Whole-brain Mapping of Inputs and Outputs of Specific Orbitofrontal Cortical Neurons in Mice.

The orbitofrontal cortex (ORB), a region crucial for stimulus-reward association, decision-making, and flexible behaviors, extensively connects with other brain areas. However, brain-wide inputs to projection-defined ORB neurons and the distribution of inhibitory neurons postsynaptic to neurons in specific ORB subregions remain poorly characterized. Here we mapped the inputs of five types of projection-specific ORB neurons and ORB outputs to two types of inhibitory neurons. We found that different projection-defined ORB neurons received inputs from similar cortical and thalamic regions, albeit with quantitative variations, particularly in somatomotor areas and medial groups of the dorsal thalamus. By counting parvalbumin (PV) or somatostatin (SST) interneurons innervated by neurons in specific ORB subregions, we found a higher fraction of PV neurons in sensory cortices and a higher fraction of SST neurons in subcortical regions targeted by medial ORB neurons. These results provide insights into understanding and investigating the function of specific ORB neurons.

Whole-brain spatial organization of hippocampal single-neuron projectomes.

Mapping single-neuron projections is essential for understanding brain-wide connectivity and diverse functions of the hippocampus (HIP). Here, we reconstructed 10,100 single-neuron projectomes of mouse HIP and classified 43 projectome subtypes with distinct projection patterns. The number of projection targets and axon-tip distribution depended on the soma location along HIP longitudinal and transverse axes. Many projectome subtypes were enriched in specific HIP subdomains defined by spatial transcriptomic profiles. Furthermore, we delineated comprehensive wiring diagrams for HIP neurons projecting exclusively within the HIP formation (HPF) and for those projecting to both intra- and extra-HPF targets. Bihemispheric projecting neurons generally projected to one pair of homologous targets with ipsilateral preference. These organization principles of single-neuron projectomes provide a structural basis for understanding the function of HIP neurons.

The Secondary Motor Cortex-striatum Circuit Contributes to Suppressing Inappropriate Responses in Perceptual Decision Behavior.

The secondary motor cortex (M2) encodes choice-related information and plays an important role in cue-guided actions. M2 neurons innervate the dorsal striatum (DS), which also contributes to decision-making behavior, yet how M2 modulates signals in the DS to influence perceptual decision-making is unclear. Using mice performing a visual Go/No-Go task, we showed that inactivating M2 projections to the DS impaired performance by increasing the false alarm (FA) rate to the reward-irrelevant No-Go stimulus. The choice signal of M2 neurons correlated with behavioral performance, and the inactivation of M2 neurons projecting to the DS reduced the choice signal in the DS. By measuring and manipulating the responses of direct or indirect pathway striatal neurons defined by M2 inputs, we found that the indirect pathway neurons exhibited a shorter response latency to the No-Go stimulus, and inactivating their early responses increased the FA rate. These results demonstrate that the M2-to-DS pathway is crucial for suppressing inappropriate responses in perceptual decision behavior.

Influence of Recent Trial History on Interval Timing.

Interval timing is involved in a variety of cognitive behaviors such as associative learning and decision-making. While it has been shown that time estimation is adaptive to the temporal context, it remains unclear how interval timing behavior is influenced by recent trial history. Here we found that, in mice trained to perform a licking-based interval timing task, a decrease of inter-reinforcement interval in the previous trial rapidly shifted the time of anticipatory licking earlier. Optogenetic inactivation of the anterior lateral motor cortex (ALM), but not the medial prefrontal cortex, for a short time before reward delivery caused a decrease in the peak time of anticipatory licking in the next trial. Electrophysiological recordings from the ALM showed that the response profiles preceded by short and long inter-reinforcement intervals exhibited task-engagement-dependent temporal scaling. Thus, interval timing is adaptive to recent experience of the temporal interval, and ALM activity during time estimation reflects recent experience of interval.

A sensitive mitochondrial thermometry 2.0 and the availability of thermogenic capacity of brown adipocyte.

The temperature of a living cell is a crucial parameter for cellular events, such as cell division, gene expressions, enzyme activities and metabolism. We previously developed a quantifiable mitochondrial thermometry 1.0 based on rhodamine B methyl ester (RhB-ME) and rhodamine 800 (Rh800), and the theory for mitochondrial thermogenesis. Given that the synthesized RhB-ME is not readily available, thus, a convenient mitochondrial thermometry 2.0 based on tetra-methyl rhodamine methyl ester (TMRM) and Rh800 for the thermogenic study of brown adipocyte was further evolved. The fluorescence of TMRM is more sensitive (∼1.4 times) to temperature than that of RhB-ME, then the TMRM-based mito-thermometry 2.0 was validated and used for the qualitatively dynamic profiles for mitochondrial thermogenic responses and mitochondrial membrane potential in living cells simultaneously. Furthermore, our results demonstrated that the heterogenous thermogenesis evoked by ß3 adrenoceptor agonist only used overall up to ∼46% of the thermogenic capacity evoked by CCCP stimulation. On the other hand, the results demonstrated that the maximum thermogenesis evoked by NE and oligomycin A used up to ∼79% of the thermogenic capacity, which suggested the maximum thermogenic capacity under physiological conditions by inhibiting the proton-ATPase function of the mitochondrial complex V, such as under the cold activation of sympathetic nerve and the co-release of sympathetic transmitters.

Direct and indirect pathway neurons in ventrolateral striatum differentially regulate licking movement and nigral responses.

Drinking behavior in rodents is characterized by stereotyped, rhythmic licking movement, which is regulated by the basal ganglia. It is unclear how direct and indirect pathways control the lick bout and individual spout contact. We find that inactivating D1 and D2 receptor-expressing medium spiny neurons (MSNs) in the ventrolateral striatum (VLS) oppositely alters the number of licks in a bout. D1- and D2-MSNs exhibit different patterns of lick-sequence-related activity and different phases of oscillation time-locked to the lick cycle. On the timescale of a lick cycle, transient inactivation of D1-MSNs during tongue protrusion reduces spout contact probability, whereas transiently inactivating D2-MSNs has no effect. On the timescale of a lick bout, inactivation of D1-MSNs (D2-MSNs) causes rate increase (decrease) in a subset of basal ganglia output neurons that decrease firing during licking. Our results reveal the distinct roles of D1- and D2-MSNs in regulating licking at both coarse and fine timescales.

Short-Term Influence of Recent Trial History on Perceptual Choice Changes with Stimulus Strength.

Perceptual decisions, especially for difficult stimuli, can be influenced by choices and outcomes in previous trials. However, it is not well understood how stimulus strength modulates the temporal characteristics as well as the magnitude of trial history influence. We addressed this question using a contrast detection task in freely moving mice. We found that, at lower as compared to higher stimulus contrast, the current choice of the mice was more influenced by choices and outcomes in the past trials and the influence emerged from a longer history. To examine the neural basis of stimulus strength-dependent history influence, we recorded from the secondary motor cortex (M2), a prefrontal region that plays an important role in cue-guided actions and memory-guided behaviors. We found that more M2 neurons conveyed information about choices on the past two trials at lower than at higher contrast. Furthermore, history-trial activity in M2 was important for decoding upcoming choice at low contrast. Thus, trial history influence of perceptual choice is adaptive to the strength of sensory evidence, which may be important for action selection in a dynamic environment.

Application of a dye-based mitochondrion-thermometry to determine the receptor downstream of prostaglandin E2 involved in the regulation of hepatocyte metabolism.

Temperature distributions inside a living cell reflect the thermodynamics and functions of cellular components. We used a newly-developed method of mitochondrial thermometry based on Rhodamine B methyl ester, which equilibrates as a thermosensitive mixture of nonfluorescent and fluorescent resonance forms. Prostaglandin E2 (PGE2) is released from hepatic non-parenchymal Kupffer cells and acts as an inflammatory factor to impact various functions of hepatocytes. The activity of PGE2 on energy mechanism of hepatocytes has not been fully elucidated and in particular, which PGE2 receptor mediates the functions has been elusive. We identified EP4 as the major receptor of PGE2 via our mitochondrion-thermometry approach and then substantiated this receptor's role in hepatic metabolism. We discovered that PGE2 is able to decrease intracellular temperature of hepatocytes, via increasing some lipogenic genes' expressions, hampering lipolysis and mitochondrial ß-oxidation, reducing intracellular ATP level and elevating cAMP level through EP4 receptor. The redox status of hepatocytes represented by FAD vs FAD + NADH ratio is influenced by PGE2 in an EP4 receptor-dependent manner. Collectively, these data demonstrate that PGE2 regulates metabolism of hepatocytes mainly through EP4 receptor.

Phase-specific Surround suppression in Mouse Primary Visual Cortex Correlates with Figure Detection Behavior Based on Phase Discontinuity.

In the primary visual cortex (V1), neuronal responses to stimuli within the receptive field (RF) are modulated by stimuli in the RF surround. A common effect of surround modulation is surround suppression, which is dependent on the feature difference between stimuli within and surround the RF and is suggested to be involved in the perceptual phenomenon of figure-ground segregation. In this study, we examined the relationship between feature-specific surround suppression of V1 neurons and figure detection behavior based on figure-ground feature difference. We trained freely moving mice to perform a figure detection task using figure and ground gratings that differed in spatial phase. The performance of figure detection increased with the figure-ground phase difference, and was modulated by stimulus contrast. Electrophysiological recordings from V1 in head-fixed mice showed that the increase in phase difference between stimuli within and surround the RF caused a reduction in surround suppression, which was associated with an increase in V1 neural discrimination between stimuli with and without RF-surround phase difference. Consistent with the behavioral performance, the sensitivity of V1 neurons to RF-surround phase difference could be influenced by stimulus contrast. Furthermore, inhibiting V1 by optogenetically activating either parvalbumin (PV)- or somatostatin (SOM)-expressing inhibitory neurons both decreased the behavioral performance of figure detection. Thus, the phase-specific surround suppression in V1 represents a neural correlate of figure detection behavior based on figure-ground phase discontinuity.

Dye-based mito-thermometry and its application in thermogenesis of brown adipocytes.

Mitochondrion is the main intracellular site for thermogenesis and attractive energy expenditure targeting for obesity therapy. Here, we develop a method of mitochondrial thermometry based on Rhodamine B methyl ester, which equilibrates as a thermosensitive mixture of nonfluorescent and fluorescent resonance forms. Using this approach, we are able to demonstrate that the efficacy of norepinephrine-induced thermogenesis is low, and measure the maximum transient rate of temperature increase in brown adipocytes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s41048-017-0039-6) contains supplementary material, which is available to authorized users.

Sympathetic transmitters control thermogenic efficacy of brown adipocytes by modulating mitochondrial complex V.

Obesity is a worldwide epidemic and results from excessive energy intake or inefficient energy expenditure. It is promising to utilize the thermogenic function of brown adipose tissue for obesity intervention. However, the mechanisms controlling the efficacy of norepinephrine-induced thermogenesis in brown adipocytes remain elusive. Here we demonstrate that norepinephrine (NE) induces low-efficacy thermogenesis, evoking both heterogeneous changes (ΔΨm and ΔpH) and homogenous responses, one of which is that NE stimulation causes large amounts of ATP consumption in brown adipocytes. We reveal that the proton-ATPase activity of mitochondrial complex V is a key factor that antagonizes proton leakage by UCP1 and determines the efficacy of NE-induced thermogenesis in brown adipocytes. Furthermore, to avoid unnecessary and undesired heat production, we reveal that ATP is a necessary sympathetic cotransmitter for the high efficacy and specificity of NE-induced thermogenesis in brown adipocytes as it increases intracellular calcium concentrations and upregulates the ATP synthase activity of complex V. Thus, we demonstrate the modulation mechanism of thermogenic efficacy in brown adipocytes. These findings imply new strategies to partially or fully utilize the thermogenic capacity of brown adipocytes to identify therapeutic targets for the treatment of obesity and diabetes.

Altered visual cortical processing in a mouse model of MECP2 duplication syndrome.

As an epigenetic modulator of gene expression, Methyl-CpG binding protein 2 (MeCP2) is essential for normal neurological function. Dysfunction of MeCP2 is associated with a variety of neurological disorders. MECP2 gene duplication in human causes neuropsychiatric symptoms such as mental retardation and autism. MeCP2 overexpression in mice results in neurobehavioural disorders, dendritic abnormalities, and synaptic defects. However, how gain of MeCP2 function influences cortical processing of sensory information remains unclear. In this study, we examined visual processing in a mouse model of MECP2 duplication syndrome (MECP2 Tg1 mouse) at 8 and 14 weeks, which were before and after the onset of behavioural symptoms, respectively. In vivo extracellular recordings from primary visual cortex (V1) showed that neurons in Tg1 mice at both adult ages preferred higher spatial frequencies (SFs) than those in wild-type (WT) littermate controls, and the semi-saturation contrasts of neurons were lower in Tg1 mice at 8 weeks but not at 14 weeks. Behavioural experiments showed that the performance for visual detection at high SFs and low contrasts was higher in MECP2 Tg1 mice. Thus, MeCP2 gain-of-function in mice leads to higher visual acuity and contrast sensitivity, both at the levels of cortical response and behavioural performance.

Cellular model of neuronal atrophy induced by DYNC1I1 deficiency reveals protective roles of RAS-RAF-MEK signaling.

Neuronal atrophy is a common pathological feature occurred in aging and neurodegenerative diseases. A variety of abnormalities including motor protein malfunction and mitochondrial dysfunction contribute to the loss of neuronal architecture; however, less is known about the intracellular signaling pathways that can protect against or delay this pathogenic process. Here, we show that the DYNC1I1 deficiency, a neuron-specific dynein intermediate chain, causes neuronal atrophy in primary hippocampal neurons. With this cellular model, we are able to find that activation of RAS-RAF-MEK signaling protects against neuronal atrophy induced by DYNC1I1 deficiency, which relies on MEK-dependent autophagy in neuron. Moreover, we further reveal that BRAF also protects against neuronal atrophy induced by mitochondrial impairment. These findings demonstrate protective roles of the RAS-RAF-MEK axis against neuronal atrophy, and imply a new therapeutic target for clinical intervention.

GASZ and mitofusin-mediated mitochondrial functions are crucial for spermatogenesis.

Nuage is an electron-dense cytoplasmic structure in germ cells that contains ribonucleoproteins and participates in piRNA biosynthesis. Despite the observation that clustered mitochondria are associated with a specific type of nuage called intermitochondrial cement (pi-body), the importance of mitochondrial functions in nuage formation and spermatogenesis is yet to be determined. We show that a germ cell-specific protein GASZ contains a functional mitochondrial targeting signal and is largely localized at mitochondria both endogenously in germ cells and in somatic cells when ectopically expressed. In addition, GASZ interacts with itself at the outer membrane of mitochondria and promotes mitofusion in a mitofusin/MFN-dependent manner. In mice, deletion of the mitochondrial targeting signal reveals that mitochondrial localization of GASZ is essential for nuage formation, mitochondrial clustering, transposon repression, and spermatogenesis. MFN1 deficiency also leads to defects in mitochondrial activity and male infertility. Our data thus reveal a requirement for GASZ and MFN-mediated mitofusion during spermatogenesis.

EEG changes as heat stress reactions in rats irradiated by high intensity 35 GHz millimeter waves.

As the application of millimeter waves for civilian and military use increases, the possibility of overexposure to millimeter waves will also increase. This paper attempts to evaluate stress reactions evoked by 35 GHz millimeter waves. The stress reactions in Sprague-Dawley (SD) rats were quantitatively studied by analyzing electroencephalogram (EEG) changes induced by overexposure to 35 GHz millimeter waves. The relative changes in average energy of the EEG and its wavelet decompositions were used for extracting the stress reaction indicators. Incident average power densities (IAPDs) of 35 GHz millimeter waves from 0.5 W cm(-2) to 7.5 W cm(-2) were employed to investigate the relation between irradiation dose and the stress reactions in the rats. Different stress reaction periods evoked by irradiation were quantitatively evaluated by EEG results. The results illustrate that stress reactions are more intense during the first part of the irradiation than during the later part. The skin temperature increase produced by millimeter wave irradiation is the principle reason for stress reactions and skin injuries. As expected, at the higher levels of irradiation, the reaction time decreases and the reaction intensity increases.

[Secondary structure changes of insulin induced by PEF exposure at different temperatures using Raman spectra and theoretical model analysis].

Recently, biological effects induced by weak electromagnetic fields have been a public concern. Our previous study found temperature and electromagnetic field co-effects on insulin conformation. Therefore, in the present study, Raman spectroscopy was employed to investigate the secondary structure changes of insulin molecule induced by pulsed electric field (PEF) exposure at various temperatures. The content changes in alpha helix of insulin were obtained. Then, protein helix-random coil transition model was used to quantitatively study the experimental results. The theoretical model could figure out the effect of PEF on alpha helix contents of insulin at different temperatures. The protein secondary structure transits from helix to random coil evoked by PEF exposure and change of thermodynamic environment, which could explain the reason for the decline of alpha helix content of insulin caused by PEF exposure together with temperature rising. The results offer experimental basis and theoretical reference for further study of the mechanism of nonthermal effects of weak electromagnetic fields on biological molecule secondary structure.

[EEG changes and stress reactions in rat induced by millimeter wave].

The present paper is aimed to study the processes of stress reaction and their judgment bases in rat induced by 35 GHz millimeter wave quantitatively. The relative change in the average energy of each EEG frequency band decomposed by wavelet analysis was calculated for extracting the stress indicator for the purpose. The rat would experience quiet period, guarding period, deadlock period and prostrating period in sequence. The judgment bases of different stress periods in rat induced by millimeter wave were obtained by analyzing the skin temperature, skin injury and changes of blood biochemical indexes during each stress period. The stress period changed from quiet period to guarding period when the skin temperature of irradiated area reached the thermal pain threshold. It was from guarding period to deadlock period when the skin had gotten serious injury. Then the rat reaction sensitivity decreased, and injury of its visceral organs occurred. The rat got to prostrating period when the sustained irradiation caused the rat's visceral organs to get more serious injury. The further sustained irradiation finally induced death of the rat.

[Comparison of digital filter and wavelet transform for extracting electroencephalogram rhythm].

It is very important to extract electroencephalogram (EEG) rhythm in clinical diagnoses. Digital filter and wavelet transform are used to extract the rhythm from a piece of EEG at the sampling rate of 2 kHz. The Daubechies order 4 wavelet (db4) was used to decompose the EEG at 8 levels. According to the filter characteristic of wavelet decomposition, the reconstructions of aS, d8, d7, d6 and d5 component are nearly corresponding to the rhythms of delta, theta, alpha, gamma separately. The 6 order ellipse infinite impulse response (IIR) filter is also used to decompose the EEG. As the quality factor of wavelet decomposition filter is constant, the wavelet transform obtains better extracted rhythm than the digital filter. Furthermore, the wavelet transform method can be used to extract the low frequency rhythm from wide frequency band.

[Raman spectra of murine skin irradiated by high power millimeter wave].

An experimental study on Raman spectroscopy of normal murine skin and the skin irradiated by high power millimeter wave (HPMM) is presented. It is showed that the Raman spectra of normal skin mainly originate from collagen, and the characteristic peaks are 857, 936 and 1 658 cm(-1). The result showed that after irradiation by HPMM, the relative intensity of the characteristic peaks at 857 and 936 cm(-1) of Raman spectra was decreased. This meant that the collagen was destroyed and even daimaged. It was probably indicated that the skin tissue was damaged and could not be restored. The result also showed that the intensity of the characteristic peak at 1658 cm(-1) of the skin tissue irradiated by millimeter wave with the duration of 20 s decreased. It was considered that the protein in the skin was destroyed. Those results were consistent with macroscopic observation results.