Rapid odor processing by layer 2 subcircuits in lateral entorhinal cortex.

Olfactory information is encoded in lateral entorhinal cortex (LEC) by two classes of layer 2 (L2) principal neurons: fan and pyramidal cells. However, the functional properties of L2 cells and how they contribute to odor coding are unclear. Here, we show in awake mice that L2 cells respond to odors early during single sniffs and that LEC is essential for rapid discrimination of both odor identity and intensity. Population analyses of L2 ensembles reveal that rate coding distinguishes odor identity, but firing rates are only weakly concentration dependent and changes in spike timing can represent odor intensity. L2 principal cells differ in afferent olfactory input and connectivity with inhibitory circuits and the relative timing of pyramidal and fan cell spikes provides a temporal code for odor intensity. Downstream, intensity is encoded purely by spike timing in hippocampal CA1. Together, these results reveal the unique processing of odor information by LEC subcircuits and highlight the importance of temporal coding in higher olfactory areas.

Interhemispheric Callosal Projections Sharpen Frequency Tuning and Enforce Response Fidelity in Primary Auditory Cortex.

Sensory cortical areas receive glutamatergic callosal projections that link information processing between brain hemispheres. In primary auditory cortex (A1), ipsilateral principal cells from a particular tonotopic region project to neurons in matching frequency space of the contralateral cortex. However, the role of interhemispheric projections in shaping cortical responses to sound and frequency tuning in awake animals is unclear. Here, we use translaminar single-unit recordings and optogenetic approaches to probe how callosal inputs modulate spontaneous and tone-evoked activity in A1 of awake mice. Brief activation of callosal inputs drove either short-latency increases or decreases in firing of individual neurons. Across all cortical layers, the majority of responsive regular spiking (RS) cells received short-latency inhibition, whereas fast spiking (FS) cells were almost exclusively excited. Consistent with the callosal-evoked increases in FS cell activity in vivo, brain slice recordings confirmed that parvalbumin (PV)-expressing cells received stronger callosal input than pyramidal cells or other interneuron subtypes. Acute in vivo silencing of the contralateral cortex generally increased spontaneous firing across cortical layers and linearly transformed responses to pure tones via both divisive and additive operations. The net effect was a decrease in signal-to-noise ratio for evoked responses and a broadening of frequency tuning curves. Together, these results suggest that callosal input regulates both the salience and tuning sharpness of tone responses in A1 via PV cell-mediated feedforward inhibition.

Arousal regulates frequency tuning in primary auditory cortex.

Changes in arousal influence cortical sensory representations, but the synaptic mechanisms underlying arousal-dependent modulation of cortical processing are unclear. Here, we use 2-photon Ca2+ imaging in the auditory cortex of awake mice to show that heightened arousal, as indexed by pupil diameter, broadens frequency-tuned activity of layer 2/3 (L2/3) pyramidal cells. Sensory representations are less sparse, and the tuning of nearby cells more similar when arousal increases. Despite the reduction in selectivity, frequency discrimination by cell ensembles improves due to a decrease in shared trial-to-trial variability. In vivo whole-cell recordings reveal that mechanisms contributing to the effects of arousal on sensory representations include state-dependent modulation of membrane potential dynamics, spontaneous firing, and tone-evoked synaptic potentials. Surprisingly, changes in short-latency tone-evoked excitatory input cannot explain the effects of arousal on the broadness of frequency-tuned output. However, we show that arousal strongly modulates a slow tone-evoked suppression of recurrent excitation underlying lateral inhibition [H. K. Kato, S. K. Asinof, J. S. Isaacson, Neuron, 95, 412-423, (2017)]. This arousal-dependent "network suppression" gates the duration of tone-evoked responses and regulates the broadness of frequency tuning. Thus, arousal can shape tuning via modulation of indirect changes in recurrent network activity.

Network-Level Control of Frequency Tuning in Auditory Cortex.

Lateral inhibition is a fundamental circuit operation that sharpens the tuning properties of cortical neurons. This operation is classically attributed to an increase in GABAergic synaptic input triggered by non-preferred stimuli. Here we use in vivo whole-cell recording and two-photon Ca2+ imaging in awake mice to show that lateral inhibition shapes frequency tuning in primary auditory cortex via an unconventional mechanism: non-preferred tones suppress both excitatory and inhibitory synaptic inputs onto layer 2/3 cells ("network suppression"). Moreover, optogenetic inactivation of inhibitory interneurons elicits a paradoxical increase in inhibitory synaptic input. These results indicate that GABAergic interneurons regulate cortical activity indirectly via the suppression of recurrent excitation. Furthermore, the network suppression underlying lateral inhibition was blocked by inactivation of somatostatin-expressing interneurons (SOM cells), but not parvalbumin-expressing interneurons (PV cells). Together, these findings reveal that SOM cells govern lateral inhibition and control cortical frequency tuning through the regulation of reverberating recurrent circuits.

Fear Learning Regulates Cortical Sensory Representations by Suppressing Habituation.

Projections from auditory cortex to the amygdala are thought to contribute to the induction of auditory fear learning. In addition, fear conditioning has been found to enhance cortical responses to conditioned tones, suggesting that cortical plasticity contributes to fear learning. However, the functional role of auditory cortex in the retrieval of fear memories is unclear and how fear learning regulates cortical sensory representations is not well understood. To address these questions, we use acute optogenetic silencing and chronic two-photon calcium imaging in mouse auditory cortex during fear learning. Longitudinal imaging of neuronal ensemble activity reveals that discriminative fear learning modulates cortical sensory representations via the suppression of cortical habituation.

Flexible Sensory Representations in Auditory Cortex Driven by Behavioral Relevance.

Animals require the ability to ignore sensory stimuli that have no consequence yet respond to the same stimuli when they become useful. However, the brain circuits that govern this flexibility in sensory processing are not well understood. Here we show in mouse primary auditory cortex (A1) that daily passive sound exposure causes a long-lasting reduction in representations of the experienced sound by layer 2/3 pyramidal cells. This habituation arises locally in A1 and involves an enhancement in inhibition and selective upregulation in the activity of somatostatin-expressing inhibitory neurons (SOM cells). Furthermore, when mice engage in sound-guided behavior, pyramidal cell excitatory responses to habituated sounds are enhanced, whereas SOM cell responses are diminished. Together, our results demonstrate the bidirectional modulation of A1 sensory representations and suggest that SOM cells gate cortical information flow based on the behavioral relevance of the stimulus.

Somatostatin cells regulate sensory response fidelity via subtractive inhibition in olfactory cortex.

Diverse types of local GABAergic interneurons shape the cortical representation of sensory information. Here we show how somatostatin-expressing interneurons (SOM cells) contribute to odor coding in mouse olfactory cortex. We find that odor-tuned SOM cells regulate principal cells through a purely subtractive operation that is independent of odor identity or intensity. This operation enhances the salience of odor-evoked activity without changing cortical odor tuning. SOM cells inhibit both principal cells and fast-spiking interneurons, indicating that subtractive inhibition reflects the interplay of multiple classes of interneurons.

Broadcasting of cortical activity to the olfactory bulb.

Odor representations are initially formed in the olfactory bulb, which contains a topographic glomerular map of odor molecular features. The bulb transmits sensory information directly to piriform cortex, where it is encoded by distributed ensembles of pyramidal cells without spatial order. Intriguingly, piriform cortex pyramidal cells project back to the bulb, but the information contained in this feedback projection is unknown. Here, we use imaging in awake mice to directly monitor activity in the presynaptic boutons of cortical feedback fibers. We show that the cortex provides the bulb with a rich array of information for any individual odor and that cortical feedback is dependent on brain state. In contrast to the stereotyped, spatial arrangement of olfactory bulb glomeruli, cortical inputs tuned to different odors commingle and indiscriminately target individual glomerular channels. Thus, the cortex modulates early odor representations by broadcasting sensory information diffusely onto spatially ordered bulbar circuits.

Single dendrite-targeting interneurons generate branch-specific inhibition.

Microcircuits composed of dendrite-targeting inhibitory interneurons and pyramidal cells (PCs) are fundamental elements of cortical networks, however, the impact of individual interneurons on pyramidal dendrites is unclear. Here, we combine paired recordings and calcium imaging to determine the spatial domain over which single dendrite-targeting interneurons influence PCs in olfactory cortex. We show that a major action of individual interneurons is to inhibit dendrites in a branch-specific fashion.

Parvalbumin-expressing interneurons linearly control olfactory bulb output.

In the olfactory bulb, odor representations by principal mitral cells are modulated by local inhibitory circuits. While dendrodendritic synapses between mitral and granule cells are typically thought to be a major source of this modulation, the contributions of other inhibitory neurons remain unclear. Here we demonstrate the functional properties of olfactory bulb parvalbumin-expressing interneurons (PV cells) and identify their important role in odor coding. Using paired recordings, we find that PV cells form reciprocal connections with the majority of nearby mitral cells, in contrast to the sparse connectivity between mitral and granule cells. In vivo calcium imaging in awake mice reveals that PV cells are broadly tuned to odors. Furthermore, selective PV cell inactivation enhances mitral cell responses in a linear fashion while maintaining mitral cell odor preferences. Thus, dense connections between mitral and PV cells underlie an inhibitory circuit poised to modulate the gain of olfactory bulb output.

Cortical feedback control of olfactory bulb circuits.

Olfactory cortex pyramidal cells integrate sensory input from olfactory bulb mitral and tufted (M/T) cells and project axons back to the bulb. However, the impact of cortical feedback projections on olfactory bulb circuits is unclear. Here, we selectively express channelrhodopsin-2 in olfactory cortex pyramidal cells and show that cortical feedback projections excite diverse populations of bulb interneurons. Activation of cortical fibers directly excites GABAergic granule cells, which in turn inhibit M/T cells. However, we show that cortical inputs preferentially target short axon cells that drive feedforward inhibition of granule cells. In vivo, activation of olfactory cortex that only weakly affects spontaneous M/T cell firing strongly gates odor-evoked M/T cell responses: cortical activity suppresses odor-evoked excitation and enhances odor-evoked inhibition. Together, these results indicate that although cortical projections have diverse actions on olfactory bulb microcircuits, the net effect of cortical feedback on M/T cells is an amplification of odor-evoked inhibition.

Dynamic sensory representations in the olfactory bulb: modulation by wakefulness and experience.

How are sensory representations in the brain influenced by the state of an animal? Here we use chronic two-photon calcium imaging to explore how wakefulness and experience shape odor representations in the mouse olfactory bulb. Comparing the awake and anesthetized state, we show that wakefulness greatly enhances the activity of inhibitory granule cells and makes principal mitral cell odor responses more sparse and temporally dynamic. In awake mice, brief repeated odor experience leads to a gradual and long-lasting (months) weakening of mitral cell odor representations. This mitral cell plasticity is odor specific, recovers gradually over months, and can be repeated with different odors. Furthermore, the expression of this experience-dependent plasticity is prevented by anesthesia. Together, our results demonstrate the dynamic nature of mitral cell odor representations in awake animals, which is constantly shaped by recent odor experience.

How inhibition shapes cortical activity.

Cortical processing reflects the interplay of synaptic excitation and synaptic inhibition. Rapidly accumulating evidence is highlighting the crucial role of inhibition in shaping spontaneous and sensory-evoked cortical activity and thus underscores how a better knowledge of inhibitory circuits is necessary for our understanding of cortical function. We discuss current views of how inhibition regulates the function of cortical neurons and point to a number of important open questions.

A major role for intracortical circuits in the strength and tuning of odor-evoked excitation in olfactory cortex.

In primary sensory cortices, there are two main sources of excitation: afferent sensory input relayed from the periphery and recurrent intracortical input. Untangling the functional roles of these two excitatory pathways is fundamental for understanding how cortical neurons process sensory stimuli. Odor representations in the primary olfactory (piriform) cortex depend on excitatory sensory afferents from the olfactory bulb. However, piriform cortex pyramidal cells also receive dense intracortical excitatory connections, and the relative contribution of these two pathways to odor responses is unclear. Using a combination of in vivo whole-cell voltage-clamp recording and selective synaptic silencing, we show that the recruitment of intracortical input, rather than olfactory bulb input, largely determines the strength of odor-evoked excitatory synaptic transmission in rat piriform cortical neurons. Furthermore, we find that intracortical synapses dominate odor-evoked excitatory transmission in broadly tuned neurons, whereas bulbar synapses dominate excitatory synaptic responses in more narrowly tuned neurons.

Linking neuronal ensembles by associative synaptic plasticity.

Synchronized activity in ensembles of neurons recruited by excitatory afferents is thought to contribute to the coding information in the brain. However, the mechanisms by which neuronal ensembles are generated and modified are not known. Here we show that in rat hippocampal slices associative synaptic plasticity enables ensembles of neurons to change by incorporating neurons belonging to different ensembles. Associative synaptic plasticity redistributes the composition of different ensembles recruited by distinct inputs such as to specifically increase the similarity between the ensembles. These results show that in the hippocampus, the ensemble of neurons recruited by a given afferent projection is fluid and can be rapidly and persistently modified to specifically include neurons from different ensembles. This linking of ensembles may contribute to the formation of associative memories.

Pyramidal cells in piriform cortex receive convergent input from distinct olfactory bulb glomeruli.

Pyramidal cells in piriform cortex integrate sensory information from multiple olfactory bulb mitral and tufted (M/T) cells. However, whether M/T cells belonging to different olfactory bulb glomeruli converge onto individual cortical cells is unclear. Here we use calcium imaging in an olfactory bulb-cortex slice preparation to provide direct evidence that neurons in piriform cortex receive convergent synaptic input from different glomeruli. We show that the combined activity of distinct glomerular pathways recruits ensembles of pyramidal cells that are not activated by the individual pathways alone. This cooperative recruitment of cortical neurons only occurs over a narrow time window and is a feature intrinsic to the olfactory cortex that can be explained by the integration of converging, subthreshold synaptic input. Cooperative recruitment enhances the differences between cortical representations of partially overlapping input patterns and may contribute to the initial steps of olfactory discrimination.

From dendrite to soma: dynamic routing of inhibition by complementary interneuron microcircuits in olfactory cortex.

Diverse inhibitory pathways shape cortical information processing; however, the relevant interneurons recruited by sensory stimuli and how they impact principal cells are unclear. Here we show that two major interneuron circuits govern dynamic inhibition in space and time within the olfactory cortex. Dendritic-targeting layer 1 interneurons receive strong input from the olfactory bulb and govern early-onset feedforward inhibition. However, this circuit is only transiently engaged during bursts of olfactory bulb input. In contrast, somatic-targeting layer 3 interneurons, recruited exclusively by recurrent excitation from pyramidal cells, produce late-onset feedback inhibition. Our results reveal two complementary interneuron circuits enforcing widespread inhibition, which shifts from the apical dendrites to somata of pyramidal cells during bursts of sensory input.

Odor representations in mammalian cortical circuits.

Spatial and temporal activity patterns of olfactory bulb projection neurons underlie the initial representations of odors in the brain. However, olfactory perception ultimately requires the integration of olfactory bulb output in higher cortical brain regions. Recent studies reveal that odor representations are sparse and highly distributed in the rodent primary olfactory (piriform) cortex. Furthermore, odor-evoked inhibition is far more widespread and broadly tuned than excitation in piriform cortex pyramidal cells. Other recent studies highlight how olfactory sensory inputs are integrated within pyramidal cell dendrites and that feedback projections from piriform cortex to olfactory bulb interneurons are a source of synaptic plasticity.

Calcium-permeable AMPA receptors mediate glutamatergic signaling in neural precursor cells of the postnatal olfactory bulb.

Neural precursor cells (NPCs) in the mammalian olfactory bulb give rise to local inhibitory neurons that integrate into existing circuitry throughout adult life. However, the functional properties of neurotransmitter receptors expressed by NPCs are not well understood. In this study, we use patch-clamp recording and calcium imaging to explore the properties of glutamate receptors expressed by NPCs in the olfactory bulb subependymal layer. We find that calcium-permeable AMPA receptors (AMPARs) are the major receptor type underlying glutamatergic signaling in olfactory bulb NPCs. We also show that when transmitter uptake is reduced, glutamate spillover from distant nerve terminals in the olfactory bulb can activate nonsynaptic NPC AMPARs and generate increases in intracellular calcium. Together, these results suggest that Ca(2+) influx via AMPARs may contribute to calcium-dependent processes that govern NPC differentiation and maturation.

Postsynaptic mechanisms govern the differential excitation of cortical neurons by thalamic inputs.

Thalamocortical (TC) afferents relay sensory input to the cortex by making synapses onto both excitatory regular-spiking principal cells (RS cells) and inhibitory fast-spiking interneurons (FS cells). This divergence plays a crucial role in coordinating excitation with inhibition during the earliest steps of somatosensory processing in the cortex. Although the same TC afferents contact both FS and RS cells, FS cells receive larger and faster excitatory inputs from individual TC afferents. Here, we show that this larger thalamic excitation of FS cells occurs via GluR2-lacking AMPA receptors (AMPARs), and results from a fourfold larger quantal amplitude compared with the thalamic inputs onto RS cells. Thalamic afferents also activate NMDA receptors (NMDARs) at synapses onto both cells types, yet RS cell NMDAR currents are slower and pass more current at physiological membrane potentials. Because of these synaptic specializations, GluR2-lacking AMPARs selectively maintain feedforward inhibition of RS cells, whereas NMDARs contribute to the spiking of RS cells and hence to cortical recurrent excitation. Thus, thalamic afferent activity diverges into two routes that rely on unique complements of postsynaptic AMPARs and NMDARs to orchestrate the dynamic balance of excitation and inhibition as sensory input enters the cortex.