Learning-induced bidirectional enhancement of inhibitory synaptic metaplasticity.

Training rodents in a particularly difficult olfactory-discrimination (OD) task results in the acquisition of the ability to perform the task well, termed 'rule learning'. In addition to enhanced intrinsic excitability and synaptic excitation in piriform cortex pyramidal neurons, rule learning results in increased synaptic inhibition across the whole cortical network to the point where it precisely maintains the balance between inhibition and excitation. The mechanism underlying such precise inhibitory enhancement remains to be explored. Here, we use brain slices from transgenic mice (VGAT-ChR2-EYFP), enabling optogenetic stimulation of single GABAergic neurons and recordings of unitary synaptic events in pyramidal neurons. Quantal analysis revealed that learning-induced enhanced inhibition is mediated by increased quantal size of the evoked inhibitory events. Next, we examined the plasticity of synaptic inhibition induced by long-lasting, intrinsically evoked spike firing in post-synaptic neurons. Repetitive depolarizing current pulses from depolarized (-70 mV) or hyperpolarized (-90 mV) membrane potentials induced long-term depression (LTD) and long-term potentiation (LTP) of synaptic inhibition, respectively. We found a profound bidirectional increase in the ability to induce both LTD, mediated by L-type calcium channels, and LTP, mediated by R-type calcium channels after rule learning. Blocking the GABAB receptor reversed the effect of intrinsic stimulation at -90 mV from LTP to LTD. We suggest that learning greatly enhances the ability to modify the strength of synaptic inhibition of principal neurons in both directions. Such plasticity of synaptic plasticity allows fine-tuning of inhibition on each particular neuron, thereby stabilizing the network while maintaining the memory of the rule. KEY POINTS: Olfactory discrimination rule learning results in long-lasting enhancement of synaptic inhibition on piriform cortex pyramidal neurons. Quantal analysis of unitary inhibitory synaptic events, evoked by optogenetic minimal stimulation, revealed that enhanced synaptic inhibition is mediated by increased quantal size. Surprisingly, metaplasticity of synaptic inhibition, induced by intrinsically evoked repetitive spike firing, is increased bidirectionally. The susceptibility to both long-term depression (LTD) and long-term potentiation (LTP) of inhibition is enhanced after learning. LTD of synaptic inhibition is mediated by L-type calcium channels and LTP by R-type calcium channels. LTP is also dependent on activation of GABAB receptors. We suggest that learning-induced changes in the metaplasticity of synaptic inhibition enable the fine-tuning of inhibition on each particular neuron, thereby stabilizing the network while maintaining the memory of the rule.

Semantic context-dependent neural representations of odors in the human piriform cortex revealed by 7T MRI.

Olfactory perception depends not only on olfactory inputs but also on semantic context. Although multi-voxel activity patterns of the piriform cortex, a part of the primary olfactory cortex, have been shown to represent odor perception, it remains unclear whether semantic contexts modulate odor representation in this region. Here, we investigated whether multi-voxel activity patterns in the piriform cortex change when semantic context modulates odor perception and, if so, whether the modulated areas communicate with brain regions involved in semantic and memory processing beyond the piriform cortex. We also explored regional differences within the piriform cortex, which are influenced by olfactory input and semantic context. We used 2 × 2 combinations of word labels and odorants that were perceived as congruent and measured piriform activity with a 1-mm isotropic resolution using 7T MRI. We found that identical odorants labeled with different words were perceived differently. This labeling effect was observed in multi-voxel activity patterns in the piriform cortex, as the searchlight decoding analysis distinguished identical odors with different labels for half of the examined stimulus pairs. Significant functional connectivity was observed between parts of the piriform cortex that were modulated by labels and regions associated with semantic and memory processing. While the piriform multi-voxel patterns evoked by different olfactory inputs were also distinguishable, the decoding accuracy was significant for only one stimulus pair, preventing definitive conclusions regarding the locational differences between areas influenced by word labels and olfactory inputs. These results suggest that multi-voxel patterns of piriform activity can be modulated by semantic context, possibly due to communication between the piriform cortex and the semantic and memory regions.

Sexually dimorphic control of affective state processing and empathic behaviors.

Recognizing the affective states of social counterparts and responding appropriately fosters successful social interactions. However, little is known about how the affective states are expressed and perceived and how they influence social decisions. Here, we show that male and female mice emit distinct olfactory cues after experiencing distress. These cues activate distinct neural circuits in the piriform cortex (PiC) and evoke sexually dimorphic empathic behaviors in observers. Specifically, the PiC → PrL pathway is activated in female observers, inducing a social preference for the distressed counterpart. Conversely, the PiC → MeA pathway is activated in male observers, evoking excessive self-grooming behaviors. These pathways originate from non-overlapping PiC neuron populations with distinct gene expression signatures regulated by transcription factors and sex hormones. Our study unveils how internal states of social counterparts are processed through sexually dimorphic mechanisms at the molecular, cellular, and circuit levels and offers insights into the neural mechanisms underpinning sex differences in higher brain functions.

Appetite-regulating hormones modulate odor perception and odor-evoked activity in hypothalamus and olfactory cortices.

Odors guide food seeking, and food intake modulates olfactory function. This interaction is mediated by appetite-regulating hormones like ghrelin, insulin, and leptin, which alter activity in the rodent olfactory bulb, but their effects on downstream olfactory cortices have not yet been established in humans. The olfactory tract connects the olfactory bulb to the cortex through 3 main striae, terminating in the piriform cortex (PirC), amygdala (AMY), olfactory tubercule (OT), and anterior olfactory nucleus (AON). Here, we test the hypothesis that appetite-regulating hormones modulate olfactory processing in the endpoints of the olfactory tract and the hypothalamus. We collected odor-evoked functional magnetic resonance imaging (fMRI) responses and plasma levels of ghrelin, insulin, and leptin from human subjects (n = 25) after a standardized meal. We found that a hormonal composite measure, capturing variance relating positively to insulin and negatively to ghrelin, correlated inversely with odor intensity ratings and fMRI responses to odorized vs. clean air in the hypothalamus, OT, and AON. No significant correlations were found with activity in PirC or AMY, the endpoints of the lateral stria. Exploratory whole-brain analyses revealed significant correlations near the diagonal band of Broca and parahippocampal gyrus. These results demonstrate that high (low) blood plasma concentrations of insulin (ghrelin) decrease perceived odor intensity and odor-evoked activity in the cortical targets of the medial and intermediate striae of the olfactory tract, as well as the hypothalamus. These findings expand our understanding of the cortical mechanisms by which metabolic hormones in humans modulate olfactory processing after a meal.

Experience-dependent evolution of odor mixture representations in piriform cortex.

Rodents can learn from exposure to rewarding odors to make better and quicker decisions. The piriform cortex is thought to be important for learning complex odor associations; however, it is not understood exactly how it learns to remember discriminations between many, sometimes overlapping, odor mixtures. We investigated how odor mixtures are represented in the posterior piriform cortex (pPC) of mice while they learn to discriminate a unique target odor mixture against hundreds of nontarget mixtures. We find that a significant proportion of pPC neurons discriminate between the target and all other nontarget odor mixtures. Neurons that prefer the target odor mixture tend to respond with brief increases in firing rate at odor onset compared to other neurons, which exhibit sustained and/or decreased firing. We allowed mice to continue training after they had reached high levels of performance and find that pPC neurons become more selective for target odor mixtures as well as for randomly chosen repeated nontarget odor mixtures that mice did not have to discriminate from other nontargets. These single unit changes during overtraining are accompanied by better categorization decoding at the population level, even though behavioral metrics of mice such as reward rate and latency to respond do not change. However, when difficult ambiguous trial types are introduced, the robustness of the target selectivity is correlated with better performance on the difficult trials. Taken together, these data reveal pPC as a dynamic and robust system that can optimize for both current and possible future task demands at once.

Acute Fasting Modulates Food-Seeking Behavior and Neural Signaling in the Piriform Cortex.

It is well known that the state of hunger can modulate hormones and hypothalamic neural circuits to drive food-seeking behavior and consumption. However, the role the sensory cortex plays in regulating foraging is much less explored. Here, we investigated whether acute fasting in mice can alter an odor-guided foraging behavior and how it can alter neurons and synapses in the (olfactory) piriform cortex (PC). Acute hunger enhances the motivation of a mouse to search for food pellets and increases food intake. The foraging behavior strongly activates the PC, as revealed by c-Fos immunostaining. The activation of PC is accompanied by an increase in excitation-inhibition ratio of synaptic density. Fasting also enhances the phosphorylation of AMP kinase, a biochemical energy regulator. Taken together, our results uncover a new regulatory brain region and implicate the PC in controlling foraging behavior.

Retrieval of olfactory fear memory alters cell proliferation and expression of pCREB and pMAPK in the corticomedial amygdala and piriform cortex.

The brain forms robust associations between odors and emotionally salient memories, making odors especially effective at triggering fearful or traumatic memories. Using Pavlovian olfactory fear conditioning (OFC), a variant of the traditional tone-shock paradigm, this study explored the changes involved in its processing. We assessed the expression of neuronal plasticity markers phosphorylated cyclic adenosine monophosphate response element binding protein (pCREB) and phosphorylated mitogen-activated protein kinase (pMAPK) 24 h and 14 days following OFC, in newborn neurons (EdU+) and in brain regions associated with olfactory memory processing; the olfactory bulb, piriform cortex, amygdale, and hippocampus. Here, we show that all proliferating neurons in the dentate gyrus of the hippocampus and glomerular layer of the olfactory bulb were colocalized with pCREB at 24 h and 14 days post-conditioning, and the number of proliferating neurons at both time points were statistically similar. This suggests the occurrence of long-term potentiation within the neurons of this pathway. Finally, OFC significantly increased the density of pCREB- and pMAPK-positive immunoreactive neurons in the medial and cortical subnuclei of the amygdala and the posterior piriform cortex, suggesting their key involvement in its processing. Together, our investigation identifies changes in neuroplasticity within critical neural circuits responsible for olfactory fear memory.

Central organization of a high-dimensional odor space.

Animals recognize groups and mixtures of odors as a unitary object. This ability is crucial to generalize known odors to newly encountered ones despite variations. However, it remains largely unknown how multitudes of odors are represented and organized in the higher brain regions to support odor object recognition. Here we discuss recent advances uncovering the population odor responses in the rodent piriform cortex and the Drosophila mushroom body, and highlight the emerging principles on the organization, mechanism, stereotypy, and experience-dependence of central odor representations.

Upstream γ-synchronization enhances odor processing in downstream neurons.

γ-oscillatory activity is ubiquitous across brain areas. Numerous studies have suggested that γ-synchrony is likely to enhance the transmission of sensory information. However, direct causal evidence is still lacking. Here, we test this hypothesis in the mouse olfactory system, where local GABAergic granule cells (GCs) in the olfactory bulb shape mitral/tufted cell (MTC) excitatory output from the olfactory bulb. By optogenetically modulating GC activity, we successfully dissociate MTC γ-synchronization from its firing rates. Recording of odor responses in downstream piriform cortex neurons shows that increasing MTC γ-synchronization enhances cortical neuron odor-evoked firing rates, reduces response variability, and improves odor ensemble representation. These gains occur despite a reduction in MTC firing rates. Furthermore, reducing MTC γ-synchronization without changing the MTC firing rates, by suppressing GC activity, degrades piriform cortex odor-evoked responses. These findings provide causal evidence that increased γ-synchronization enhances the transmission of sensory information between two brain regions.

Top-down feedback enables flexible coding strategies in the olfactory cortex.

In chemical sensation, multiple models have been proposed to explain how odors are represented in the olfactory cortex. One hypothesis is that the combinatorial identity of active neurons within sniff-related time windows is critical, whereas another model proposes that it is the temporal structure of neural activity that is essential for encoding odor information. We find that top-down feedback to the main olfactory bulb dictates the information transmitted to the piriform cortex and switches between these coding strategies. Using a detailed network model, we demonstrate that feedback control of inhibition influences the excitation-inhibition balance in mitral cells, restructuring the dynamics of piriform cortical cells. This results in performance improvement in odor discrimination tasks. These findings present a framework for early olfactory computation, where top-down feedback to the bulb flexibly shapes the temporal structure of neural activity in the piriform cortex, allowing the early olfactory system to dynamically switch between two distinct coding models.

Fast and slow feedforward inhibitory circuits for cortical odor processing.

Feedforward inhibitory circuits are key contributors to the complex interplay between excitation and inhibition in the brain. Little is known about the function of feedforward inhibition in the primary olfactory (piriform) cortex. Using in vivo two-photon-targeted patch clamping and calcium imaging in mice, we find that odors evoke strong excitation in two classes of interneurons - neurogliaform (NG) cells and horizontal (HZ) cells - that provide feedforward inhibition in layer 1 of the piriform cortex. NG cells fire much earlier than HZ cells following odor onset, a difference that can be attributed to the faster odor-driven excitatory synaptic drive that NG cells receive from the olfactory bulb. As a result, NG cells strongly but transiently inhibit odor-evoked excitation in layer 2 principal cells, whereas HZ cells provide more diffuse and prolonged feedforward inhibition. Our findings reveal unexpected complexity in the operation of inhibition in the piriform cortex.

A hypothesis concerning distinct schemes of olfactory activation evoked by perceived versus nonperceived input.

SignificanceThe authors propose that odors are consciously perceived or not, depending on whether the olfactory cortex succeeds in activating the endopiriform nucleus-a structure that, in turn, is capable of activating multiple downstream brain areas. The authors further propose that the cellular mechanisms of endopiriform nucleus activation are an attenuated form of cellular events that occur during epileptic seizure initiation. If correct, the authors' hypothesis could help explain the mechanisms of action of certain general anesthetics.

Smell-induced gamma oscillations in human olfactory cortex are required for accurate perception of odor identity.

Studies of neuronal oscillations have contributed substantial insight into the mechanisms of visual, auditory, and somatosensory perception. However, progress in such research in the human olfactory system has lagged behind. As a result, the electrophysiological properties of the human olfactory system are poorly understood, and, in particular, whether stimulus-driven high-frequency oscillations play a role in odor processing is unknown. Here, we used direct intracranial recordings from human piriform cortex during an odor identification task to show that 3 key oscillatory rhythms are an integral part of the human olfactory cortical response to smell: Odor induces theta, beta, and gamma rhythms in human piriform cortex. We further show that these rhythms have distinct relationships with perceptual behavior. Odor-elicited gamma oscillations occur only during trials in which the odor is accurately perceived, and features of gamma oscillations predict odor identification accuracy, suggesting that they are critical for odor identity perception in humans. We also found that the amplitude of high-frequency oscillations is organized by the phase of low-frequency signals shortly following sniff onset, only when odor is present. Our findings reinforce previous work on theta oscillations, suggest that gamma oscillations in human piriform cortex are important for perception of odor identity, and constitute a robust identification of the characteristic electrophysiological response to smell in the human brain. Future work will determine whether the distinct oscillations we identified reflect distinct perceptual features of odor stimuli.

Cell assembly formation and structure in a piriform cortex model.

The piriform cortex is rich in recurrent excitatory synaptic connections between pyramidal neurons. We asked how such connections could shape cortical responses to olfactory lateral olfactory tract (LOT) inputs. For this, we constructed a computational network model of anterior piriform cortex with 2000 multicompartment, multiconductance neurons (500 semilunar, 1000 layer 2 and 500 layer 3 pyramids; 200 superficial interneurons of two types; 500 deep interneurons of three types; 500 LOT afferents), incorporating published and unpublished data. With a given distribution of LOT firing patterns, and increasing the strength of recurrent excitation, a small number of firing patterns were observed in pyramidal cell networks: first, sparse firings; then temporally and spatially concentrated epochs of action potentials, wherein each neuron fires one or two spikes; then more synchronized events, associated with bursts of action potentials in some pyramidal neurons. We suggest that one function of anterior piriform cortex is to transform ongoing streams of input spikes into temporally focused spike patterns, called here "cell assemblies", that are salient for downstream projection areas.

Olfactory Information Storage Engages Subcortical and Cortical Brain Regions That Support Valence Determination.

The olfactory bulb (OB) delivers sensory information to the piriform cortex (PC) and other components of the olfactory system. OB-PC synapses have been reported to express short-lasting forms of synaptic plasticity, whereas long-term potentiation (LTP) of the anterior PC (aPC) occurs predominantly by activating inputs from the prefrontal cortex. This suggests that brain regions outside the olfactory system may contribute to olfactory information processing and storage. Here, we compared functional magnetic resonance imaging BOLD responses triggered during 20 or 100 Hz stimulation of the OB. We detected BOLD signal increases in the anterior olfactory nucleus (AON), PC and entorhinal cortex, nucleus accumbens, dorsal striatum, ventral diagonal band of Broca, prelimbic-infralimbic cortex (PrL-IL), dorsal medial prefrontal cortex, and basolateral amygdala. Significantly stronger BOLD responses occurred in the PrL-IL, PC, and AON during 100 Hz compared with 20 Hz OB stimulation. LTP in the aPC was concomitantly induced by 100 Hz stimulation. Furthermore, 100 Hz stimulation triggered significant nuclear immediate early gene expression in aPC, AON, and PrL-IL. The involvement of the PrL-IL in this process is consistent with its putative involvement in modulating behavioral responses to odor experience. Furthermore, these results indicate that OB-mediated information storage by the aPC is embedded in a connectome that supports valence evaluation.

Spatial maps in piriform cortex during olfactory navigation.

Odours are a fundamental part of the sensory environment used by animals to guide behaviours such as foraging and navigation1,2. Primary olfactory (piriform) cortex is thought to be the main cortical region for encoding odour identity3-8. Here, using neural ensemble recordings in freely moving rats performing an odour-cued spatial choice task, we show that posterior piriform cortex neurons carry a robust spatial representation of the environment. Piriform spatial representations have features of a learned cognitive map, being most prominent near odour ports, stable across behavioural contexts and independent of olfactory drive or reward availability. The accuracy of spatial information carried by individual piriform neurons was predicted by the strength of their functional coupling to the hippocampal theta rhythm. Ensembles of piriform neurons concurrently represented odour identity as well as spatial locations of animals, forming an odour-place map. Our results reveal a function for piriform cortex in spatial cognition and suggest that it is well-suited to form odour-place associations and guide olfactory-cued spatial navigation.

Age-Dependent Contributions of NMDA Receptors and L-Type Calcium Channels to Long-Term Depression in the Piriform Cortex.

In the hippocampus, the contributions of N-methyl-D-aspartate receptors (NMDARs) and L-type calcium channels (LTCCs) to neuronal transmission and synaptic plasticity change with aging, underlying calcium dysregulation and cognitive dysfunction. However, the relative contributions of NMDARs and LTCCs in other learning encoding structures during aging are not known. The piriform cortex (PC) plays a significant role in odor associative memories, and like the hippocampus, exhibits forms of long-term synaptic plasticity. Here, we investigated the expression and contribution of NMDARs and LTCCs in long-term depression (LTD) of the PC associational fiber pathway in three cohorts of Sprague Dawley rats: neonatal (1-2 weeks), young adult (2-3 months) and aged (20-25 months). Using a combination of slice electrophysiology, Western blotting, fluorescent immunohistochemistry and confocal imaging, we observed a shift from an NMDAR to LTCC mediation of LTD in aged rats, despite no difference in the amount of LTD expression. These changes in plasticity are related to age-dependent differential receptor expression in the PC. LTCC Cav1.2 expression relative to postsynaptic density protein 95 is increased in the associational pathway of the aged PC layer Ib. Enhanced LTCC contribution in synaptic depression in the PC may contribute to altered olfactory function and learning with aging.

Basolateral amygdala to posterior piriform cortex connectivity ensures precision in learned odor threat.

Odor perception can both evoke emotional states and be shaped by emotional or hedonic states. The amygdala complex plays an important role in recognition of, and response to, hedonically valenced stimuli, and has strong, reciprocal connectivity with the primary olfactory (piriform) cortex. Here, we used differential odor-threat conditioning in rats to test the role of basolateral amygdala (BLA) input to the piriform cortex in acquisition and expression of learned olfactory threat responses. Using local field potential recordings, we demonstrated that functional connectivity (high gamma band coherence) between the BLA and posterior piriform cortex (pPCX) is enhanced after differential threat conditioning. Optogenetic suppression of activity within the BLA prevents learned threat acquisition, as do lesions of the pPCX prior to threat conditioning (without inducing anosmia), suggesting that both regions are critical for acquisition of learned odor threat responses. However, optogenetic BLA suppression during testing did not impair threat response to the CS+ , but did induce generalization to the CS-. A similar loss of stimulus control and threat generalization was induced by selective optogenetic suppression of BLA input to pPCX. These results suggest an important role for amygdala-sensory cortical connectivity in shaping responses to threatening stimuli.

Long-Range Respiratory and Theta Oscillation Networks Depend on Spatial Sensory Context.

Neural oscillations can couple networks of brain regions, especially at lower frequencies. The nasal respiratory rhythm, which elicits robust olfactory bulb oscillations, has been linked to episodic memory, locomotion, and exploration, along with widespread oscillatory coherence. The piriform cortex is implicated in propagating the olfactory-bulb-driven respiratory rhythm, but this has not been tested explicitly in the context of both hippocampal theta and nasal respiratory rhythm during exploratory behaviors. We investigated systemwide interactions during foraging behavior, which engages respiratory and theta rhythms. Local field potentials from the olfactory bulb, piriform cortex, dentate gyrus, and CA1 of hippocampus, primary visual cortex, and nasal respiration were recorded simultaneously from male rats. We compared interactions among these areas while rats foraged using either visual or olfactory spatial cues. We found high coherence during foraging compared with home cage activity in two frequency bands that matched slow and fast respiratory rates. Piriform cortex and hippocampus maintained strong coupling at theta frequency during periods of slow respiration, whereas other pairs showed coupling only at the fast respiratory frequency. Directional analysis shows that the modality of spatial cues was matched to larger influences in the network by the respective primary sensory area. Respiratory and theta rhythms also coupled to faster oscillations in primary sensory and hippocampal areas. These data provide the first evidence of widespread interactions among nasal respiration, olfactory bulb, piriform cortex, and hippocampus in awake freely moving rats, and support the piriform cortex as an integrator of respiratory and theta activity.SIGNIFICANCE STATEMENT Recent studies have shown widespread interactions between the nasally driven respiratory rhythm and neural oscillations in hippocampus and neocortex. With this study, we address how the respiratory rhythm interacts with ongoing slow brain rhythms across olfactory, hippocampal, and visual systems in freely moving rats. Patterns of network connectivity change with behavioral state, with stronger interactions at fast and slow respiratory frequencies during foraging as compared with home cage activity. Routing of interactions between sensory cortices depends on the modality of spatial cues present during foraging. Functional connectivity and cross-frequency coupling analyses suggest strong bidirectional interactions between olfactory and hippocampal systems related to respiration and point to the piriform cortex as a key area for mediating respiratory and theta rhythms.

Plasticity of olfactory bulb inputs mediated by dendritic NMDA-spikes in rodent piriform cortex.

The piriform cortex (PCx) is essential for learning of odor information. The current view postulates that odor learning in the PCx is mainly due to plasticity in intracortical (IC) synapses, while odor information from the olfactory bulb carried via the lateral olfactory tract (LOT) is 'hardwired.' Here, we revisit this notion by studying location- and pathway-dependent plasticity rules. We find that in contrast to the prevailing view, synaptic and optogenetically activated LOT synapses undergo strong and robust long-term potentiation (LTP) mediated by only a few local NMDA-spikes delivered at theta frequency, while global spike timing-dependent plasticity (STDP) protocols failed to induce LTP in these distal synapses. In contrast, IC synapses in apical and basal dendrites undergo plasticity with both NMDA-spikes and STDP protocols but to a smaller extent compared with LOT synapses. These results are consistent with a self-potentiating mechanism of odor information via NMDA-spikes that can form branch-specific memory traces of odors that can further associate with contextual IC information via STDP mechanisms to provide cognitive and emotional value to odors.