrieval (9, 10). Memory retrieval is also evoked by activating a cellular engram, a group of neurons active during memory formation and retrieval (11–18). The central locus of CC in mammals, the amygdala (19), is located in a relatively inaccessible area beneath the cortex (20). Thus, although numerous longitudinal imaging studies have documented experience-dependent changes in the structure of spines of cortical and hippocampal neurons (21, 22), few imaging studies have directly examined synaptic changes that occur in the amygdala during associative memory formation.Instead, synaptic changes that occur in the amygdala during CC (23) have been studied primarily using indirect measures of synaptic strength, such as the ratio of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor/N-methyl D-aspartate (AMPA/NMDA) currents in excitatory postsynaptic currents (EPSCs). Increases in AMPA/NMDA ratio in amygdalar neurons following auditory fear conditioning (FC), a type of CC (24–27), indicate that associative memory formation coincides with increases in synaptic strength. In addition, imaging experiments in brain regions beyond the amygdala have shown diverse effects following CC. For example, following contextual fear conditioning, engram neurons in the CA1 region of the hippocampus that receive inputs from CA3 engram neurons displayed spines that were larger and more densely packed than nonengram cells (28). Furthermore, experiments in which neuronal morphology was directly observed before and after FC found that neurons in the frontal association (29) and primary motor cortex (30) showed a decrease in the number of spines, whereas neurons in the auditory cortex showed an increase in spine number with memory formation (31).To obtain previously unavailable insight into memory formation within the central locus of associative memory storage, we developed a paradigm combining in vivo labeling and imaging with informatics and analysis tools. We used this paradigm to map synaptic changes that occur over time in the intact brain of a living vertebrate during memory formation. We imaged the pallium of teleost fish, which contains the putative homolog of the mammalian amygdala based on anatomy (32), gene expression (33), and function (34). The pallium is on the surface of the brain (35), and zebrafish larvae are highly transparent, allowing for intact, whole-brain imaging using selective plane illumination microscopy (SPIM) without the need for invasive intervention (36). In addition, while most studies of learning in zebrafish have used adults (37–40), at least one study showed that larval zebrafish can learn to associate a place with a positive valence US (41). These attributes suggest that larval zebrafish may be an ideal model organism for studying synaptic changes during memory formation due to CC. We have engaged this challenge by combining purpose-built experimental tools with data management software that enables transparent analyses of large and heterogeneous datasets. All data were characterized and stored at the time of creation in a customized data management system designed to conform to findability, accessibility, interoperability, and reusability (i.e., FAIR principles) (see Materials and Methods) (42).     

A structural basis for enhancement of long-term associative memory in single dendritic spines regulated by PKC

Using both scanning confocal and electron microscopic morphometric measurements, we analyzed single dendritic spines of CA1 pyramidal cells in the hippocampi of water maze-trained rats vs. controls. Two days after completion of all training, we observed a memory-specific increase in the number of mushroom spines-all of which make synaptic contacts-but not in the numbers of filopodia or stubby or thin spines, as quantified with double-blind protocols in both scanning confocal and electron microscopic images. This memory-specific increase of mushroom spine number was enhanced by the PKC activator and candidate Alzheimer's disease therapeutic bryostatin, blocked by the PKCalpha-isozyme blocker Ro 31-8220, and accompanied by increases in the number of "perforated" postsynaptic densities, increased numbers of presynaptic vesicles, and the increased occurrence of double-synapse presynaptic boutons associated with the mushroom spines. These and other confocally imaged immunohistochemical results described here involving PKC substrates indicate that individual mushroom spines provide structural storage sites for long-term associative memory and sites for memory-specific synaptogenesis that involve PKC-regulated changes of spine shape, as well as PKC-regulated changes of pre- and postsynaptic ultrastructure.     

Persistence of fear memory across time requires the basolateral amygdala complex

Mammals evolved a potent fear-motivated defensive system capable of single-trial fear learning that shows no forgetting over the lifespan of the animal. The basolateral amygdala complex (BLA) is considered an essential component of this conditional fear learning system. However, recent studies challenge this view and suggest that plasticity within other brain regions (i.e., central nucleus of the amygdala) may be crucial for fear conditioning. In the present study, we examine the mnemonic limits of contextual fear conditioning in the absence of the BLA using overtraining and by measuring remote fear memories. After excitotoxic lesions of the BLA were created, animals underwent overtraining and were tested at recent and remote memory intervals. Here we show that animals with BLA lesions can learn normal levels of fear. However, this fear memory loses its adaptive features: it is acquired slowly and shows substantial forgetting when remote memory is tested. Collectively, these findings suggest that fear-related plasticity acquired by brain regions outside of the BLA, unlike those acquired in the intact animals, do so for a relatively time-limited period.     

Laminar and columnar auditory cortex in avian brain

The mammalian neocortex mediates complex cognitive behaviors, such as sensory perception, decision making, and language. The evolutionary history of the cortex, and the cells and circuitry underlying similar capabilities in nonmammals, are poorly understood, however. Two distinct features of the mammalian neocortex are lamination and radially arrayed columns that form functional modules, characterized by defined neuronal types and unique intrinsic connections. The seeming inability to identify these characteristic features in nonmammalian forebrains with earlier methods has often led to the assumption of uniqueness of neocortical cells and circuits in mammals. Using contemporary methods, we demonstrate the existence of comparable columnar functional modules in laminated auditory telencephalon of an avian species (Gallus gallus). A highly sensitive tracer was placed into individual layers of the telencephalon within the cortical region that is similar to mammalian auditory cortex. Distribution of anterograde and retrograde transportable markers revealed extensive interconnections across layers and between neurons within narrow radial columns perpendicular to the laminae. This columnar organization was further confirmed by visualization of radially oriented axonal collaterals of individual intracellularly filled neurons. Common cell types in birds and mammals that provide the cellular substrate of columnar functional modules were identified. These findings indicate that laminar and columnar properties of the neocortex are not unique to mammals and may have evolved from cells and circuits found in more ancient vertebrates. Specific functional pathways in the brain can be analyzed in regard to their common phylogenetic origins, which introduces a previously underutilized level of analysis to components involved in higher cognitive functions.     

Motility of dendritic spines in visual cortex in vivo: changes during the critical period and effects of visual deprivation

Cortical dendritic spines are highly motile postsynaptic structures onto which most excitatory synapses are formed. It has been postulated that spine dynamics might reflect synaptic plasticity of cortical neurons. To test this hypothesis, we have investigated spine dynamics during the critical period in mouse visual cortex in vivo with and without sensory deprivation. The motility of spines on apical dendrites of layer 5 neurons was assayed by time-lapse two-photon microscopy. Spines were motile at the ages examined, postnatal days (P)21-P42, although motility decreased between P21 and P28 and then remained stable through P42. Binocular deprivation from before the time of eye-opening up-regulated spine motility during the peak of the critical period (P28), without affecting average spine length, class distribution, or density. Deprivation at the start of the critical period had no effect on spine motility, whereas continued deprivation through the end of the critical period appeared to reduce spine motility slightly. We conclude that spine motility might be involved in critical-period plasticity and that reduction of activity during the critical period enhances spine dynamics.     

Fear and safety learning differentially affect synapse size and dendritic translation in the lateral amygdala

Fear learning is associated with changes in synapse strength in the lateral amygdala (LA). To examine changes in LA dendritic spine structure with learning, we used serial electron microscopy to re-construct dendrites after either fear or safety conditioning. The spine apparatus, a smooth endoplasmic reticulum (sER) specialization found in very large spines, appeared more frequently after fear conditioning. Fear conditioning was associated with larger synapses on spines that did not contain a spine apparatus, whereas safety conditioning resulted in smaller synapses on these spines. Synapses on spines with a spine apparatus were smaller after safety conditioning but unchanged with fear conditioning, suggesting a ceiling effect. There were more polyribosomes and multivesicular bodies throughout the dendrites from fear conditioned rats, indicating increases in both protein synthesis and degradation. Polyribosomes were associated with the spine apparatus under both training conditions. We conclude that LA synapse size changes bidirectionally with learning and that the spine apparatus has a central role in regulating synapse size and local translation.     

Spectro-temporal modulation transfer function of single voxels in the human auditory cortex measured with high-resolution fMRI

Are visual and auditory stimuli processed by similar mechanisms in the human cerebral cortex? Images can be thought of as light energy modulations over two spatial dimensions, and low-level visual areas analyze images by decomposition into spatial frequencies. Similarly, sounds are energy modulations over time and frequency, and they can be identified and discriminated by the content of such modulations. An obvious question is therefore whether human auditory areas, in direct analogy to visual areas, represent the spectro-temporal modulation content of acoustic stimuli. To answer this question, we measured spectro-temporal modulation transfer functions of single voxels in the human auditory cortex with functional magnetic resonance imaging. We presented dynamic ripples, complex broadband stimuli with a drifting sinusoidal spectral envelope. Dynamic ripples are the auditory equivalent of the gratings often used in studies of the visual system. We demonstrate selective tuning to combined spectro-temporal modulations in the primary and secondary auditory cortex. We describe several types of modulation transfer functions, extracting different spectro-temporal features, with a high degree of interaction between spectral and temporal parameters. The overall low-pass modulation rate preference of the cortex matches the modulation content of natural sounds. These results demonstrate that combined spectro-temporal modulations are represented in the human auditory cortex, and suggest that complex signals are decomposed and processed according to their modulation content, the same transformation used by the visual system.     

Serotonin-mushroom body circuit modulating the formation of anesthesia-resistant memory in Drosophila

Pavlovian olfactory learning in Drosophila produces two genetically distinct forms of intermediate-term memories: anesthesia-sensitive memory, which requires the amnesiac gene, and anesthesia-resistant memory (ARM), which requires the radish gene. Here, we report that ARM is specifically enhanced or inhibited in flies with elevated or reduced serotonin (5HT) levels, respectively. The requirement for 5HT was additive with the memory defect of the amnesiac mutation but was occluded by the radish mutation. This result suggests that 5HT and Radish protein act on the same pathway for ARM formation. Three supporting lines of evidence indicate that ARM formation requires 5HT released from only two dorsal paired medial (DPM) neurons onto the mushroom bodies (MBs), the olfactory learning and memory center in Drosophila: (i) DPM neurons were 5HT-antibody immunopositive; (ii) temporal inhibition of 5HT synthesis or release from DPM neurons, but not from other serotonergic neurons, impaired ARM formation; (iii) knocking down the expression of d5HT1A serotonin receptors in α/β MB neurons, which are innervated by DPM neurons, inhibited ARM formation. Thus, in addition to the Amnesiac peptide required for anesthesia-sensitive memory formation, the two DPM neurons also release 5HT acting on MB neurons for ARM formation.     

Learning-related long-term potentiation of inhibitory synapses in the cerebellar cortex

Despite the widespread distribution of inhibitory synapses throughout the central nervous system, plasticity of inhibitory synapses related to associative learning has never been reported. In the cerebellum, the neural correlate of fear memory is provided by a long-term potentiation (LTP) of the excitatory synapse between the parallel fibers (PFs) and the Purkinje cell (PC). In this article, we provide evidence that inhibitory synapses in the cerebellar cortex also are affected by fear conditioning. Whole-cell patch-clamp recordings of spontaneous and miniature GABAergic events onto the PC show that the frequency but not the amplitude of these events is significantly greater up to 24 h after the conditioning. Adequate levels of excitation and inhibition are required to maintain the temporal fidelity of a neuronal network. Such fidelity can be evaluated by determining the time window for multiple input coincidence detection. We found that, after fear learning, PCs are able to integrate excitatory inputs with greater probability within short delays, but the width of the whole window is unchanged. Therefore, excitatory LTP provides a more effective detection, and inhibitory potentiation serves to maintain the time resolution of the system.     

Disruption of the expression of the proprotein convertase PC7 reduces BDNF production and affects learning and memory in mice

PC7 belongs to the proprotein convertase family, whose members are implicated in the cleavage of secretory precursors. The in vivo function of PC7 is unknown. Herein, we find that the precursor proBDNF is processed into mature BDNF in COS-1 cells coexpressing proBDNF with either PC7 or Furin. Conversely, the processing of proBDNF into BDNF is markedly reduced in the absence of either Furin or PC7 in mouse primary hepatocytes. In vivo we observe that BDNF and PC7 mRNAs are colocalized in mouse hippocampus and amygdala and that mature BDNF protein levels are reduced in these brain areas in PC7 KO mice but not in the hippocampus of PC1/3 KO mice. Various behavioral tests reveal that in PC7 KO mice spatial memory is intact and plasticity of responding is mildly abnormal. Episodic and emotional memories are severely impaired, but both are rescued with the tyrosine receptor kinase B agonist 7,8-dihydroxyflavone. Altogether, these results support an in vivo role for PC7 in the regulation of certain types of cognitive performance, in part via proBDNF processing. Because polymorphic variants of human PC7 are being characterized, it will be important in future studies to determine their effects on additional physiological and behavioral processes.Nine secretory proprotein convertases (PCs) play major roles in regulating multiple cellular and extracellular processes both in health and disease states (reviewed in refs. 1 and 2). The convertases PC1/3, PC2, Furin, PC4, PC5/6, PACE4, and PC7 cleave their substrates after single or pairs of basic amino acids, SKI-1/S1P processes protein precursors after nonbasic residues, and PCSK9 has no known substrates other than itself (1). Studies that have analyzed tissue expression, levels, regulation, ontogeny, phenotypes of model animals lacking one or more PCs, and human/mouse natural mutations are starting to provide clues as to the specific roles of these enzymes in cells and whole-animal physiological and pathological processes.The type I membrane-bound PC7 is the most ancient member of the mammalian basic amino acid-specific PC family, and it exhibits the closest homology to yeast kexin (3). Human PC7 is synthesized as an N-glycosylated zymogen (proPC7) that, like most other PCs, undergoes autocatalytic cleavage in the endoplasmic reticulum at RAKR₁₄₁↓SV. Mature PC7 can reach the cell surface by an unconventional route from the endoplasmic reticulum (4), but it also accumulates in the trans Golgi network (TGN) and can cycle between the cell surface and TGN via endosomes, in part by virtue of a Pro-Leu-Cys₇₂₆ motif in its cytosolic tail (5). The tail also contains two cysteine residues, Cys₆₉₉ and Cys₇₀₄, which are palmitoylated (3, 4, 6), that may assist in this process. Confocal and electron microscopy studies have revealed that PC7 localizes to vesicles located immediately beneath the plasma membrane (4, 7). No soluble shed forms of PC7 have been detected. Enzymatic activity assays using only the soluble luminal/extracellular domain of PC7 (sol.PC7) and fluorogenic substrates have indicated that this Ca²⁺-dependent enzyme exhibits a neutral pH optimum and a cleavage specificity similar to that of Furin, cleaving within the general motif (R/K)-2X_n-R↓ where n = 0–2 (8, 9).Only the membrane-bound PC7 induces the processing of proepidermal growth factor into a ∼115-kDa transmembrane form (10). PC7 is abundant in neurons (11) but is also expressed in microglia (12). It has been shown that PC7 exerts an important function in MHC class I-mediated antigen presentation (7), befitting its high expression within the immune system (3). Finally, PC7 is unique because it is able to shed the human transferrin receptor 1 (TfR1) into a soluble form by cleavage at KTECER₁₀₀↓LA within endosomes (13).The physiological importance of the PCs is illustrated by the early death or major phenotypes observed in mice lacking one or more convertase (1, 14). Although generated several years ago, deletion of PC7 is the only PC KO mouse for which no overt phenotype(s) has been described (15). In contrast, PC7 knockdown in Xenopus is embryonic lethal. These embryos lack eyes and brain and exhibit abnormal anterior neural development (16). Unless this knockdown is due to an off-target effect, amphibian PC7 seems to fulfill essential neuronal functions that, in mammals, may be either nonessential or redundantly assumed by other PCs. Notably, the regulation of the PC7 gene (PCSK7) has not been examined (3, 17).In the present work we describe behavioral alterations in mice lacking PC7. Our results show that PC7 KO mice have lower levels of BDNF in the hippocampus and amygdala than WT mice and exhibit learning and memory impairments. Reduced BDNF levels are likely responsible for some of these deficits, because they are rescued by an agonist to the BDNF receptor tyrosine receptor kinase B (TrkB). Therefore, PC7 seems to play unique roles in the CNS, at least in part, through regulating levels of BDNF.     

Glucocorticoids interact with the hippocampal endocannabinoid system in impairing retrieval of contextual fear memory

There is extensive evidence that glucocorticoid hormones impair the retrieval of memory of emotionally arousing experiences. Although it is known that glucocorticoid effects on memory retrieval impairment depend on rapid interactions with arousal-induced noradrenergic activity, the exact mechanism underlying this presumably nongenomically mediated glucocorticoid action remains to be elucidated. Here, we show that the hippocampal endocannabinoid system, a rapidly activated retrograde messenger system, is involved in mediating glucocorticoid effects on retrieval of contextual fear memory. Systemic administration of corticosterone (0.3-3 mg/kg) to male Sprague-Dawley rats 1 h before retention testing impaired the retrieval of contextual fear memory without impairing the retrieval of auditory fear memory or directly affecting the expression of freezing behavior. Importantly, a blockade of hippocampal CB1 receptors with AM251 prevented the impairing effect of corticosterone on retrieval of contextual fear memory, whereas the same impairing dose of corticosterone increased hippocampal levels of the endocannabinoid 2-arachidonoylglycerol. We also found that antagonism of hippocampal β-adrenoceptor activity with local infusions of propranolol blocked the memory retrieval impairment induced by the CB receptor agonist WIN55,212-2. Thus, these findings strongly suggest that the endocannabinoid system plays an intermediary role in regulating rapid glucocorticoid effects on noradrenergic activity in impairing memory retrieval of emotionally arousing experiences.     

Arc expression and neuroplasticity in primary auditory cortex during initial learning are inversely related to neural activity

Models of learning-dependent sensory cortex plasticity require local activity and reinforcement. An alternative proposes that neural activity involved in anticipation of a sensory stimulus, or the preparatory set, can direct plasticity so that changes could occur in regions of sensory cortex lacking activity. To test the necessity of target-induced activity for initial sensory learning, we trained rats to detect a low-frequency sound. After learning, Arc expression and physiologically measured neuroplasticity were strong in a high-frequency auditory cortex region with very weak target-induced activity in control animals. After 14 sessions, Arc and neuroplasticity were aligned with target-induced activity. The temporal and topographic correspondence between Arc and neuroplasticity suggests Arc may be intrinsic to the neuroplasticity underlying perceptual learning. Furthermore, not all neuroplasticity could be explained by activity-dependent models but can be explained if the neural activity involved in the preparatory set directs plasticity.     

Differential roles of the dopamine 1-class receptors,D1R and D5R,in hippocampal dependent memory

Activation of the hippocampal dopamine 1-class receptors (D1R and D5R) are implicated in contextual fear conditioning (CFC). However, the specific role of the D1R versus D5R in hippocampal dependent CFC has not been investigated. Generation of D1R- and D5R-specific in situ hybridization probes showed that D1R and D5R mRNA expression was greatest in the dentate gyrus (DG) of the hippocampus. To identify the role of each receptor in CFC we generated spatially restricted KO mice that lack either the D1R or D5R in DG granule cells. DG D1R KOs displayed significant fear memory deficits, whereas DG D5R KOs did not. Furthermore, D1R KOs but not D5R KOs, exhibited generalized fear between two similar but different contexts. In the familiar home cage context, c-Fos expression was relatively low in the DG of control mice, and it increased upon exposure to a novel context. This level of c-Fos expression in the DG did not further increase when a footshock was delivered in the novel context. In DG D1R KOs, DG c-Fos levels in the home cage was higher than that of the control mice, but it did not further increase upon exposure to a novel context and remained at the same level upon a shock delivery. In contrast, the levels of DG c-Fos expression was unaffected by the deletion of DG D5R neither in the home cage nor upon a shock delivery. These results suggest that DG D1Rs, but not D5Rs, contribute to the formation of distinct contextual representations of novel environments.The hippocampus is crucial for aversive Pavlovian conditioning, such as contextual fear conditioning (CFC) (1, 2). In CFC, the conditioned stimulus (context) is paired with the unconditioned stimulus (footshock), and after pairing, the context serves as a cue to predict a potential footshock (3, 4). Although the role of dopamine has been studied in the context of reward learning (5), evidence suggests that midbrain dopaminergic neurons are also important for aversive Pavlovian conditioning (6–9). In line with this evidence, hippocampal encoding of novel and contextual information is linked to dopamine release via excitation of dopamine neurons of the midbrain (5, 10, 11). Additionally, delivery of aversive stimuli, such as a footshock, results in increased dopaminergic neuron activity (12). Moreover, inactivation of hippocampal D1Rs and D5Rs attenuates contextual fear memory (13). Thus, it follows that delivery of an aversive stimulus activates midbrain dopamine neurons that project to the hippocampus, which is crucial for encoding novel contextual cues (12, 14, 15). Activation of hippocampal D1Rs and D5Rs may then strengthen the encoding of novel contextual information during CFC.The precise role of subregion-specific D1R or D5R activation in hippocampal-dependent learning and memory is unknown. This is in part due to the inability to discriminate between and spatially restrict D1R from D5R function (16–18), which is an important caveat because each receptor is involved in modulating distinct neuronal processes (19–22). Indeed, there is a lack of consensus of D1R and D5R expression patterns in the rodent hippocampus (23–27). Moreover, pharmacological findings are at odds with D1R and D5R global KO studies, which show that neither D1Rs nor D5Rs are required for fear conditioning (16, 17). Therefore, to reconcile these disparate findings and to test the necessity of D1R and D5R activation for CFC, it is necessary to functionally isolate and spatially restrict hippocampal D1R and D5R activity.In this study, we found that D1Rs and D5Rs exhibit overlapping expression in dentate gyrus (DG) granule cells. DG D1R activation is necessary to increase c-Fos expression in the DG and CA3 to enhance novel contextual encoding. Moreover, DG D1R activation decreases generalization of the conditioned fear response to novel contexts. However, we found no role for DG D5Rs in modulating DG c-Fos expression or contextual fear learning and memory. In using our subregion-specific KO mice, we show that the hippocampal dopamine signal plays a definitive role in CFC.     

Dynamics of subjective contour formation in the early visual cortex

To elucidate the roles of visual areas V1 and V2 and their interaction in early perceptual processing, we studied the responses of V1 and V2 neurons to statically displayed Kanizsa figures. We found evidence that V1 neurons respond to illusory contours of the Kanizsa figures. The illusory contour signals in V1 are weaker than in V2, but are significant, particularly in the superficial layers. The population averaged response to illusory contours emerged 100 msec after stimulus onset in the superficial layers of V1, and around 120--190 msec in the deep layers. The illusory contour response in V2 began earlier, occurring at 70 msec in the superficial layers and at 95 msec in the deep layers. The temporal sequence of the events suggests that the computation of illusory contours involves intercortical interaction, and that early perceptual organization is likely to be an interactive process.