Spatiotemporal development of the neuronal accumulation of amyloid precursor protein and the amyloid plaque formation in the brain of 3xTg-AD mice.

The amyloid plaque is a hallmark of Alzheimer's disease. The accumulation of the amyloid precursor protein (APP) in the neuronal structure is assumed to lead to amyloid plaque formation through the excessive production of ß-amyloid protein. To study the relationship between the neuronal accumulation of APP and amyloid plaque formation, we histologically analyzed their development in the different brain regions in 3xTg-AD mice, which express Swedish mutated APP (APPSWE) in the neurons. Observation throughout the brain revealed APPSWE-positive somata in the broad regions. Quantitative model analysis showed that the somatic accumulation of APPSWE developed firstly in the hippocampus from a very early age (<1 month) and proceeded slower in the isocortex. In line with this, the hippocampus was the first region to form amyloid plaques at the age of 9-12 months, while amyloid plaques were rarely observed in the isocortex. Females had more APPSWE-positive somata and plaques than males. Furthermore, amyloid plaques were observed in the lateral septum and pontine grey, which did not contain APPSWE-positive somata but only the APPSWE-positive fibers. These results suggested that neuronal accumulation of APPSWE, both in somatodendritic and axonal domains, is closely related to the formation of amyloid plaques.

Multiple factors contribute to flight behaviors during fear conditioning.

Shifting defensive mode from one to another by the imminence of threat is crucial for survival. The transition of defensive mode from freezing to flight is observed during the modified fear conditioning, however, the flight during fear conditioning is not well characterized. To characterize the flight behaviors during the fear conditioning, we conducted experiments in male mice focusing on the influence of the context, the intensity of the unconditioned stimulus and conditioned stimulus (CS), the schedule of conditioning, and the state of the subject. Flight behaviors triggered by salient CS showed characteristics of fear-potentiated defensive behaviors depending on the conditioned context, while repetitive conditioning enhanced the expression of the flight and developed an association between the CS and the flight. The salient auditory stimulus was the primary factor to trigger flight behaviors. Also, the spaced conditioning increased the expression of flight behaviors. Taken together, the flight behavior during fear conditioning is not a simple conditioned response nor simple fear-potentiated behavior, but a complicated mixture of multiple components of defensive behaviors. The transition of defensive mode could be induced by the integration of multiple innate and learned components of fear or anxiety.

Pain-related neuronal ensembles in the primary somatosensory cortex contribute to hyperalgesia and anxiety.

The mechanism by which acute pain or itch information at the periphery is processed in the primary somatosensory cortex (S1) remains unclear. To elucidate this, we used a viral-mediated targeted-recombination-in-active population system to target S1 neuronal ensembles that are active during pain or itch sensations. We induced the expression of excitatory or inhibitory designer receptors exclusively activated by designer drugs in pain- or itch-related S1 neurons. We identified neuronal populations in mice that regulate the sensory components of pain and itch in the S1 hind paw region. Notably, the neuronal circuit between pain-related S1 neurons and the parafascicular nucleus contributed to hyperalgesia and anxiety-like behavior. We propose that S1 plays an essential role in sensory and affective responses to noxious stimuli, such as pain.

Transthyretin Is Commonly Upregulated in the Hippocampus of Two Stress-Induced Depression Mouse Models.

Chronic stress can affect gene expression in the hippocampus, which alters neural and cerebrovascular functions, thereby contributing to the development of mental disorders such as depression. Although several differentially expressed genes in the depressed brain have been reported, gene expression changes in the stressed brain remain underexplored. Therefore, this study examines hippocampal gene expression in two mouse models of depression induced by forced swim stress (FSS) and repeated social defeat stress (R-SDS). Transthyretin (Ttr) was commonly upregulated in the hippocampus of both mouse models, as determined by microarray, RT-qPCR, and Western blot analyses. Evaluation of the effects of overexpressed Ttr in the hippocampus using adeno-associated virus-mediated gene transfer revealed that TTR overexpression induced depression-like behavior and upregulation of Lcn2 and several proinflammatory genes (Icam1 and Vcam1) in the hippocampus. Upregulation of these inflammation-related genes was confirmed in the hippocampus obtained from mice vulnerable to R-SDS. These results suggest that chronic stress upregulates Ttr expression in the hippocampus and that Ttr upregulation may be involved in the induction of depression-like behavior.

Modified Fear Conditioning for Inducing Flight Behaviors in Mice.

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active defensive behavior, such as jumping or rapid darting, is expressed under high threat imminence or actual threat, whereas passive defensive behavior, such as freezing, is expressed when the threat is predicted, but the threat imminence is relatively low. In classical fear conditioning, subjects typically exhibit freezing as a conditioned defensive response, with little expression of active defensive behavior in most cases. Here, we introduce a modified fear conditioning procedure for mice to observe the transition from freezing to flight and vice versa, involving five repetitive pairings of conditioned stimuli (CS; continuous tone, 8 kHz, 95 dB SPL (sound pressure levels)) and unconditioned stimuli (US; foot shock, 0.4 or 0.9 mA, 1.0 s) over two days. This modified fear conditioning procedure requires a relatively large number of conditioning sessions and conditioning days but does not necessitate a high-intensity foot shock for modest expression of flight behavior. Using the same context for conditioning and salient CS presentations is essential to elicit flight behaviors. This modified fear conditioning procedure is a reliable method for observing active defensive behaviors in mice, providing an opportunity to elucidate the fine mechanisms and characteristics of such behaviors in a fearful context.

[Characterization of the dorsal raphe-periaqueductal grey DAT neurons innervating onto the extended amygdala].

It has been known that a number of tyrosine hydroxylase (TH)-positive neurons, which are regarded as dopaminergic (DA) neurons, exist in the dorsal raphe (DR). These DA neurons in the DR and periaqueductal gray (PAG) region (DADR-PAG neurons) are thought to belong to the A10 cluster, which is known to be heterogeneous. This DA population projects to the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) and has been reported to modulate various affective behaviors. The DA transporter (DAT) neurons, which are well overlapping with DA neurons, in the DR-PAG region are also expected to be heterogeneous. However, even though the heterogeneity of DA/DATDR-PAG neurons has been suggested, the characteristics of each DA/DATDR-PAG neuron subpopulation are not well investigated. In this paper, we summarize the previous reports investigating the heterogeneity of DA/DATDR-PAG neurons and the functional importance of DA/DATDR-PAG neurons on various affective behaviors and introduce our recent findings that DATDR-PAG neurons consist of two subpopulations: TH+/vasoactive intestinal peptide (VIP)- putative DA neurons and TH-/VIP+ putative glutamatergic neurons.

5-HT and α-m-5-HT attenuate excitatory synaptic transmissions onto the lateral amygdala principal neurons via presynaptic 5-HT1B receptors.

Accumulating evidence suggests that the serotonergic (5-HT) system in the amygdala has significant effects on affective states. Dysregulation of the 5-HT system in the basolateral amygdaloid complex causes affective disorders. To search for therapeutic targets, subtype specification of 5-HT receptors is crucial. The present study was undertaken to identify the 5-HT receptor subtype responsible for the 5-HT-mediated suppression of excitatory transmission to principal neurons (PNs) in the lateral amygdala (LA). Whole-cell recordings were performed to record excitatory post synaptic currents (EPSCs) in acute rat brain slices. We confirmed that 5-HT and α-m-5-HT, a broad 5-HT2 receptor agonist, attenuated EPSCs in LA PNs. The extent of suppressions by 5-HT and α-m-5-HT remained unchanged in the presence of ritanserin, a broad 5-HT2 receptor antagonist. In the presence of NAS-181, a selective 5-HT1B receptor antagonist, the extent of EPSC suppressions by 5-HT and α-m-5-HT was diminished. CP93129, a selective 5-HT1B receptor agonist, attenuated EPSCs in LA PNs, and this effect was abolished in the presence of NAS-181. Additionally, the paired-pulse ratio of EPSCs was increased by CP93129. Thus, our results indicate that 5-HT and α-m-5-HT attenuate excitatory transmissions to LA PNs via presynaptic 5-HT1B receptors.

Vocalization during agonistic encounter in Mongolian gerbils: Impact of sexual experience.

Behaviors and vocalizations associated with aggression are essential for animals to survive, reproduce, and organize social hierarchy. Mongolian gerbils (Meriones unguiculatus) are highly aggressive and frequently emit calls. We took advantage of these features to study the relationship between vocalizations and aggressive behaviors in virgin and sexually experienced male and female Mongolian gerbils through the same-sex resident-intruder test. Both sexes of resident gerbils exhibited aggressive responses toward intruders. Multiparous females exhibited the most aggressive responses among the four groups. We also confirmed two groups of vocalizations during the encounters: high-frequency (>24.6 kHz) and low-frequency (<24.6 kHz). At the timing of high-frequency vocalizations observed during the tests, the vast majority (96.2%) of the behavioral interactions were non-agonistic. While, at the timing of low-frequency vocalizations observed during the tests, around half (45%) of the behavioral interactions were agonistic. Low-frequency vocalizations were observed mainly during encounters in which multiparous females were involved. These results suggest that high- and low-frequency vocalizations relate to non-agonistic and agonistic interactions, respectively. In addition to affecting aggressive behavior, sexual experience also affects vocalization during encounters. These findings provide new insights into the modulatory effects of sex and sexual experience on vocalizations during agonistic encounters.

Histochemical Characterization of the Dorsal Raphe-Periaqueductal Grey Dopamine Transporter Neurons Projecting to the Extended Amygdala.

The dorsal raphe (DR) nucleus contains many tyrosine hydroxylase (TH)-positive neurons which are regarded as dopaminergic (DA) neurons. These DA neurons in the DR and periaqueductal gray (PAG) region (DADR-PAG neurons) are a subgroup of the A10 cluster, which is known to be heterogeneous. This DA population projects to the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) and has been reported to modulate various affective behaviors. To characterize, the histochemical features of DADR-PAG neurons projecting to the CeA and BNST in mice, the current study combined retrograde labeling with Fluoro-Gold (FG) and histological techniques, focusing on TH, dopamine transporter (DAT), vasoactive intestinal peptide (VIP), and vesicular glutamate transporter 2 (VGlut2). To identify putative DA neurons, DAT-Cre::Ai14 mice were used. It was observed that DATDR-PAG neurons consisted of the following two subpopulations: TH+/VIP- and TH-/VIP+ neurons. The DAT+/TH-/VIP+ subpopulation would be non-DA noncanonical DAT neurons. Anterograde labeling of DATDR-PAG neurons with AAV in DAT-Cre mice revealed that the fibers exclusively innervated the lateral part of the CeA and the oval nucleus of the BNST. Retrograde labeling with FG injections into the CeA or BNST revealed that the two subpopulations similarly innervated these regions. Furthermore, using VGlut2-Cre::Ai14 mice, it was turned out that the TH-/VIP+ subpopulations innervating both CeA and BNST were VGlut2-positive neurons. These two subpopulations of DATDR-PAG neurons, TH+/VIP- and TH-/VIP+, might differentially interfere with the extended amygdala, thereby modulating affective behaviors.

Auditory cortical activity elicited by infrared laser irradiation from the outer ear in Mongolian gerbils.

Infrared neural stimulation has been studied for its potential to replace an electrical stimulation of a cochlear implant. No studies, however, revealed how the technic reliably evoke auditory cortical activities. This research investigated the effects of cochlear laser stimulation from the outer ear on auditory cortex using brain imaging of activity-dependent changes in mitochondrial flavoprotein fluorescence signal. An optic fiber was inserted into the gerbil's ear canal to stimulate the lateral side of the cochlea with an infrared laser. Laser stimulation was found to activate the identified primary auditory cortex. In addition, the temporal profile of the laser-evoked responses was comparable to that of the auditory responses. Our results indicate that infrared laser irradiation from the outer ear has the capacity to evoke, and possibly manipulate, the neural activities of the auditory cortex and may substitute for the present cochlear implants in future.

Serotonergic control of GABAergic inhibition in the lateral amygdala.

Much evidence implicates the serotonergic regulation of the amygdala in anxiety. Thus the present study was undertaken to characterize the influence of serotonin (5-HT) on principal neurons (PNs) of the rat lateral amygdala (LA), using whole cell recordings in vitro. Because inhibition is a major determinant of PN activity, we focused on the control of GABAergic transmission by 5-HT. IPSCs were elicited by local electrical stimulation of LA in the presence of glutamate receptor antagonists. We found that 5-HT reduces GABAA inhibitory postsynaptic currents (IPSCs) via presynaptic 5-HT1B receptors. While the presynaptic inhibition of GABA release also attenuated GABAB currents, this effect was less pronounced than for GABAA currents because 5-HT also induced a competing postsynaptic enhancement of GABAB currents. That is, GABAB currents elicited by pressure application of GABA or baclofen were enhanced by 5-HT. In addition, we obtained evidence suggesting that 5-HT differentially regulates distinct subsets of GABAergic synapses. Indeed, GABAA IPSCs were comprised of two components: a relatively 5-HT-insensitive IPSC that had a fast time course and a 5-HT-sensitive component that had a slower time course. Because the relative contribution of these two components varied depending on whether neurons were recorded at proximity versus at a distance from the stimulating electrodes, we speculate that distinct subtypes of local-circuit cells contribute the two contingents of GABAergic synapses. Overall, our results indicate that 5-HT is a potent regulator of synaptic inhibition in LA.NEW & NOTEWORTHY We report that 5-HT, acting via presynaptic 5-HT1B receptors, attenuates GABAA IPSCs by reducing GABA release in the lateral amygdala (LA). In parallel, 5-HT enhances GABAB currents postsynaptically, such that GABAB inhibitory postsynaptic currents (IPSCs) are relatively preserved from the presynaptic inhibition of GABA release. We also found that the time course of 5-HT-sensitive and -insensitive GABAA IPSCs differ. Together, these results indicate that 5-HT is a potent regulator of synaptic inhibition in LA.

Three forebrain structures directly inform the auditory midbrain of echolocating bats.

Echolocating bats emit various types of vocalizations for navigation and communication, and need to pay attention to vocal sounds. Projections from forebrain centers to auditory centers are involved in the attention to vocalizations, with the inferior colliculus (IC) being the main target of the projections. Here, using a retrograde tracer, we demonstrate that three forebrain structures, namely, the medial prefrontal cortex (mPFC), amygdala, and auditory cortex (AC), send direct descending projections to the central nucleus of IC. We found that all three structures projected to the bilateral IC. A comparison of the patterns of retrogradely labeled cells across animals suggests that the ipsilateral AC-IC projection is topographically organized, whereas mPFC-IC or amygdala-IC projections did not show clear topographic organization. Together with evidence from previous studies, these results suggest that three descending projections to the IC form loops between the forebrain and IC to make attention to various vocal sounds.

Hearing sensitivity evaluated by the auditory brainstem response in Miniopterus fuliginosus.

This study evaluated the hearing sensitivity of Miniopterus fuliginosus, a frequency-modulating (FM) bat species, by measuring the auditory brainstem responses in the inferior colliculus. The average audiogram was U-shaped. The mean threshold decreased gradually as the frequency increased from 16 to 40 kHz and then decreased rapidly as the frequency reached 46 kHz, with the peak sensitivity occurring at the terminal portion of the echolocation pulse between frequencies of 44 and 56 kHz. The shape of audiogram of M. fuliginosus is consistent with other FM bats, and is compared with its vocalization behavior.

Organization of subcortical auditory nuclei of Japanese house bat (Pipistrellus abramus) identified with cytoarchitecture and molecular expression.

The auditory system of echolocating bats shows remarkable specialization likely related to analyzing echoes of sonar pulses. However, significant interspecies differences have been observed in the organization of auditory pathways among echolocating bats, and the homology of auditory nuclei with those of non-echolocating species has not been established. Here, in order to establish the homology and specialization of auditory pathways in echolocating bats, the expression of markers for glutamatergic, GABAergic, and glycinergic phenotypes in the subcortical auditory nuclei of Japanese house bat (Pipistrellus abramus) was evaluated. In the superior olivary complex, we identified the medial superior olive and superior paraolivary nuclei as expressing glutamatergic and GABAergic phenotypes, respectively, suggesting these nuclei are homologous with those of rodents. In the nuclei of the lateral lemniscus (NLL), the dorsal nucleus was found to be purely GABAergic, the intermediate nucleus was a mixture of glutamatergic and inhibitory neurons, the compact part of the ventral nucleus was purely glycinergic, and the multipolar part of the ventral nucleus expressed both GABA and glycine. In the inferior colliculus (IC), the central nucleus was found to be further subdivided into dorsal and ventral parts according to differences in the density of terminals and the morphology of large GABAergic neurons, suggesting specialization to sonar pulse structure. Medial geniculate virtually lacked GABAergic neurons, suggesting that the organization of the tectothalamic pathway is similar with that of rodents. Taken together, our findings revealed that specialization primarily occurs with regard to nuclei size and organization of the NLL and IC.

Organization of projection from brainstem auditory nuclei to the inferior colliculus of Japanese house bat (Pipistrellus abramus).

OBJECTIVES: Echolocating bats show remarkable specialization which is related to analysis of echoes of biosonars in subcortical auditory brainstem pathways. The inferior colliculus (IC) receives inputs from all lower brainstem auditory nuclei, i.e., cochlear nuclei, nuclei of the lateral lemniscus, and superior olivary complex, and create de novo responses to sound, which is considered crucial for echolocation. Inside the central nucleus of the IC (ICC), small domains which receive specific combination of extrinsic inputs are the basis of integration of sound information. In addition to extrinsic inputs, each domain is interconnected by local IC neurons but the cell types related to the interconnection are not well-understood. The primary objective of the current study is to examine whether the ascending inputs are reorganized and terminate in microdomains inside the ICC. METHODS: We made injection of a retrograde tracer into different parts of the ICC, and analyzed distribution of retrogradely labeled cells in the auditory brainstem of Japanese house bat (Pipistrellus abramus). RESULTS: Pattern of ascending projections from brainstem nuclei was similar to other bat species. Percentages of labeled cells in several nuclei were correlated each other. Furthermore, within the IC, we identified that large GABAergic (LG) and glutamatergic neurons made long-range connection. CONCLUSIONS: Synaptic organization of IC of Japanese house bat shows specialization which is likely to relate for echolocation. Input nuclei to the IC make clusters which terminate in specific part of the ICC, implying the presence of microdomains. LG neurons have roles for binding IC microdomains.

Acoustic characteristics used by Japanese macaques for individual discrimination.

The vocalizations of primates contain information about speaker individuality. Many primates, including humans, are able to distinguish conspecifics based solely on vocalizations. The purpose of this study was to investigate the acoustic characteristics used by Japanese macaques in individual vocal discrimination. Furthermore, we tested human subjects using monkey vocalizations to evaluate species specificity with respect to such discriminations. Two monkeys and five humans were trained to discriminate the coo calls of two unfamiliar monkeys. We created a stimulus continuum between the vocalizations of the two monkeys as a set of probe stimuli (whole morph). We also created two sets of continua in which only one acoustic parameter, fundamental frequency (f0) or vocal tract characteristic (VTC), was changed from the coo call of one monkey to that of another while the other acoustic feature remained the same (f0 morph and VTC morph, respectively). According to the results, the reaction times both of monkeys and humans were correlated with the morph proportion under the whole morph and f0 morph conditions. The reaction time to the VTC morph was correlated with the morph proportion in both monkeys, whereas the reaction time in humans, on average, was not correlated with morph proportion. Japanese monkeys relied more consistently on VTC than did humans for discriminating monkey vocalizations. Our results support the idea that the auditory system of primates is specialized for processing conspecific vocalizations and suggest that VTC is a significant acoustic feature used by Japanese macaques to discriminate conspecific vocalizations.

Role of vocal tract characteristics in individual discrimination by Japanese macaques (Macaca fuscata).

The Japanese macaque (Macaca fuscata) exhibits a species-specific communication sound called the "coo call" to locate group members and maintain within-group contact. Monkeys have been demonstrated to be capable of discriminating between individuals based only on their voices, but there is still debate regarding how the fundamental frequencies (F0) and filter properties of the vocal tract characteristics (VTC) contribute to individual discrimination in nonhuman primates. This study was performed to investigate the acoustic keys used by Japanese macaques in individual discrimination. Two animals were trained with standard Go/NoGo operant conditioning to distinguish the coo calls of two unfamiliar monkeys. The subjects were required to continue depressing a lever until the stimulus changed from one monkey to the other. The test stimuli were synthesized by combining the F0s and VTC from each individual. Both subjects released the lever when the VTC changed, whereas they did not when the F0 changed. The reaction times to the test stimuli were not significantly different from that to the training stimuli that shared the same VTC. Our data suggest that vocal tract characteristics are important for the identification of individuals by Japanese macaques.