"Brain-breath" interactions: respiration-timing-dependent impact on functional brain networks and beyond.

Breathing is a natural daily action that one cannot do without, and it sensitively and intensely changes under various situations. What if this essential act of breathing can impact our overall well-being? Recent studies have demonstrated that breathing oscillations couple with higher brain functions, i.e., perception, motor actions, and cognition. Moreover, the timing of breathing, a phase transition from exhalation to inhalation, modulates specific cortical activity and accuracy in cognitive tasks. To determine possible respiratory roles in attentional and memory processes and functional neural networks, we discussed how breathing interacts with the brain that are measured by electrophysiology and functional neuroimaging: (i) respiration-dependent modulation of mental health and cognition; (ii) respiratory rhythm generation and respiratory pontomedullary networks in the brainstem; (iii) respiration-dependent effects on specific brainstem regions and functional neural networks (e.g., glutamatergic PreBötzinger complex neurons, GABAergic parafacial neurons, adrenergic C1 neurons, parabrachial nucleus, locus coeruleus, temporoparietal junction, default-mode network, ventral attention network, and cingulo-opercular salience network); and (iv) a potential application of breathing manipulation in mental health care. These outlines and considerations of "brain-breath" interactions lead to a better understanding of the interoceptive and cognitive mechanisms that underlie brain-body interactions in health conditions and in stress-related and neuropsychiatric disorders.

Hippocampal ensemble dynamics and memory performance are modulated by respiration during encoding.

During offline brain states, such as sleep and memory consolidation, respiration coordinates hippocampal activity. However, the role of breathing during online memory traces remains unclear. Here, we show that respiration can be recruited during online memory encoding. Optogenetic manipulation was used to control activation of the primary inspiratory rhythm generator PreBötzinger complex (PreBötC) in transgenic mice. When intermittent PreBötC-induced apnea covered the object exploration time during encoding, novel object detection was impaired. Moreover, the mice did not exhibit freezing behavior during presentation of fear-conditioned stimuli (CS+) when PreBötC-induced apnea occurred at the exact time of encoding. This apnea did not evoke changes in CA3 cell ensembles between presentations of CS+ and conditioned inhibition (CS-), whereas in normal breathing, CS+ presentations produced dynamic changes. Our findings demonstrate that components of central respiratory activity (e.g., frequency) during online encoding strongly contribute to shaping hippocampal ensemble dynamics and memory performance.

Respiration-timing-dependent changes in activation of neural substrates during cognitive processes.

We previously showed that cognitive performance declines when the retrieval process spans an expiratory-to-inspiratory (EI) phase transition (an onset of inspiration). To identify the neural underpinning of this phenomenon, we conducted functional magnetic resonance imaging (fMRI) while participants performed a delayed matching-to-sample (DMTS) recognition memory task with a short delay. Respiration during the task was monitored using a nasal cannula. Behavioral data replicated the decline in memory performance specific to the EI transition during the retrieval process, while an extensive array of frontoparietal regions were activated during the encoding, delay, and retrieval processes of the task. Within these regions, when the retrieval process spanned the EI transition, activation was reduced in the anterior cluster of the right temporoparietal junction (TPJa, compared to cases when the retrieval process spanned the inspiratory-to-expiratory phase transition) and the left and right middle frontal gyrus, dorsomedial prefrontal cortex, and somatosensory areas (compared to cases when the retrieval process did not span any phase transition). These results in task-related activity may represent respiratory interference specifically in information manipulation rather than memory storage. Our findings demonstrate a cortical-level effect of respiratory phases on cognitive processes and highlight the importance of the timing of breathing for successful performance.

Respiratory fluctuations in pupil diameter are not maintained during cognitive tasks.

Pupil diameter fluctuation throughout the respiratory cycle is autonomically controlled in the resting state, as pupils dilate during inspiration and constrict during expiration. Furthermore, pupil size is differentially modulated by cognitive states between task engagement and disengagement. To determine whether respiratory-dependent fluctuations in pupil size are maintained during a cognitive task, we employed healthy human subjects performing a delayed matching-to-sample task with a short delay and measured their pupil sizes and R wave-to-R wave intervals (RRIs). We detected respiratory fluctuations in pupil size and the RRI during the delay period immediately before the discrimination stage of the task. During the discrimination stage, the cognitive state with the higher task engagement yielded more pupil dilation. However, respiratory fluctuations in pupil size were abolished, whereas those in the RRI were still discernible during the discrimination stage. Our results suggest that an alternative control mechanism involving the cognitive state associated with task engagement overrides the respiratory-related autonomic control of pupil diameter.

Respiratory modulation of cognitive performance during the retrieval process.

Recent research suggests that cognitive performance might be altered by the respiratory-synchronized activity generated in the brain. Previous human studies, however, have yielded inconsistent results when assessing task performance during distinct respiratory phases (inspiratory phase vs. expiratory phase). We therefore tested whether cognitive performance was regulated based on the timing of breathing components (e.g., expiratory-to-inspiratory (EI) phase transition) during the retrieval process. To determine the role of respiration in performance, the present study employed healthy subjects (n = 18) in a delayed matching-to-sample visual recognition task where a test cue was given in the respiratory phase-locked (Phased) or regularly paced (Non-phased) presentation paradigm. During the Phased session but not during the Non-phased session, the response time (RT) of the task increased by 466 ms (p = 0.003), and accuracy decreased by 21.4% (p = 0.004) when the retrieval process encompassed the EI transition. Breathing-dependent changes were particularly prominent when the EI transition occurred during the middle step of the retrieval process. Meanwhile, changes in the RT and accuracy were not observed when the retrieval process encompassed the inspiratory-to-expiratory phase transition. This is the first time that a certain phase transition in the respiratory cycle has been shown to modulate performance on a time scale of several seconds in a cognitive task. We propose that attenuation of these breathing-dependent cognitive fluctuations might be crucial for the maintenance and stability of successful performance in daily life and sports.

Spatial information is preferentially processed by the distal part of CA3: Implication for memory retrieval.

For the past decades, CA3 was considered as a single functional entity. However, strong differences between the proximal (close to the dentate gyrus) and the distal (close to CA2) parts of CA3 in terms of connectivity patterns, gene expression and electrophysiological properties suggest that it is not the case. We recently showed that proximal CA3 (together with distal CA1) preferentially deals with non-spatial information [1]. In contrast to proximal CA3, distal CA3 mainly receives and predominantly projects to spatially tuned areas. Here, we tested if distal CA3 preferentially processes spatial information, which would suggest a segregation of the spatial information along the proximodistal axis of CA3. We used a high-resolution imaging technique based on the detection of the expression of the immediate-early gene Arc, commonly used to map activity in the medial temporal lobe. We showed that distal CA3 is strongly recruited in a newly designed delayed nonmatching-to-location task with high memory demands in rats, while proximal CA3 is not. These results indicate a functional segregation of CA3 that mirrors the one reported in CA1, and suggest the existence of a distal CA3- proximal CA1 spatial subnetwork. These findings bring further evidence for the existence of 'specialized' spatial and non-spatial subnetworks segregated along the proximodistal axis of the hippocampus and put forward the 'segregated' view of information processing in the hippocampus as a reasonable alternative to the well-accepted 'integrated' view, according to which spatial and non-spatial information are systematically integrated in the hippocampus to form episodic memory.

Spatial information is preferentially processed by the distal part of CA3: implication for memory retrieval.

For the past decades, CA3 was considered as a single functional entity. However, strong differences between the proximal (close to the dentate gyrus) and the distal (close to CA2) parts of CA3 in terms of connectivity patterns, gene expression and electrophysiological properties suggest that it is not the case. We recently showed that proximal CA3 (together with distal CA1) preferentially deals with non-spatial information [1]. In contrast to proximal CA3, distal CA3 mainly receives and predominantly projects to spatially tuned areas. Here, we tested if distal CA3 preferentially processes spatial information, which would suggest a segregation of the spatial information along the proximodistal axis of CA3. We used a high-resolution imaging technique based on the detection of the expression of the immediate-early gene Arc, commonly used to map activity in the medial temporal lobe. We showed that distal CA3 is strongly recruited in a newly designed delayed nonmatching-to-location task with high memory demands in rats, while proximal CA3 is not. These results indicate a functional segregation of CA3 that mirrors the one reported in CA1, and suggest the existence of a distal CA3- proximal CA1 spatial subnetwork. These findings bring further evidence for the existence of 'specialized' spatial and non-spatial subnetworks segregated along the proximodistal axis of the hippocampus and put forward the 'segregated' view of information processing in the hippocampus as a reasonable alternative to the well-accepted 'integrated' view, according to which spatial and non-spatial information are systematically integrated in the hippocampus to form episodic memory.

Regional Specific Evidence for Memory-Load Dependent Activity in the Dorsal Subiculum and the Lateral Entorhinal Cortex.

The subiculum and the lateral entorhinal cortex (LEC) are the main output areas of the hippocampus which contribute to spatial and non-spatial memory. The proximal part of the subiculum (bordering CA1) receives heavy projections from the perirhinal cortex and the distal part of CA1 (bordering the subiculum), both known for their ties to object recognition memory. However, the extent to which the proximal subiculum contributes to non-spatial memory is still unclear. Comparatively, the involvement of the LEC in non-spatial information processing is quite well known. However, very few studies have investigated its role within the frame of memory function. Thus, it is not known whether its contribution depends on memory load. In addition, the deep layers of the EC have been shown to be predictive of subsequent memory performance, but not its superficial layers. Hence, here we tested the extent to which the proximal part of the subiculum and the superficial and deep layers of the LEC contribute to non-spatial memory, and whether this contribution depends on the memory load of the task. To do so, we imaged brain activity at cellular resolution in these areas in rats performing a delayed nonmatch to sample task based on odors with two different memory loads (5 or 10 odors). This imaging technique is based on the detection of the RNA of the immediate-early gene Arc, which is especially tied to synaptic plasticity and behavioral demands, and is commonly used to map activity in the medial temporal lobe. We report for the first time that the proximal part of the subiculum is recruited in a memory-load dependent manner and the deep layers of the LEC engaged under high memory load conditions during the retrieval of non-spatial memory, thus shedding light on the specific networks contributing to non-spatial memory retrieval.

Encoding and reactivation patterns predictive of successful memory performance are topographically organized along the longitudinal axis of the hippocampus.

An ongoing debate in human memory research is whether the encoding and the retrieval of memory engage the same part of the hippocampus and the same cells, or whether encoding preferentially involves the anterior part of the hippocampus and retrieval its posterior part. Here, we used a human to rat translational behavioral approach combined to high-resolution molecular imaging to address this issue. We showed that successful memory performance is predicted by encoding and reactivation patterns only in the dorsal part of the rat hippocampus (posterior part in humans), but not in the ventral part (anterior part in humans). Our findings support the view that the encoding and the retrieval processes per se are not segregated along the longitudinal axis of the hippocampus, but that activity predictive of successful memory is and concerns specifically the dorsal part of the hippocampus. In addition, we found evidence that these processes are likely to be mediated by the activation/reactivation of the same cells at this level. Given the translational character of the task, our results suggest that both the encoding and the retrieval processes take place in the same cells of the posterior part of the human hippocampus.

Proximodistal segregation of nonspatial information in CA3: preferential recruitment of a proximal CA3-distal CA1 network in nonspatial recognition memory.

A prevailing view in memory research is that CA3 principally supports spatial processes. However, few studies have investigated the contribution of CA3 to nonspatial memory function. Interestingly, the proximal part of CA3 (close to the dentate gyrus) predominantly projects to distal CA1 (away from the dentate gyrus), which preferentially processes nonspatial information. Moreover, the cytoarchitecture and connectivity patterns in the proximal and distal parts of CA3 strongly differ, suggesting a functional segregation in this area. Here, we tested whether CA3 is recruited during nonspatial recognition memory, and whether nonspatial information is differentially represented along the proximodistal axis of CA3. Furthermore, we investigated whether the pattern of activation within CA3 would mirror that of CA1. We used a high-resolution imaging technique specifically designed to analyze brain activity in distant areas that is based on the detection of the expression of the immediate-early gene Arc, used as a marker of neuronal activation. We showed that proximal CA3 is strongly recruited during a nonspatial delayed nonmatching-to-sample recognition memory task in rats, while distal CA3 is not. In addition, distal CA1 was more activated than proximal CA1 in the same task. These findings suggest a functional segregation of CA3 that mirrors that of CA1, and potentially indicate the existence of a proximal CA3-distal CA1 hippocampal subnetwork that would preferentially process nonspatial information during recognition memory.

Mapping memory function in the medial temporal lobe with the immediate-early gene Arc.

For the past two decades an increasing number of studies have underlined the crucial role of the immediate - early gene Arc in plasticity processes thought to sustain memory function. Because of the high spatial and temporal resolution of this technique, the detection of Arc products appears to have become a new standard for the mapping of cognitive processes. To date, most Arc studies have focused on identifying the contribution of the hippocampal subfields CA1 and CA3 to spatial processes. In contrast, few have investigated their role in non-spatial memory, or the role of other medial temporal lobe (MTL) areas in spatial and non-spatial memory. This short review describes recent studies focusing on these issues. After a brief overview of Arc's functions, we report a set of studies that put to the test some well-accepted theories in recognition memory. First, we describe data indicating that the parahippocampal areas may not be strictly segregated into spatial and non-spatial streams, as originally described. Second, we report findings revealing a functional segregation along the dorsoventral axis in CA1, but not in CA3. Finally, we bring evidence for a segregation of CA3 along the proximodistal axis and discuss the involvement of a proximal CA3-distal CA1 network during non-spatial memory. In summary, 'Arc imaging' appears to be a powerful tool to identify neural substrates of cognitive processes, not only in the hippocampus but also in the remaining of the MTL. Moreover, because of its fundamental role in synaptic processes, it offers a rare and exciting opportunity to further bridge plasticity processes and memory function.

Quantitative gene-expression analysis of the ligand-receptor system for classical neurotransmitters and neuropeptides in hippocampal CA1, CA3, and dentate gyrus.

We have shown quantitative expression levels of genes coding for the "ligand-receptor system" for classical neurotransmitters and neuropeptides in hippocampal subregions CA1, CA3, and dentate gyrus (DG). Using a combination of DNA microarray and quantitative PCR methods, we found that the three subregions have relatively similar expression patterns of ionotropic receptors for classical neurotransmitters. Expression of ionotropic receptors for glutamate and GABA represents more than 90% of all ionotropic receptors for classical neurotransmitters, and the expression ratio between ionotropic receptors for glutamate and GABA is constant (1.2:1-1.6:1) in each subregion. Meanwhile, the three subregions have different expression patterns of neuropeptide receptors. Furthermore, there are asymmetric expression patterns between neuropeptides and their receptors. Expression of Cck, Npy, Sst, and Penk1 represents 90% of neuropeptides derived locally in the hippocampus, whereas expression of these four neuropeptide receptors accounts for 50% of G protein-coupled receptors for neuropeptides. We propose that CA1, CA3, and DG have different modalities based on the ligand-receptor system, particularly the "neuropeptidergic system." Our quantitative gene-expression analysis provides fundamental data to support functional differences between the three hippocampal subregions regarding ligand-receptor interactions.

Gene expression profiling of neuropeptides in mouse cerebellum, hippocampus, and retina.

OBJECTIVE: We examined gene expression profiling in single neuron types and small regions of the nervous system. METHODS: The RNAs were extracted from mouse cerebellar Purkinje cells, granule cell layer, hippocampal CA1 and CA3 pyramidal cell layers, and three layers of the retina (outer nuclear layer, inner nuclear layer, and ganglion cell layer) were dissected by laser capture microdissection. The gene expression profiling of each sample was examined by Affymetrix GeneChip and real-time reverse transcription polymerase chain reaction. We studied the gene expression of 62 neuropeptide and hormone genes and 387 G-protein-coupled receptor (GPCR) genes. RESULTS: Among them, cholecystokinin and neuropeptide Y genes were the most widely expressed. The gene expression of cholecystokinin was very high in the hippocampus, suggesting that cholecystokinin transcripts might have unknown roles in the hippocampus. More than 10 neuropeptide genes were expressed in the ganglion cell layer of the retina, whereas the outer nuclear layer of the retina did not express a considerable amount of neuropeptide mRNAs. In total 12 GPCR genes were found in all tissues examined, and half were orphans (6 of 12). CONCLUSION: The high ratio of orphan GPCR genes suggests our limited knowledge of the ligand-receptor system in the nervous system. These results provide basic information for studying the function of neuropeptides.

Thinking outside the pyramidal cell: unexplored contributions of interneurons and neuropeptide Y to estrogen-induced synapse formation in the hippocampus.

Since the first finding that 17beta-estradiol (E) can regulate CA1 pyramidal cell synapse formation, subsequent studies have explored many potential E-dependent mechanisms occurring within CA1 pyramidal cells. Fewer studies have focused on E-dependent processes outside of the pyramidal cell that may influence events activity of the pyramidal cells. This review considers hippocampal interneurons, which can potently regulate the excitability of simultaneously firing pyramidal cells. In particular, we discuss neuropeptide Y (NPY) expression by these interneurons because our published findings show that NPY expression is increased by E in a subset of interneurons which coincidentally exhibit E-regulated increase in GABA synthesis and are uniquely situated anatomically such that they may regulate synaptic activity. Here we review the role of different phenotypes of CA1 interneurons, and we propose a model in which E-stimulated NPY gene expression and the release of NPY by interneurons inhibits glutamate release presynaptically and alters glutamate-dependent synaptic events in the rat hippocampus during adulthood.

Estrogen and ovariectomy regulate mRNA and protein of glutamic acid decarboxylases and cation-chloride cotransporters in the adult rat hippocampus.

17beta-Estradiol spatiotemporally regulates the gamma-aminobutyric acid (GABAergic) tone in the adult hippocampus. However, the complex estrogenic effect on the GABAergic system is still unclear. In adult central nervous system (CNS) neurons, GABA can induce both inhibitory and excitatory actions, which are predominantly controlled by the cation-chloride cotransporters NKCC1 and KCC2. We therefore studied the estrogenic regulation of two glutamate decarboxylase (GAD) isoforms, GAD65 and GAD67, as well as NKCC1 and KCC2 in the adult female rat hippocampus by immunohistochemistry and in situ hybridization. First, we focused on the duration after ovariectomy (OVX) and its effects on GAD65 protein levels. The basal number of GAD65-immunoreactive cells decreased after long-term (10 days) OVX compared to short-term (3 days) OVX. We found that, only after long-term OVX but not after short-term OVX, estradiol increased the number of GAD65-immunoreactive cells in the CA1 pyramidal cell layer. Furthermore, estradiol did not alter the GAD65-immunoreactive cell population in any other CA1 subregion. Second, we therefore focused on long-term OVX and the estrogenic regulation of GAD and cation-chloride cotransporter mRNA levels. In the pyramidal cell layer, estradiol affected GAD65, GAD67 and NKCC1 mRNA levels, but not KCC2 mRNA levels. Both GAD65 and NKCC1 mRNA levels increased within 24 h after estradiol treatment, followed by a subsequent increase in GAD67 mRNA levels. These findings suggest that basal levels of estrogen might contribute to a balance between the excitatory and inhibitory synaptic transmission onto CA1 pyramidal cells by regulating perisomatic GAD and NKCC1 expression in the adult hippocampus.