Hierarchical Elemental Odor Coding for Fine Discrimination Between Enantiomer Odors or Cancer-Characteristic Odors.

Odors trigger various emotional responses such as fear of predator odors, aversion to disease or cancer odors, attraction to male/female odors, and appetitive behavior to delicious food odors. Odor information processing for fine odor discrimination, however, has remained difficult to address. The olfaction and color vision share common features that G protein-coupled receptors are the remote sensors. As different orange colors can be discriminated by distinct intensity ratios of elemental colors, such as yellow and red, odors are likely perceived as multiple elemental odors hierarchically that the intensities of elemental odors are in order of dominance. For example, in a mixture of rose and fox-unique predator odors, robust rose odor alleviates the fear of mice to predator odors. Moreover, although occult blood odor is stronger than bladder cancer-characteristic odor in urine samples, sniffer mice can discriminate bladder cancer odor in occult blood-positive urine samples. In forced-choice odor discrimination tasks for pairs of enantiomers or pairs of body odors vs. cancer-induced body odor disorders, sniffer mice discriminated against learned olfactory cues in a wide range of concentrations, where correct choice rates decreased in the Fechner's law, as perceptual ambiguity increased. In this mini-review, we summarize the current knowledge of how the olfactory system encodes and hierarchically decodes multiple elemental odors to control odor-driven behaviors.

Entorhinal Layer II Calbindin-Expressing Neurons Originate Widespread Telencephalic and Intrinsic Projections.

In the present study we provide the first systematic and quantitative hodological study of the calbindin-expressing (CB+) principal neurons in layer II of the entorhinal cortex and compared the respective projections of the lateral and medial subdivisions of the entorhinal cortex. Using elaborate quantitative retrograde tracing, complemented by anterograde tracing, we report that the layer II CB+ population comprises neurons with diverse, mainly excitatory projections. At least half of them originate local intrinsic and commissural projections which distribute mainly to layer I and II. We further show that long-range CB+ projections from the two entorhinal subdivisions differ substantially in that MEC projections mainly target field CA1 of the hippocampus, whereas LEC CB+ projections distribute much more widely to a substantial number of known forebrain targets. This connectional difference between the CB+ populations in LEC and MEC is reminiscent of the overall projection pattern of the two entorhinal subdivisions.

Imaging local brain activity of multiple freely moving mice sharing the same environment.

Electrophysiological field potential dynamics have been widely used to investigate brain functions and related psychiatric disorders. Considering recent demand for its applicability to freely moving subjects, especially for animals in a group and socially interacting with each other, here we propose a new method based on a bioluminescent voltage indicator LOTUS-V. Using our fiber-free recording method based on the LOTUS-V, we succeeded in capturing dynamic change of brain activity in freely moving mice. Because LOTUS-V is the ratiometric indicator, motion and head-angle artifacts were not significantly detected. Taking advantage of our method as a fiber-free system, we further succeeded in simultaneously recording from multiple independently-locomotive mice that were freely interacting with one another. Importantly, this enabled us to find that the primary visual cortex, a center of visual processing, was activated during the interaction of mice. This methodology may further facilitate a wide range of studies in neurobiology and psychiatry.

Behavioral evidence for the use of functional categories during group reversal task performance in monkeys.

A functional category is a set of stimuli that are regarded as equivalent independently of their physical properties and elicit the same behavioral responses. Major psychological theories suggest the ability to form and utilize functional categories as a basis of higher cognition that markedly increases behavioral flexibility. Vaughan claimed the category use in pigeons on the basis of partition, a mathematical criterion for equivalence, however, there have been some criticisms that the evidence he showed was insufficient. In this study, by using a group reversal task, a procedure originally used by Vaughan, we aimed to gather further evidence to prove the category use in animals. Macaque monkeys, which served as subjects in our study, could efficiently perform the task not only with familiar stimulus sets as Vaughan demonstrated but also with novel sets, and furthermore the task performance was stable even when the number of stimuli in a set was increased, which we consider as further evidence for the category use in animals. In addition, by varying the timing of the reversal, we found that a category formation takes place soon after encountering new stimuli, i.e. in a few blocks of trial after a novel stimulus set was introduced.

Intrinsic Projections of Layer Vb Neurons to Layers Va, III, and II in the Lateral and Medial Entorhinal Cortex of the Rat.

Layer V of the entorhinal cortex (EC) receives input from the hippocampus and originates main entorhinal outputs. The deep-sublayer Vb, immunopositive for the transcription factor Ctip2, is thought to be the main recipient of hippocampal projections, whereas the superficial-sublayer LVa, immunonegative for Ctip2, originates the main outputs of EC. This disrupts the proposed role of EC as mediating hippocampal-cortical interactions. With the use of specific (trans)synaptic tracing approaches, we report that, in medial entorhinal cortex, layer Vb neurons innervate neurons in layers Va, II, and III. A similar circuitry exists in the lateral entorhinal cortex. We conclude that EC-layer Vb neurons mediate two circuits in the hippocampus-memory system: (1) a hippocampal output circuit to telencephalic areas by projecting to layer Va and (2) a feedback projection, sending information back to the EC-hippocampal loop via neurons in layers II and III.

Sex Differences in Risk Preference and c-Fos Expression in Paraventricular Thalamic Nucleus of Rats During Gambling Task.

Different biological requirements between males and females may cause sex differences in decision preference when choosing between taking a risk to get a higher gain or taking a lower but sure gain. Several studies have tested this assumption in rats, however the conclusion remains controversial because the previous real-world like gambling tasks contained a learning component to track a global payoff of probabilistic outcome in addition to risk preference. Therefore, we modified a simple gambling task allowing us to exclude such learning effect, and investigated the sex difference in risk preference of rats and its neural basis. The task required water deprived rats to choose between a risky option which provided four drops of water or no reward at a 50% random chance vs. a sure option which provided predictable amount x (x = 1, 2, 3, 4). The amount and the risk were explicitly instructed so that different choice conditions could be tested trial by trial without re-learning of reward contingency. Although both sexes correctly chose the sure option with the same level of accuracy when the sure option provided the best offer (x = 4), they exhibited different choice performances when two options had the same expected value (x = 2). Males and females both preferred to take risky choices than sure choices (risk seeking), but males were more risk seeking than females. Outcome-history analysis of their choice pattern revealed that females reduced their risk preference after losing risky choices, whereas males did not. Rather, as losses continued, reaction time for subsequent risky choices got shorter in males. Given that significant sex difference features mainly emerged after negative experiences, male and female rats may evaluate an unsuccessful outcome of their decision in different manners. Furthermore, c-Fos expression in the paraventricular nucleus of the thalamus (PV) was higher in the gambling task than for the control task in males while c-fos levels did not differ in females. The present study provides a clear evidence of sex differences in risk preference in rats and suggests that the PV is a candidate region contributing to sex differences in risky decision making.

Ventrolateral Prefrontal Cortex Updates Chosen Value According to Choice Set Size.

Having chosen an item typically increases the subjective value of the chosen item, and people generally enjoy making choices from larger choice sets. However, having too many items to choose from can reduce the value of chosen items-for example, because of conflict or choice difficulty. In this study, we investigated the effects of choice set size on behavioral and neural value updating (revaluation) of the chosen item. In the scanner, participants selected items from choice sets of various sizes (one, two, four, or eight items). After they chose an item, participants rerated the chosen item, and we quantified revaluation by taking the difference of postchoice minus prechoice ratings. Revaluation of chosen items increased up to choice sets of four alternatives but then decreased again for items chosen from choice sets of eight alternatives, revealing both a linear and a quadratic effect of choice set size. At the time of postchoice rating, activation of the ventrolateral pFC (VLPFC) reflected the influence of choice set size on parametric revaluation, without significant relation to either prechoice or postchoice ratings tested separately. Additional analyses revealed relations of choice set size to anterior cingulate and insula activity during actual choice and increased coupling of both regions to revaluation-related VLPFC during postchoice rating. These data suggest that the VLPFC plays a central role in a network that relates choice set size to updating the value of chosen items and integrates choice overload with value-enhancing effects of larger choice sets.

Robust, highly customizable, and economical multi-channel electrode for chronic multi-unit recording in behaving animals.

Multi-unit recording has been one of the most widely used techniques to investigate the correlation between multiple neuronal activities and behavior. However, a common problem of currently used multi-channel electrodes is their physical weakness. In this study, we developed a novel multi-channel electrode with sufficient physical strength to penetrate a thickened dura mater. This electrode consists of low-cost materials and is easily fabricated, and it enables recording without removing dura mater, thereby reducing the risk of inflammation, infection, or brain herniation. The low-cost multi-channel electrode developed in this study would be a useful tool for chronic recording in behaving animals.

Increased transgene expression level of rabies virus vector for transsynaptic tracing.

Viral vectors that can infect neurons transsynaptically and can strongly express foreign genes are useful for investigating the organization of neural circuits. We previously developed a propagation-competent rabies virus (RV) vector based on a highly attenuated HEP-Flury strain (rHEP5.0-CVSG), which selectively infects neurons and propagates between synaptically connected neurons in a retrograde direction. Its relatively low level of transgene expression, however, makes immunostaining necessary to visualize the morphological features of infected neurons. To increase the transgene expression level of this RV vector, in this study we focused on two viral proteins: the large protein (L) and matrix protein (M). We first attempted to enhance the expression of L, which is a viral RNA polymerase, by deleting the extra transcription unit and shortening the intergenic region between the G and L genes. This viral vector (rHEP5.0-GctL) showed increased transgene expression level with efficient transsynaptic transport. We next constructed an RV vector with a rearranged gene order (rHEP5.0-GML) with the aim to suppress the expression of M, which plays a regulatory role in virus RNA synthesis. Although this vector showed high transgene expression level, the efficiency of transsynaptic transport was low. To further evaluate the usability of rHEP5.0-GctL as a transsynaptic tracer, we inserted a fluorescent timer as a transgene, which changes the color of its fluorescence from blue to red over time. This viral vector enabled us the differentiation of primary infected neurons from secondary infected neurons in terms of the fluorescence wavelength. We expect this propagation-competent RV vector to be useful for elucidating the complex organization of the central nervous system.

Parahippocampal and retrosplenial connections of rat posterior parietal cortex.

The posterior parietal cortex has been implicated in spatial functions, including navigation. The hippocampal and parahippocampal region and the retrosplenial cortex are crucially involved in navigational processes and connections between the parahippocampal/retrosplenial domain and the posterior parietal cortex have been described. However, an integrated account of the organization of these connections is lacking. Here, we investigated parahippocampal connections of each posterior parietal subdivision and the neighboring secondary visual cortex using conventional retrograde and anterograde tracers as well as transsynaptic retrograde tracing with a modified rabies virus. The results show that posterior parietal as well as secondary visual cortex entertain overall sparse connections with the parahippocampal region but not with the hippocampal formation. The medial and lateral dorsal subdivisions of posterior parietal cortex receive sparse input from deep layers of all parahippocampal areas. Conversely, all posterior parietal subdivisions project moderately to dorsal presubiculum, whereas rostral perirhinal cortex, postrhinal cortex, caudal entorhinal cortex and parasubiculum all receive sparse posterior parietal input. This indicated that the presubiculum might be a major liaison between parietal and parahippocampal domains. In view of the close association of the presubiculum with the retrosplenial cortex, we included the latter in our analysis. Our data indicate that posterior parietal cortex is moderately connected with the retrosplenial cortex, particularly with rostral area 30. The relative sparseness of the connectivity with the parahippocampal and retrosplenial domains suggests that posterior parietal cortex is only a modest actor in forming spatial representations underlying navigation and spatial memory in parahippocampal and retrosplenial cortex. © 2017 Wiley Periodicals, Inc.

Comparative Overview of Visuospatial Working Memory in Monkeys and Rats.

Neural mechanisms of working memory, particularly its visuospatial aspect, have long been studied in non-human primates. On the other hand, rodents are becoming more important in systems neuroscience, as many of the innovative research methods have become available for them. There has been a question on whether primates and rodents have similar neural backgrounds for working memory. In this article, we carried out a comparative overview of the neural mechanisms of visuospatial working memory in monkeys and rats. In monkeys, a number of lesion studies indicate that the brain region most responsible for visuospatial working memory is the ventral dorsolateral prefrontal cortex (vDLPFC), as the performance in the standard tests for visuospatial working memory, such as delayed response and delayed alternation tasks, are impaired by lesions in this region. Single-unit studies revealed a characteristic firing pattern in neurons in this area, a sustained delay activity. Further studies indicated that the information maintained in the working memory, such as cue location and response direction in a delayed response, is coded in the sustained delay activity. In rats, an area comparable to the monkey vDLPFC was found to be the dorsal part of the medial prefrontal cortex (mPFC), as the delayed alternation in a T-maze is impaired by its lesion. Recently, the sustained delay activity similar to that found in monkeys has been found in the dorsal mPFC of rats performing the delayed response task. Furthermore, anatomical studies indicate that the vDLPFC in monkeys and the dorsal mPFC in rats have much in common, such as that they are both the major targets of parieto-frontal projections. Thus lines of evidence indicate that in both monkeys and rodents, the PFC plays a critical role in working memory.

Representation of Functional Category in the Monkey Prefrontal Cortex and Its Rule-Dependent Use for Behavioral Selection.

Humans, monkeys, and other animals are considered to have the cognitive ability to use functional categories--that is, stimulus groups based on functional equivalence independent of physical properties. To investigate the underlying neural mechanisms of the use of functional categories, we recorded single-unit activity in the prefrontal cortex of monkeys performing a behavioral task in which the rule-dependent usage of functional category was needed to select an appropriate response. We found a neural correlate of functional categories on the single-neuron level and found that category information is coded independently of other task-relevant information such as rule and contingency information. Analysis of the time course of the information activation suggested that contingency information used for action selection is derived by integrating incoming category information with rule information maintained throughout a session. Such neural computation can be considered as the neural background of flexible behavioral control based on category and rule.

A novel method for quantifying similarities between oscillatory neural responses in wavelet time-frequency power profiles.

Quantifying similarities and differences between neural response patterns is an important step in understanding neural coding in sensory systems. It is difficult, however, to compare the degree of similarity among transient oscillatory responses. We developed a novel method of wavelet correlation analysis for quantifying similarity between transient oscillatory responses, and tested the method with olfactory cortical responses. In the anterior piriform cortex (aPC), the largest area of the primary olfactory cortex, odors induce inhibitory activities followed by transient oscillatory local field potentials (osci-LFPs). Qualitatively, the resulting time courses of osci-LFPs for identical odors were modestly different. We then compared several methods for quantifying the similarity between osci-LFPs for identical or different odors. Using fast Fourier transform band-pass filters, a conventional method demonstrated high correlations of the 0-2Hz components for both identical and different odors. None of the conventional methods tested demonstrated a clear correlation between osci-LFPs. However, wavelet correlation analysis resolved a stimulus dependency of 2-45Hz osci-LFPs in the aPC output layer, and produced experience-dependent high correlations in the input layer between some of the identical or different odors. These results suggest that redundancy in the neural representation of sensory information may change in the aPC. This wavelet correlation analysis may be useful for quantifying the similarities of transient oscillatory neural responses.

Neck collar for restraining head and body movements in rats for behavioral task performance and simultaneous neural activity recording.

BACKGROUND: Head fixation has been one of the major methods in behavioral neurophysiology because it allows precision in stimulus application and behavioral assessment. Most neural recordings in awake monkeys have been obtained under head fixation, which is nowadays also being used in awake rodents. However, head fixation devices in rats often become unstable within several months, which increases risks for inflammation, infection, and necrosis of the bone and surrounding tissue. NEW METHOD: In this study we developed a novel non-invasive "neck collar system" for restraining the head and body movements of behaving rats. RESULTS: The attachment of the neck collar for 2-3 months did not affect the animals' health and welfare. Rats under neck-collar fixation could learn a behavioral task (standard delayed licking task) with the same efficiency as those under standard head fixation. They could also learn a more complicated task (delayed pro/anti-licking task) under neck-collar fixation and afterwards transfer their learning to the task under standard head fixation. Furthermore, we were able to record single-unit activity in rats under neck-collar fixation during the performance of the standard delayed licking task. COMPARISON WITH EXISTING METHOD(S): This system consists of economical materials and is easily constructed, and it enables head-restraint without surgery, thus eliminating the risk of inflammation or infection. CONCLUSIONS: We consider the neck-collar fixation developed in this study would be useful for restraining the head of a behaving rodent.

Discrete coding of stimulus value, reward expectation, and reward prediction error in the dorsal striatum.

To investigate how the striatum integrates sensory information with reward information for behavioral guidance, we recorded single-unit activity in the dorsal striatum of head-fixed rats participating in a probabilistic Pavlovian conditioning task with auditory conditioned stimuli (CSs) in which reward probability was fixed for each CS but parametrically varied across CSs. We found that the activity of many neurons was linearly correlated with the reward probability indicated by the CSs. The recorded neurons could be classified according to their firing patterns into functional subtypes coding reward probability in different forms such as stimulus value, reward expectation, and reward prediction error. These results suggest that several functional subgroups of dorsal striatal neurons represent different kinds of information formed through extensive prior exposure to CS-reward contingencies.

Effects of G-gene Deletion and Replacement on Rabies Virus Vector Gene Expression.

The glycoprotein-gene (G gene) -deleted rabies virus (RV) vector is a powerful tool to examine the function and structure of neural circuits. We previously reported that the deletion of the G gene enhances the transgene expression level of the RV vector. However, the mechanism of this enhancement remains to be clarified. We presume that there are two possible factors for this enhancement. The first factor is the glycoprotein of RV, which shows cytotoxicity; thus, may cause a dysfunction in the translation process of infected cells. The second possible factor is the enhanced expression of the L gene, which encodes viral RNA polymerase. In the RV, it is known that the gene expression level is altered depending on the position of the gene. Since G-gene deletion displaces the L gene in the genome, the expression of the L gene and viral transcription may be enhanced. In this study, we compared the transgene expression level and viral transcription of three recombinant RV vectors. The effect of glycoprotein was examined by comparing the viral gene expression of G-gene-intact RV and G-gene-replaced RV. Despite the fact that the L-gene transcription level of these two RV vectors was similar, the G-gene-replaced RV vector showed higher viral transcription and transgene expression level than the G-gene-intact RV vector. To examine the effect of the position of the L gene, we compared the viral gene expression of the G-gene-deleted RV and G-gene-replaced RV. The G-gene-deleted RV vector showed higher L-gene transcription, viral transcription, and transgene expression level than the G-gene-replaced RV vector. These results indicate that G-gene deletion enhances the transgene expression level through at least two factors, the absence of glycoprotein and enhancement of L-gene expression. These findings enable investigators to design a useful viral vector that shows a controlled desirable transgene expression level in applications.

Dopaminergic and serotonergic modulation of anterior insular and orbitofrontal cortex function in risky decision making.

Systemic manipulations have shown that dopamine and serotonin systems are involved in risky decision making. However, how they work within the regions that implement risky choices remains unclear. The present study investigated the role of dopamine and serotonin in the rat anterior insular cortex (AIC) and orbitofrontal cortex (OFC), which make different contributions to risky decision making. We examined the effects of local injection of the D1 (SCH23390), D2 (eticlopride), 5-HT1A (WAY100635) and 5-HT2A (M100907) receptor antagonists into the AIC or OFC on risk preference in a gambling task. We found that different dopamine and serotonin receptor subtypes in the AIC and OFC differentially influence risky decision making: intra-AIC injection of D2R or 5-HT1AR blockers increased risk preference whereas intra-OFC injection of the 5-HT1AR blocker decreased it. Risk preference was not altered by intra-AIC injection of D1R and 5-HT2AR blockers or by intra-OFC injection of D1R, D2R, and 5-HT2AR blockers. Furthermore, additional analyses revealed that dopamine and serotonin signaling in the AIC have outcome history-dependent effects on risk taking: intra-AIC injection of the D2R blocker increased risk preference particularly after winning in a previous risky choice, whereas intra-AIC injection of the 5-HT1AR blocker increased risk preference after losing.

Neural activity associated with enhanced facial attractiveness by cosmetics use.

Previous psychological studies have shown that make-up enhances facial attractiveness. Although neuroimaging evidence indicates that the orbitofrontal cortex (OFC) shows greater activity for faces of attractive people than for those of unattractive people, there is no direct evidence that the OFC also shows greater activity for the face of an individual wearing make-up than for the same face without make-up. Using functional magnetic resonance imaging (fMRI), we investigated neural activity while subjects viewed 144 photographs of the same faces with and without make-up (48 with make-up, 48 without make-up, and 48 scrambled photographs) and assigned these faces an attractiveness rating. The behavioral data showed that the faces with make-up were rated as more attractive than those without make-up. The imaging data revealed that the left OFC and the right hippocampus showed greater activity for faces with make-up than for those without make-up. Furthermore, the activities of the right anterior cingulate cortex, left hippocampus, and left OFC increased with increasing facial attractiveness resulting from cosmetics use. These results provide direct evidence of the neural underpinnings of cosmetically enhanced facial attractiveness.

Organization of multisynaptic inputs to the dorsal and ventral dentate gyrus: retrograde trans-synaptic tracing with rabies virus vector in the rat.

Behavioral, anatomical, and gene expression studies have shown functional dissociations between the dorsal and ventral hippocampus with regard to their involvement in spatial cognition, emotion, and stress. In this study we examined the difference of the multisynaptic inputs to the dorsal and ventral dentate gyrus (DG) in the rat by using retrograde trans-synaptic tracing of recombinant rabies virus vectors. Three days after the vectors were injected into the dorsal or ventral DG, monosynaptic neuronal labeling was present in the entorhinal cortex, medial septum, diagonal band, and supramammillary nucleus, each of which is known to project to the DG directly. As in previous tracing studies, topographical patterns related to the dorsal and ventral DG were seen in these regions. Five days after infection, more of the neurons in these regions were labeled and labeled neurons were also seen in cortical and subcortical regions, including the piriform and medial prefrontal cortices, the endopiriform nucleus, the claustrum, the cortical amygdala, the medial raphe nucleus, the medial habenular nucleus, the interpeduncular nucleus, and the lateral septum. As in the monosynaptically labeled regions, a topographical distribution of labeled neurons was evident in most of these disynaptically labeled regions. These data indicate that the cortical and subcortical inputs to the dorsal and ventral DG are conveyed through parallel disynaptic pathways. This second-order input difference in the dorsal and ventral DG is likely to contribute to the functional differentiation of the hippocampus along the dorsoventral axis.

Rabies virus vector transgene expression level and cytotoxicity improvement induced by deletion of glycoprotein gene.

The glycoprotein (G) of rabies virus (RV) is required for binding to neuronal receptors and for viral entry. G-deleted RV vector is a powerful tool for investigating the organization and function of the neural circuits. It gives the investigator the ability to genetically target initial infection to particular neurons and to control trans-synaptic propagation. In this study we have quantitatively evaluated the effect of G gene deletion on the cytotoxicity and transgene expression level of the RV vector. We compared the characteristics of the propagation-competent RV vector (rHEP5.0-CVSG-mRFP) and the G-deleted RV vector (rHEP5.0-ΔG-mRFP), both of which are based on the attenuated HEP-Flury strain and express monomeric red fluorescent protein (mRFP) as a transgene. rHEP5.0-ΔG-mRFP showed lower cytotoxicity than rHEP5.0-CVSG-mRFP, and within 16 days of infection we found no change in the basic electrophysiological properties of neurons infected with the rHEP5.0-ΔG-mRFP. The mRFP expression level of rHEP5.0-ΔG-mRFP was much higher than that of rHEP5.0-CVSG-mRFP, and 3 days after infection the retrogradely infected neurons were clearly visualized by the expressed fluorescent protein without any staining. This may be due to the low cytotoxicity and/or the presumed change in the polymerase gene (L) expression level of the G-deleted RV vector. Although the mechanisms remains to be clarified, the results of this study indicate that deletion of the G gene greatly improves the usability of the RV vector for studying the organization and function of the neural circuits by decreasing the cytotoxicity and increasing the transgene expression level.