Circadian rhythm mechanism in the suprachiasmatic nucleus and its relation to the olfactory system.

Animals need sleep, and the suprachiasmatic nucleus, the center of the circadian rhythm, plays an important role in determining the timing of sleep. The main input to the suprachiasmatic nucleus is the retinohypothalamic tract, with additional inputs from the intergeniculate leaflet pathway, the serotonergic afferent from the raphe, and other hypothalamic regions. Within the suprachiasmatic nucleus, two of the major subtypes are vasoactive intestinal polypeptide (VIP)-positive neurons and arginine-vasopressin (AVP)-positive neurons. VIP neurons are important for light entrainment and synchronization of suprachiasmatic nucleus neurons, whereas AVP neurons are important for circadian period determination. Output targets of the suprachiasmatic nucleus include the hypothalamus (subparaventricular zone, paraventricular hypothalamic nucleus, preoptic area, and medial hypothalamus), the thalamus (paraventricular thalamic nuclei), and lateral septum. The suprachiasmatic nucleus also sends information through several brain regions to the pineal gland. The olfactory bulb is thought to be able to generate a circadian rhythm without the suprachiasmatic nucleus. Some reports indicate that circadian rhythms of the olfactory bulb and olfactory cortex exist in the absence of the suprachiasmatic nucleus, but another report claims the influence of the suprachiasmatic nucleus. The regulation of circadian rhythms by sensory inputs other than light stimuli, including olfaction, has not been well studied and further progress is expected.

In vivo recording of the circadian calcium rhythm in Prokineticin 2 neurons of the suprachiasmatic nucleus.

Prokineticin 2 (Prok2) is a small protein expressed in a subpopulation of neurons in the suprachiasmatic nucleus (SCN), the primary circadian pacemaker in mammals. Prok2 has been implicated as a candidate output molecule from the SCN to control multiple circadian rhythms. Genetic manipulation specific to Prok2-producing neurons would be a powerful approach to understanding their function. Here, we report the generation of Prok2-tTA knock-in mice expressing the tetracycline transactivator (tTA) specifically in Prok2 neurons and an application of these mice to in vivo recording of Ca2+ rhythms in these neurons. First, the specific and efficient expression of tTA in Prok2 neurons was verified by crossing the mice with EGFP reporter mice. Prok2-tTA mice were then used to express a fluorescent Ca2+ sensor protein to record the circadian Ca2+ rhythm in SCN Prok2 neurons in vivo. Ca2+ in these cells showed clear circadian rhythms in both light-dark and constant dark conditions, with their peaks around midday. Notably, the hours of high Ca2+ nearly coincided with the rest period of the behavioral rhythm. These observations fit well with the predicted function of Prok2 neurons as a candidate output pathway of the SCN by suppressing locomotor activity during both daytime and subjective daytime.

In vivo recording of suprachiasmatic nucleus dynamics reveals a dominant role of arginine vasopressin neurons in circadian pacesetting.

The central circadian clock of the suprachiasmatic nucleus (SCN) is a network consisting of various types of neurons and glial cells. Individual cells have the autonomous molecular machinery of a cellular clock, but their intrinsic periods vary considerably. Here, we show that arginine vasopressin (AVP) neurons set the ensemble period of the SCN network in vivo to control the circadian behavior rhythm. Artificial lengthening of cellular periods by deleting casein kinase 1 delta (CK1δ) in the whole SCN lengthened the free-running period of behavior rhythm to an extent similar to CK1δ deletion specific to AVP neurons. However, in SCN slices, PER2::LUC reporter rhythms of these mice only partially and transiently recapitulated the period lengthening, showing a dissociation between the SCN shell and core with a period instability in the shell. In contrast, in vivo calcium rhythms of both AVP and vasoactive intestinal peptide (VIP) neurons in the SCN of freely moving mice demonstrated stably lengthened periods similar to the behavioral rhythm upon AVP neuron-specific CK1δ deletion, without changing the phase relationships between each other. Furthermore, optogenetic activation of AVP neurons acutely induced calcium increase in VIP neurons in vivo. These results indicate that AVP neurons regulate other SCN neurons, such as VIP neurons, in vivo and thus act as a primary determinant of the SCN ensemble period.

Vasopressin neurons in the paraventricular hypothalamus promote wakefulness via lateral hypothalamic orexin neurons.

The sleep-wakefulness cycle is regulated by complicated neural networks that include many different populations of neurons throughout the brain. Arginine vasopressin neurons in the paraventricular nucleus of the hypothalamus (PVHAVP) regulate various physiological events and behaviors, such as body-fluid homeostasis, blood pressure, stress response, social interaction, and feeding. Changes in arousal level often accompany these PVHAVP-mediated adaptive responses. However, the contribution of PVHAVP neurons to sleep-wakefulness regulation has remained unknown. Here, we report the involvement of PVHAVP neurons in arousal promotion. Optogenetic stimulation of PVHAVP neurons rapidly induced transitions to wakefulness from both NREM and REM sleep. This arousal effect was dependent on AVP expression in these neurons. Similarly, chemogenetic activation of PVHAVP neurons increased wakefulness and reduced NREM and REM sleep, whereas chemogenetic inhibition of these neurons significantly reduced wakefulness and increased NREM sleep. We observed dense projections of PVHAVP neurons in the lateral hypothalamus with potential connections to orexin/hypocretin (LHOrx) neurons. Optogenetic stimulation of PVHAVP neuronal fibers in the LH immediately induced wakefulness, whereas blocking orexin receptors attenuated the arousal effect of PVHAVP neuronal activation drastically. Monosynaptic rabies-virus tracing revealed that PVHAVP neurons receive inputs from multiple brain regions involved in sleep-wakefulness regulation, as well as those involved in stress response and energy metabolism. Moreover, PVHAVP neurons mediated the arousal induced by novelty stress and a melanocortin receptor agonist melanotan-II. Thus, our data suggested that PVHAVP neurons promote wakefulness via LHOrx neurons in the basal sleep-wakefulness and some stressful conditions.

Cell Type-Specific Genetic Manipulation and Impaired Circadian Rhythms in Vip tTA Knock-In Mice.

The suprachiasmatic nucleus (SCN), the central circadian clock in mammals, is a neural network consisting of various types of GABAergic neurons, which can be differentiated by the co-expression of specific peptides such as vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP). VIP has been considered as a critical factor for the circadian rhythmicity and synchronization of individual SCN neurons. However, the precise mechanisms of how VIP neurons regulate SCN circuits remain incompletely understood. Here, we generated Vip tTA knock-in mice that express tetracycline transactivator (tTA) specifically in VIP neurons by inserting tTA sequence at the start codon of Vip gene. The specific and efficient expression of tTA in VIP neurons was verified using EGFP reporter mice. In addition, combined with Avp-Cre mice, Vip tTA mice enabled us to simultaneously apply different genetic manipulations to VIP and AVP neurons in the SCN. Immunostaining showed that VIP is expressed at a slightly reduced level in heterozygous Vip tTA mice but is completely absent in homozygous mice. Consistently, homozygous Vip tTA mice showed impaired circadian behavioral rhythms similar to those of Vip knockout mice, such as attenuated rhythmicity and shortened circadian period. In contrast, heterozygous mice demonstrated normal circadian behavioral rhythms comparable to wild-type mice. These data suggest that Vip tTA mice are a valuable genetic tool to express exogenous genes specifically in VIP neurons in both normal and VIP-deficient mice, facilitating the study of VIP neuronal roles in the SCN neural network.

Wireless Photometry Prototype for Tri-Color Excitation and Multi-Region Recording.

Visualizing neuronal activation and neurotransmitter release by using fluorescent sensors is increasingly popular. The main drawback of contemporary multi-color or multi-region fiber photometry systems is the tethered structure that prevents the free movement of the animals. Although wireless photometry devices exist, a review of literature has shown that these devices can only optically stimulate or excite with a single wavelength simultaneously, and the lifetime of the battery is short. To tackle this limitation, we present a prototype for implementing a fully wireless photometry system with multi-color and multi-region functions. This paper introduces an integrated circuit (IC) prototype fabricated in TSMC 180 nm CMOS process technology. The prototype includes 3-channel optical excitation, 2-channel optical recording, wireless power transfer, and wireless data telemetry blocks. The recording front end has an average gain of 107 dB and consumes 620 µW of power. The light-emitting diode (LED) driver block provides a peak current of 20 mA for optical excitation. The rectifier, the core of the wireless power transmission, operates with 63% power conversion efficiency at 13.56 MHz and a maximum of 87% at 2 MHz. The system is validated in a laboratory bench test environment and compared with state-of-the-art technologies. The optical excitation and recording front end and the wireless power transfer circuit evaluated in this paper will form the basis for a future miniaturized final device with a shank that can be used in in vivo experiments.

Towards a Tri-Color Wireless Photometry System for the Monitoring of Neuronal Activity in the Basal Forebrain and Hippocampus.

Healthy cholinergic function is important for brain function, and disruption of the system is thought to be the cause of dementia, including Alzheimer's disease. The 'Cholinergic Hypothesis' theorizes that cognitive decline is due disruption of the cholinergic system, defined by the low concentration of neurotransmitters such as acetylcholine (ACh) and neurotransmitter-releasing elements such as calcium ions (Ca2+). The ability to measure ACh and Ca2+ concentrations enables researchers to make inferences on the relationship between these indicators that play a role in the onset of neurological conditions. Current commercial devices have one or more of the following limitations: i) they are tethered making it difficult to verify in naturally behaving animal subjects, ii) they are capable of only measuring a single indicator at any given time, or iii) they have multiple shanks that penetrate the cortex. We propose a tri-color miniaturized photometry system capable of optically stimulating indicators in neurons located in the hippocampus and basal forebrain and optically reading the neurons' response. The resulting device has an average gain of 123 dB and a power consumption of 29 mW, comparable to other state-of-the-art devices.

GABA from vasopressin neurons regulates the time at which suprachiasmatic nucleus molecular clocks enable circadian behavior.

The suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, is a network structure composed of multiple types of γ-aminobutyric acid (GABA)-ergic neurons and glial cells. However, the roles of GABA-mediated signaling in the SCN network remain controversial. Here, we report noticeable impairment of the circadian rhythm in mice with a specific deletion of the vesicular GABA transporter in arginine vasopressin (AVP)-producing neurons. These mice showed disturbed diurnal rhythms of GABAA receptor-mediated synaptic transmission in SCN neurons and marked lengthening of the activity time in circadian behavioral rhythms due to the extended interval between morning and evening locomotor activities. Synchrony of molecular circadian oscillations among SCN neurons did not significantly change, whereas the phase relationships between SCN molecular clocks and circadian morning/evening locomotor activities were altered significantly, as revealed by PER2::LUC imaging of SCN explants and in vivo recording of intracellular Ca2+ in SCN AVP neurons. In contrast, daily neuronal activity in SCN neurons in vivo clearly showed a bimodal pattern that correlated with dissociated morning/evening locomotor activities. Therefore, GABAergic transmission from AVP neurons regulates the timing of SCN neuronal firing to temporally restrict circadian behavior to appropriate time windows in SCN molecular clocks.

Inhalation Frequency Controls Reformatting of Mitral/Tufted Cell Odor Representations in the Olfactory Bulb.

In mammals, olfactory sensation depends on inhalation, which controls activation of sensory neurons and temporal patterning of central activity. Odor representations by mitral and tufted (MT) cells, the main output from the olfactory bulb (OB), reflect sensory input as well as excitation and inhibition from OB circuits, which may change as sniff frequency increases. To test the impact of sampling frequency on MT cell odor responses, we obtained whole-cell recordings from MT cells in anesthetized male and female mice while varying inhalation frequency via tracheotomy, allowing comparison of inhalation-linked responses across cells. We characterized frequency effects on MT cell responses during inhalation of air and odorants using inhalation pulses and also "playback" of sniffing recorded from awake mice. Inhalation-linked changes in membrane potential were well predicted across frequency from linear convolution of 1 Hz responses; and, as frequency increased, near-identical temporal responses could emerge from depolarizing, hyperpolarizing, or multiphasic MT responses. However, net excitation was not well predicted from 1 Hz responses and varied substantially across MT cells, with some cells increasing and others decreasing in spike rate. As a result, sustained odorant sampling at higher frequencies led to increasing decorrelation of the MT cell population response pattern over time. Bulk activation of sensory inputs by optogenetic stimulation affected MT cells more uniformly across frequency, suggesting that frequency-dependent decorrelation emerges from odor-specific patterns of activity in the OB network. These results suggest that sampling behavior alone can reformat early sensory representations, possibly to optimize sensory perception during repeated sampling.SIGNIFICANCE STATEMENT Olfactory sensation in mammals depends on inhalation, which increases in frequency during active sampling of olfactory stimuli. We asked how inhalation frequency can shape the neural coding of odor information by recording from projection neurons of the olfactory bulb while artificially varying odor sampling frequency in the anesthetized mouse. We found that sampling an odor at higher frequencies led to diverse changes in net responsiveness, as measured by action potential output, that were not predicted from low-frequency responses. These changes led to a reorganization of the pattern of neural activity evoked by a given odorant that occurred preferentially during sustained, high-frequency inhalation. These results point to a novel mechanism for modulating early sensory representations solely as a function of sampling behavior.

Post-Inhibitory Rebound Spikes in Rat Medial Entorhinal Layer II/III Principal Cells: In Vivo, In Vitro, and Computational Modeling Characterization.

Medial entorhinal cortex Layer-II stellate cells (mEC-LII-SCs) primarily interact via inhibitory interneurons. This suggests the presence of alternative mechanisms other than excitatory synaptic inputs for triggering action potentials (APs) in stellate cells during spatial navigation. Our intracellular recordings show that the hyperpolarization-activated cation current (Ih) allows post-inhibitory-rebound spikes (PIRS) in mEC-LII-SCs. In vivo, strong inhibitory-post-synaptic potentials immediately preceded most APs shortening their delay and enhancing excitability. In vitro experiments showed that inhibition initiated spikes more effectively than excitation and that more dorsal mEC-LII-SCs produced faster and more synchronous spikes. In contrast, PIRS in Layer-II/III pyramidal cells were harder to evoke, voltage-independent, and slower in dorsal mEC. In computational simulations, mEC-LII-SCs morphology and Ih homeostatically regulated the dorso-ventral differences in PIRS timing and most dendrites generated PIRS with a narrow range of stimulus amplitudes. These results suggest inhibitory inputs could mediate the emergence of grid cell firing in a neuronal network.

Cell-Type-Specific Modulation of Sensory Responses in Olfactory Bulb Circuits by Serotonergic Projections from the Raphe Nuclei.

UNLABELLED: Serotonergic neurons in the brainstem raphe nuclei densely innervate the olfactory bulb (OB), where they can modulate the initial representation and processing of olfactory information. Serotonergic modulation of sensory responses among defined OB cell types is poorly characterized in vivo Here, we used cell-type-specific expression of optical reporters to visualize how raphe stimulation alters sensory responses in two classes of GABAergic neurons of the mouse OB glomerular layer, periglomerular (PG) and short axon (SA) cells, as well as mitral/tufted (MT) cells carrying OB output to piriform cortex. In PG and SA cells, brief (1-4 s) raphe stimulation elicited a large increase in the magnitude of responses linked to inhalation of ambient air, as well as modest increases in the magnitude of odorant-evoked responses. Near-identical effects were observed when the optical reporter of glutamatergic transmission iGluSnFR was expressed in PG and SA cells, suggesting enhanced excitatory input to these neurons. In contrast, in MT cells imaged from the dorsal OB, raphe stimulation elicited a strong increase in resting GCaMP fluorescence with only a slight enhancement of inhalation-linked responses to odorant. Finally, optogenetically stimulating raphe serotonergic afferents in the OB had heterogeneous effects on presumptive MT cells recorded extracellularly, with an overall modest increase in resting and odorant-evoked responses during serotonergic afferent stimulation. These results suggest that serotonergic afferents from raphe dynamically modulate olfactory processing through distinct effects on multiple OB targets, and may alter the degree to which OB output is shaped by inhibition during behavior. SIGNIFICANCE STATEMENT: Modulation of the circuits that process sensory information can profoundly impact how information about the external world is represented and perceived. This study investigates how the serotonergic system modulates the initial processing of olfactory information by the olfactory bulb, an obligatory relay between sensory neurons and cortex. We find that serotonergic projections from the raphe nuclei to the olfactory bulb dramatically enhance the responses of two classes of inhibitory interneurons to sensory input, that this effect is mediated by increased glutamatergic drive onto these neurons, and that serotonergic afferent activation alters the responses of olfactory bulb output neurons in vivo These results elucidate pathways by which neuromodulatory systems can dynamically regulate brain circuits during behavior.

Rebound spiking properties of mouse medial entorhinal cortex neurons in vivo.

The medial entorhinal cortex is the gateway between the cortex and hippocampus, and plays a critical role in spatial coding as represented by grid cell activity. In the medial entorhinal cortex, inhibitory circuits are robust, and the presence of the h-current leads to rebound potentials and rebound spiking in in vitro experiments. It has been hypothesized that these properties, combined with network oscillations, may contribute to grid cell formation. To examine the properties of in vivo rebound spikes, we performed whole-cell patch-clamp recordings in medial entorhinal cortex neurons in anaesthetized mice. We injected hyperpolarizing inputs representing inhibitory synaptic inputs along with sinusoidal oscillations and found that hyperpolarizing inputs injected at specific phases of oscillation had a higher probability of inducing subsequent spikes at the peak of the oscillation in some neurons. This effect was prominent in the cells with large sag potential, which is a marker of the h-current. In addition, larger and longer hyperpolarizing current square-pulse stimulation resulted in a larger probability of eliciting rebound spikes, though we did not observe a relationship between the amplitude or duration of hyperpolarizing current pulse stimulation and the delay of rebound spikes. Overall these results suggest that rebound spikes are observed in vivo and may play a role in generating grid cell activity in medial entorhinal cortex neurons.

In vivo cholinergic modulation of the cellular properties of medial entorhinal cortex neurons.

Extensive in vitro data and modeling studies suggest that intrinsic properties of medial entorhinal cortex (MEC) neurons contribute to the spiking behaviour of functional cell types of MEC neurons, such as grid cells, recorded in behaving animals. It remains unclear, however, how intrinsic properties of MEC neurons influence cellular dynamics in intact networks in vivo. In order to begin to bridge the gap between electrophysiological data sets from brain slices and behaving animals, in the present study we performed intracellular recordings using sharp electrodes in urethane-anaesthetized rats to elucidate the cellular dynamics of MEC neurons in vivo. We focused on the h-current-dependent sag potential during hyperpolarizing current steps, subthreshold resonance in response to oscillatory frequency sweeps (chirp stimuli), persistent spiking in response to brief depolarizing inputs and the relationship between firing frequency and input (f-I curve), each of which is sensitive to cholinergic modulation in vitro. Consistent with data from in vitro studies, cholinergic activation by systemic application of the acetylcholinesterase inhibitor, physostigmine, resulted in decreased sag amplitude, increased sag time constant and a decrease of the peak resonance frequency. The f-I curve was also modulated by physostigmine in many neurons, but persistent spiking was not observed in any of our recordings, even when picrotoxin, a GABAA blocker, was included in the internal solution of the recording pipette to reduce possible effects of network inhibition. These results suggest that intrinsic oscillatory and rate-coding mechanisms, but not intrinsic bistability, are significantly modulated by acetylcholine in the intact entorhinal network.

Testing the sorption hypothesis in olfaction: a limited role for sniff strength in shaping primary odor representations during behavior.

The acquisition of sensory information during behavior shapes the neural representation, central processing, and perception of external stimuli. In mammals, a sniff represents the basic unit of odor sampling, yet how sniffing shapes odor representations remains poorly understood. Perhaps the earliest hypothesis of the role of sniffing in olfaction arises from the fact that odorants with different physicochemical properties exhibit different patterns of deposition across the olfactory epithelium, and that these patterns are differentially affected by flow rate. However, whether sniff flow rates shape odor representations during natural sniffing remains untested, and whether animals make use of odorant sorption-airflow relationships as part of an active odor-sampling strategy remains unclear. We tested these ideas in the intact rat using a threefold approach. First, we asked whether sniff strength shapes odor representations in vivo by imaging from olfactory receptor neuron (ORN) terminals during controlled changes in inhalation flow in the anesthetized rat. Second, we asked whether sniff strength shapes odor representations by imaging from ORNs during natural sniffing in the awake rat. Third, we asked whether rats actively modulate sniff strength during an odor discrimination task. We found that, while artificial changes in flow rate can alter ORN responses, sniff strength has negligible effect on odor representations during natural sniffing, and behaving rats do not modulate flow rate to improve odor discrimination. These data suggest that modulating sniff strength does not shape odor representations sufficiently to be part of a strategy for active odor sensing in the behaving animal.

Effects of acetylcholine on neuronal properties in entorhinal cortex.

The entorhinal cortex (EC) receives prominent cholinergic innervation from the medial septum and the vertical limb of the diagonal band of Broca (MSDB). To understand how cholinergic neurotransmission can modulate behavior, research has been directed toward identification of the specific cellular mechanisms in EC that can be modulated through cholinergic activity. This review focuses on intrinsic cellular properties of neurons in EC that may underlie functions such as working memory, spatial processing, and episodic memory. In particular, the study of stellate cells (SCs) in medial entorhinal has resulted in discovery of correlations between physiological properties of these neurons and properties of the unique spatial representation that is demonstrated through unit recordings of neurons in medial entorhinal cortex (mEC) from awake-behaving animals. A separate line of investigation has demonstrated persistent firing behavior among neurons in EC that is enhanced by cholinergic activity and could underlie working memory. There is also evidence that acetylcholine plays a role in modulation of synaptic transmission that could also enhance mnemonic function in EC. Finally, the local circuits of EC demonstrate a variety of interneuron physiology, which is also subject to cholinergic modulation. Together these effects alter the dynamics of EC to underlie the functional role of acetylcholine in memory.

Behavioral state-dependent changes in the information processing mode in the olfactory system.

Changes in behavioral state are accompanied by coordinated changes in the information processing mode in the hippocampus and neocortex of the brain. We review here the recent progress in the knowledge of behavioral state-dependent changes in the information processing mode in the central olfactory system. Olfactory cortex shows state-dependent gating of afferent sensory inputs. In the olfactory bulb, granule-to-mitral dendrodendritic synaptic inhibition is enhanced and the frequency of synchronized oscillatory activity of bulbar output neurons decreases during slow-wave sleep or deeply anesthetized state. These results suggest that the information processing mode in the whole olfactory system changes in a behavioral state-dependent manner to keep the neuronal circuits functioning optimally in each behavioral state.

Dendrodendritic synapses and functional compartmentalization in the olfactory bulb.

Odorant receptor maps in the glomerular layer of the mammalian olfactory bulb are organized into zones and domains. This organization is functionally related to specific odor-induced behavioral responses. Mitral and tufted cells associated with a glomerulus (an odorant receptor channel) extend long lateral dendrites that form dendrodendritic synapses with granule cells associated with other channels. The knowledge of spatial distribution of these long dendrites with regard to the zonal and domain organization will help us to understand the logic of integration of signals from distinct odorant receptor channels in the olfactory bulb.

Behavioral state regulation of dendrodendritic synaptic inhibition in the olfactory bulb.

Behavioral states regulate how information is processed in local neuronal circuits. Here, we asked whether dendrodendritic synaptic interactions in the olfactory bulb vary with brain and behavioral states. To examine the state-dependent change of the dendrodendritic synaptic transmission, we monitored changes in field potential responses in the olfactory bulb of urethane-anesthetized and freely behaving rats. In urethane-anesthetized rats, granule-to-mitral dendrodendritic synaptic inhibition was larger and longer when slow waves were present in the electroencephalogram (slow-wave state) than during the fast-wave state. The state-dependent alternating change in the granule-to-mitral inhibition was regulated by the cholinergic system. In addition, the frequency of the spontaneous oscillatory activity of local field potentials and periodic discharges of mitral cells in the olfactory bulb shifted in synchrony with shifts in the neocortical brain state. Freely behaving rats showed multilevel changes in dendrodendritic synaptic inhibition that corresponded to diverse behavioral states; the inhibition was the largest during slow-wave sleep state, and successively smaller during light sleep, awake immobility, and awake moving states. These results provide evidence that behavioral state-dependent global changes in cholinergic tone modulate dendrodendritic synaptic inhibition and the information processing mode in the olfactory bulb.