An intra-oral flavor detection task in freely moving mice.

Flavor plays a critical role in the pleasure of food. Flavor research has mainly focused on human subjects and revealed that many brain regions are involved in flavor perception. However, animal models for elucidating the mechanisms of neural circuits are lacking. Herein, we demonstrate the use of a novel behavioral task in which mice are capable of flavor detection. When the olfactory pathways of the mice were blocked, they could not perform the task. However, behavioral accuracy was not affected when the gustatory pathway was blocked by benzocaine. These results indicate that the mice performed this detection task mainly based on the olfaction. We conclude that this novel task can contribute to research on the neural mechanisms of flavor perception.

Enhancement of Haloperidol-Induced Catalepsy by GPR143, an L-Dopa Receptor, in Striatal Cholinergic Interneurons.

Dopamine neurons play crucial roles in pleasure, reward, memory, learning, and fine motor skills and their dysfunction is associated with various neuropsychiatric diseases. Dopamine receptors are the main target of treatment for neurologic and psychiatric disorders. Antipsychotics that antagonize the dopamine D2 receptor (DRD2) are used to alleviate the symptoms of these disorders but may also sometimes cause disabling side effects such as parkinsonism (catalepsy in rodents). Here we show that GPR143, a G-protein-coupled receptor for L-3,4-dihydroxyphenylalanine (L-DOPA), expressed in striatal cholinergic interneurons enhances the DRD2-mediated side effects of haloperidol, an antipsychotic agent. Haloperidol-induced catalepsy was attenuated in male Gpr143 gene-deficient (Gpr143-/y ) mice compared with wild-type (Wt) mice. Reducing the endogenous release of L-DOPA and preventing interactions between GPR143 and DRD2 suppressed the haloperidol-induced catalepsy in Wt mice but not Gpr143-/y mice. The phenotypic defect in Gpr143-/y mice was mimicked in cholinergic interneuron-specific Gpr143-/y (Chat-cre;Gpr143flox/y ) mice. Administration of haloperidol increased the phosphorylation of ribosomal protein S6 at Ser240/244 in the dorsolateral striatum of Wt mice but not Chat-cre;Gpr143flox/y mice. In Chinese hamster ovary cells stably expressing DRD2, co-expression of GPR143 increased cell surface expression level of DRD2, and L-DOPA application further enhanced the DRD2 surface expression. Shorter pauses in cholinergic interneuron firing activity were observed after intrastriatal stimulation in striatal slice preparations from Chat-cre;Gpr143flox/y mice compared with those from Wt mice. Together, these findings provide evidence that GPR143 regulates DRD2 function in cholinergic interneurons and may be involved in parkinsonism induced by antipsychotic drugs.

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.

Cellular Profiles of Prodynorphin and Preproenkephalin mRNA-Expressing Neurons in the Anterior Olfactory Tubercle of Mice.

The olfactory tubercle (OT) is a striatal region that receives olfactory inputs. mRNAs of prodynorphin (Pdyn) and preproenkephalin (Penk), precursors of dynorphins and enkephalins, respectively, are strongly expressed in the striatum. Both produce opioid peptides with various physiological effects such as pain relief and euphoria. Recent studies have revealed that OT has anatomical and cytoarchitectonic domains that play different roles in odor-induced motivated behavior. Neuronal subtypes of the OT can be distinguished by their expression of the dopamine receptors D1 (Drd1) and D2 (Drd2). Here, we addressed whether and which type of opioid peptide precursors the D1- and D2-expressing neurons in the OT express. We used multiple fluorescence in situ hybridization for mRNAs of the opioid precursors and dopamine receptors to characterize mouse OT neurons. Pdyn was mainly expressed by Drd1-expressing cells in the dense cell layer (DCL) of the OT, whereas Penk was expressed primarily by Drd2-expressing cells in the DCL. We also confirmed the presence of a larger population of Pdyn-Penk-Drd1 co-expressing cells in the DCL of the anteromedial OT compared with the anterolateral OT. These observations will help understand whether and how dynorphins and enkephalins in the OT are involved in diverse odor-induced motivated behaviors.

Multifaceted roles of orexin neurons in mediating methamphetamine-induced changes in body temperature and heart rate.

Methamphetamine (METH), which is used to improve the alertness of narcoleptic patients, elicits autonomic physiological responses such as increases in body temperature, blood pressure and heart rate. We have shown that orexin synthesizing neurons, which have an important role in maintaining wakefulness, greatly contribute to the regulation of cardiovascular and thermoregulatory function. This regulation is partly mediated by glutamatergic as well as orexinergic signalling from the orexin neurons. These signals may also be involved in the autonomic response elicited by METH. This study aimed to determine if loss of either orexin or glutamate in orexin neurons would affect METH-induced changes in heart rate and body temperature. We used transgenic mice in which the vesicular glutamate transporter 2 gene was disrupted selectively in orexin-producing neurons (ORX;vGT2-KO), prepro-orexin knockout mice (ORX-KO), and control wild type mice (WT). We measured body temperature, heart rate and locomotor activity with a pre-implanted telemetry probe and compared the effect of METH (0.5, 2 and 5 mg/kg i.p.) on these parameters between these three groups. A low dose of METH induced hyperthermia and tachycardia responses in ORX;vGT2-KO mice, which were significant compared to ORX-KO and WT mice. The highest dose of METH induced hypothermia and bradycardia in ORX-KO mice, however, it induced hyperthermia in both WT and ORX;vGT2-KO mice. These results suggest that glutamate and orexin from orexin neurons have differential roles in mediating METH-induced changes in body temperature and heart rate.

Autistic-Like Behavior and Impairment of Serotonin Transporter and AMPA Receptor Trafficking in N-Ethylmaleimide Sensitive Factor Gene-Deficient Mice.

Autism spectrum disorder (ASD), characterized by profound impairment in social interactions and communication skills, is the most common neurodevelopmental disorder. Many studies on the mechanisms underlying the development of ASD have focused on the serotonergic system; however, these studies have failed to completely elucidate the mechanisms. We previously identified N-ethylmaleimide-sensitive factor (NSF) as a new serotonin transporter (SERT)-binding protein and described its importance in SERT membrane trafficking and uptake in vitro. In the present study, we generated Nsf +/- mice and investigated their behavioral, neurotransmitter, and neurophysiological phenotypes in vivo. Nsf +/- mice exhibited abnormalities in sociability, communication, repetitiveness, and anxiety. Additionally, Nsf loss led to a decrease in membrane SERT expression in the raphe and accumulation of glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors at the synaptic membrane surface in the hippocampal CA1 region. We found that postsynaptic density and long-term depression were impaired in the hippocampal CA1 region of Nsf +/- mice. Taken together, these findings demonstrate that NSF plays a role in synaptic plasticity and glutamatergic and serotonergic systems, suggesting a possible mechanism by which the gene is linked to the pathophysiology of autistic behaviors.

Dopamine facilitates associative memory encoding in the entorhinal cortex.

Mounting evidence shows that dopamine in the striatum is critically involved in reward-based reinforcement learning1,2. However, it remains unclear how dopamine reward signals influence the entorhinal-hippocampal circuit, another brain network that is crucial for learning and memory3-5. Here, using cell-type-specific electrophysiological recording6, we show that dopamine signals from the ventral tegmental area and substantia nigra control the encoding of cue-reward association rules in layer 2a fan cells of the lateral entorhinal cortex (LEC). When mice learned novel olfactory cue-reward associations using a pre-learned association rule, spike representations of LEC fan cells grouped newly learned rewarded cues with a pre-learned rewarded cue, but separated them from a pre-learned unrewarded cue. Optogenetic inhibition of fan cells impaired the learning of new associations while sparing the retrieval of pre-learned memory. Using fibre photometry, we found that dopamine sends novelty-induced reward expectation signals to the LEC. Inhibition of LEC dopamine signals disrupted the associative encoding of fan cells and impaired learning performance. These results suggest that LEC fan cells represent a cognitive map of abstract task rules, and that LEC dopamine facilitates the incorporation of new memories into this map.

Distribution of mRNA for GPR143, a receptor of 3,4-L-dihydroxyphenylalanine, and of immunoreactivities for nicotinic acetylcholine receptors in the nigrostriatal and mesolimbic regions.

Nicotine exerts its reinforcing actions by activating nicotinic acetylcholine receptors (nAChRs), but the detailed mechanisms remain unclear. Nicotine releases 3, 4-dihydroxyphenylalanine (DOPA), a neurotransmitter candidate in the central nervous system. Here, we investigated the distribution of GPR143, a receptor of DOPA, and nAChR subunits in the nigrostriatal and mesolimbic regions. We found GPR143 mRNA-positive cells in the striatum and nucleus accumbens. Some of them were surrounded by tyrosine hydroxylase (TH)-immunoreactive fibers. There were some GPR143 mRNA-positive cells coexpressing TH, and nAChR subunit α4 or α7 in the substantia nigra and ventral tegmental area. These findings suggest that DOPA-GPR143 signaling may be involved in the nicotine action in the nigrostriatal and mesolimbic dopaminergic systems.

Hypothetical Roles of the Olfactory Tubercle in Odor-Guided Eating Behavior.

Olfaction plays an important role in the evaluation, motivation, and palatability of food. The chemical identity of odorants is coded by a spatial combination of activated glomeruli in the olfactory bulb, which is referred to as the odor map. However, the functional roles of the olfactory cortex, a collective region that receives axonal projections from the olfactory bulb, and higher olfactory centers in odor-guided eating behaviors are yet to be elucidated. The olfactory tubercle (OT) is a component of the ventral striatum and forms a node within the mesolimbic dopaminergic pathway. Recent studies have revealed the anatomical domain structures of the OT and their functions in distinct odor-guided motivated behaviors. Another component of the ventral striatum, the nucleus accumbens, is well known for its involvement in motivation and hedonic responses for foods, which raises the possibility of functional similarities between the OT and nucleus accumbens in eating. This review first summarizes recent findings on the domain- and neuronal subtype-specific roles of the OT in odor-guided motivated behaviors and then proposes a model for the regulation of eating behaviors by the OT.

Tuning of olfactory cortex ventral tenia tecta neurons to distinct task elements of goal-directed behavior.

The ventral tenia tecta (vTT) is a component of the olfactory cortex and receives both bottom-up odor signals and top-down signals. However, the roles of the vTT in odor-coding and integration of inputs are poorly understood. Here, we investigated the involvement of the vTT in these processes by recording the activity from individual vTT neurons during the performance of learned odor-guided reward-directed tasks in mice. We report that individual vTT cells are highly tuned to a specific behavioral epoch of learned tasks, whereby the duration of increased firing correlated with the temporal length of the behavioral epoch. The peak time for increased firing among recorded vTT cells encompassed almost the entire temporal window of the tasks. Collectively, our results indicate that vTT cells are selectively activated during a specific behavioral context and that the function of the vTT changes dynamically in a context-dependent manner during goal-directed behaviors.

Region- and neuronal-subtype-specific expression of Na,K-ATPase alpha and beta subunit isoforms in the mouse brain.

Na,K-ATPase is a ubiquitous molecule contributing to the asymmetrical distribution of Na+ and K+ ions across the plasma membrane and maintenance of the membrane potential, a prerequisite of neuronal activity. Na,K-ATPase comprises three subunits (α, ß, and FXYD). The α subunit has four isoforms in mice, with three of them (α1, α2, and α3) expressed in the brain. However, the functional and biological significances of the different brain isoforms remain to be fully elucidated. Recent studies have revealed the association of Atp1a3, a gene encoding α3 subunit, with neurological disorders. To map the cellular distributions of the α subunit isoforms and their coexpression patterns, we evaluated the mRNA expression of Atp1a1, Atp1a2, and Atp1a3 by in situ hybridization in the mouse brain. Atp1a1 and Atp1a3 were expressed in neurons, whereas Atp1a2 was almost exclusively expressed in glial cells. Most neurons coexpressed Atp1a1 and Atp1a3, with highly heterogeneous expression levels across the brain regions and neuronal subtypes. We identified parvalbumin (PV)-expressing GABAergic neurons in the hippocampus, somatosensory cortex, and retrosplenial cortex as an example of a neuronal subtype expressing low Atp1a1 and high Atp1a3. The expression of Atp1b isoforms was also heterogeneous across brain regions and cellular subtypes. The PV-expressing neurons expressed a high level of Atp1b1 and a low level of Atp1b2 and Atp1b3. These findings provide basic information on the region- and neuronal-subtype-dependent expression of Na,K-ATPase α and ß subunit isoforms, as well as a rationale for the selective involvement of neurons expressing high levels of Atp1a3 in neurological disorders.

GABAergic neurons in the olfactory cortex projecting to the lateral hypothalamus in mice.

Olfaction guides goal-directed behaviours including feeding. To investigate how central olfactory neural circuits control feeding behaviour in mice, we performed retrograde tracing from the lateral hypothalamus (LH), an important feeding centre. We observed a cluster of retrogradely labelled cells distributed in the posteroventral region of the olfactory peduncle. Histochemical analyses revealed that the majority of these retrogradely labelled projection neurons expressed glutamic acid decarboxylase 65/67 (GAD65/67), but not vesicular glutamate transporter 1 (VGluT1). We named this region containing GABAergic projection neurons the ventral olfactory nucleus (VON) to differentiate it from the conventional olfactory peduncle. VON neurons were less immunoreactive for DARPP-32, a striatal neuron marker, compared to neurons in the olfactory tubercle and nucleus accumbens, which distinguished the VON from the ventral striatum. Fluorescent labelling confirmed putative synaptic contacts between VON neurons and olfactory bulb projection neurons. Rabies-virus-mediated trans-synaptic labelling revealed that VON neurons received synaptic inputs from the olfactory bulb, other olfactory cortices, horizontal limb of the diagonal band, and prefrontal cortex. Collectively, these results identify novel GABAergic projection neurons in the olfactory cortex that may integrate olfactory sensory and top-down inputs and send inhibitory output to the LH, which may modulate odour-guided LH-related behaviours.

Opposing Roles of Dopamine Receptor D1- and D2-Expressing Neurons in the Anteromedial Olfactory Tubercle in Acquisition of Place Preference in Mice.

Olfaction induces adaptive motivated behaviors. Odors associated with food induce attractive behavior, whereas those associated with dangers induce aversive behavior. We previously reported that learned odor-induced attractive and aversive behaviors accompany activation of the olfactory tubercle (OT) in a domain- and cell type-specific manner. Odor cues associated with a sugar reward induced attractive behavior and c-fos expression in the dopamine receptor D1-expressing neurons (D1 neurons) in the anteromedial OT. In contrast, odor cues associated with electrical shock induced aversive behavior and c-fos expression in the pamine receptor D2-expressing neurons (D2 neurons) in the anteromedial OT, as well as the D1 neurons in the lateral OT. Here, we investigated whether the D1 and D2 neurons in the anteromedial OT play distinct roles in attractive or aversive behaviors, using optogenetic stimulation and real-time place preference (RTPP) tests. Mice expressing ChETA (ChR2/E123T)-enhanced yellow fluorescent protein (EYFP) in the D1 neurons in the anteromedial OT spent a longer time in the photo-stimulation side of the place preference chamber than the control mice expressing EYFP. On the other hand, upon optogenetic stimulation of the D2 neurons in the anteromedial OT, the mice spent a shorter time in the photo-stimulation side than the control mice. Local neural activation in the anteromedial OT during the RTPP tests was confirmed by c-fos mRNA expression. These results suggest that the D1 and D2 neurons in the anteromedial OT play opposing roles in attractive and aversive behaviors, respectively.

Functional development of olfactory tubercle domains during weaning period in mice.

Mammals shift their feeding habits from mother's milk to environmental foods postnatally. While this weaning process accompanies the acquisition of attractive behaviour toward environmental foods, the underlying neural mechanism for the acquisition is poorly understood. We previously found that adult mouse olfactory tubercle (OT), which belongs to the olfactory cortex and ventral striatum, has functional domains that represent odour-induced motivated behaviours, and that c-fos induction occurs mainly in the anteromedial domain of OT following learned odour-induced food seeking behaviour. To address the question whether the anteromedial OT domain is involved in the postnatal acquisition of food seeking behaviour, we examined OT development during weaning of mice. Whereas at postnatal day 15 (P15), all mice were attracted to lactating mothers, P21 mice were more attracted to familiar food pellets. Mapping of c-fos induction during food seeking and eating behaviours showed that while c-fos activation was observed across wide OT domains at P15, the preferential activation of c-fos in the anteromedial domain occurred at P21 and later ages. These results indicate that preferential c-fos activation in the anteromedial OT domain occurred concomitantly with the acquisition of attractive behaviour toward food, which suggests the importance of this domain in the weaning process.

Mapping of Learned Odor-Induced Motivated Behaviors in the Mouse Olfactory Tubercle.

An odor induces food-seeking behaviors when humans and animals learned to associate the odor with food, whereas the same odor elicits aversive behaviors following odor-danger association learning. It is poorly understood how central olfactory circuits transform the learned odor cue information into appropriate motivated behaviors. The olfactory tubercle (OT) is an intriguing area of the olfactory cortex in that it contains medium spiny neurons as principal neurons and constitutes a part of the ventral striatum. The OT is therefore a candidate area for participation in odor-induced motivated behaviors. Here we mapped c-Fos activation of medium spiny neurons in different domains of the mouse OT following exposure to learned odor cues. Mice were trained to associate odor cues to a sugar reward or foot shock punishment to induce odor-guided approach behaviors or aversive behaviors. Regardless of odorant types, the anteromedial domain of the OT was activated by learned odor cues that induced approach behaviors, whereas the lateral domain was activated by learned odor cues that induced aversive behaviors. In each domain, a larger number of dopamine receptor D1 type neurons were activated than D2 type neurons. These results indicate that specific domains of the OT represent odor-induced distinct motivated behaviors rather than odor stimuli, and raise the possibility that neuronal type-specific activation in individual domains of the OT plays crucial roles in mediating the appropriate learned odor-induced motivated behaviors. Significance statement: Although animals learn to associate odor cues with various motivated behaviors, the underlying circuit mechanisms are poorly understood. The olfactory tubercle (OT), a subarea of the olfactory cortex, also constitutes the ventral striatum. Here, we trained mice to associate odors with either reward or punishment and mapped odor-induced c-Fos activation in the OT. Regardless of odorant types, the anteromedial domain was activated by approach behavior-inducing odors, whereas the lateral domain was activated by aversive behavior-inducing odors. In each domain, dopamine receptor D1 neurons were preferentially activated over D2 neurons. The results indicate that specific OT domains represent odor-induced distinct motivated behaviors rather than odor types, and suggest the importance of neuronal type-specific activation in individual domains in mediating appropriate behaviors.

Rapid induction of granule cell elimination in the olfactory bulb by noxious stimulation in mice.

Elimination of granule cells (GCs) in the olfactory bulb (OB) is not a continuous event but is rather promoted during short time windows associated with the animal's behavior. We previously showed that apoptotic GC elimination is enhanced during food eating and subsequent rest or sleep, and that top-down inputs from the olfactory cortex (OC) to the OB during the postprandial period are the crucial signal promoting GC elimination. However, whether enhanced GC elimination occurs during behaviors other than postprandial behavior is not clear. Here, we investigated whether exposure to noxious stimulation promotes apoptotic GC elimination in mice. Mice were delivered a brief electrical foot shock, during and immediately after which they showed startle and fear responses. Surprisingly, the number of apoptotic GCs increased 2-fold within 10 min after the start of foot shock delivery. This enhancement of GC apoptosis was significantly suppressed by injection of the GABAA receptor agonist muscimol in the OC, despite these muscimol-injected mice showing similar behavioral responses by foot shock as control mice. These results indicate that GC elimination is promoted in foot shock-delivered mice within a short time period of startle and fear responses. They also indicate that OC activity plays a central role in the enhanced GC elimination during this period, as is also the case in GC elimination during the postprandial period.

Reorganization of neuronal circuits of the central olfactory system during postprandial sleep.

Plastic changes in neuronal circuits often occur in association with specific behavioral states. In this review, we focus on an emerging view that neuronal circuits in the olfactory system are reorganized along the wake-sleep cycle. Olfaction is crucial to sustaining the animals' life, and odor-guided behaviors have to be newly acquired or updated to successfully cope with a changing odor world. It is therefore likely that neuronal circuits in the olfactory system are highly plastic and undergo repeated reorganization in daily life. A remarkably plastic feature of the olfactory system is that newly generated neurons are continually integrated into neuronal circuits of the olfactory bulb (OB) throughout life. New neurons in the OB undergo an extensive selection process, during which many are eliminated by apoptosis for the fine tuning of neuronal circuits. The life and death decision of new neurons occurs extensively during a short time window of sleep after food consumption (postprandial sleep), a typical daily olfactory behavior. We review recent studies that explain how olfactory information is transferred between the OB and the olfactory cortex (OC) along the course of the wake-sleep cycle. Olfactory sensory input is effectively transferred from the OB to the OC during waking, while synchronized top-down inputs from the OC to the OB are promoted during the slow-wave sleep. We discuss possible neuronal circuit mechanisms for the selection of new neurons in the OB, which involves the encoding of olfactory sensory inputs and memory trace formation during waking and internally generated activities in the OC and OB during subsequent sleep. The plastic changes in the OB and OC are well coordinated along the course of olfactory behavior during wakefulness and postbehavioral rest and sleep. We therefore propose that the olfactory system provides an excellent model in which to understand behavioral state-dependent plastic mechanisms of the neuronal circuits in the brain.

Elimination of adult-born neurons in the olfactory bulb is promoted during the postprandial period.

Granule cells (GCs) in the mouse olfactory bulb (OB) continue to be generated in adulthood, with nearly half incorporated and the remainder eliminated. Here, we show that elimination of adult-born GCs is promoted during a short time window in the postprandial period. Under restricted feeding, the number of apoptotic GCs specifically increased within a few hours after the start of feeding. This enhanced GC apoptosis occurred in association with postprandial behaviors that included grooming, resting, and sleeping, and was particularly correlated with the length of postprandial sleep. Further, deprivation of olfactory sensory experience in the local OB area potentiated the extent of GC elimination in that area during the postprandial period. Sensory experience-dependent enhancement of GC elimination also occurred during postprandial period under natural feeding condition. These results suggest that extensive structural reorganization of bulbar circuitry occurs during the postprandial period, reflecting sensory experience during preceding waking period.

Compensation of depleted neuronal subsets by new neurons in a local area of the adult olfactory bulb.

In the olfactory bulb (OB), loss of preexisting granule cells (GCs) and incorporation of adult-born new GCs continues throughout life. GCs consist of distinct subsets. Here, we examined whether the loss and incorporation of GC subsets are coordinated in the OB. We classified GCs into mGluR2-expressing and -negative subsets and selectively ablated mGluR2-expressing GCs in a local area of the OB with immunotoxin-mediated cell ablation method. The density of mGluR2-expressing GCs showed considerable recovery within several weeks after the ablation. During recovery, an mGluR2-expressing new GC subset was preferentially incorporated over an mGluR2-negative new GC subset in the area of ablation, whereas the preferential incorporation was not observed in the intact area. The area-specific preferential incorporation of mGluR2-expressing new GCs occurred for BrdU analog- and retrovirus-labeled adult-born cells as well as for neonate-derived transplanted cells. The mGluR2-expressing new GCs in the ablated area were synaptically incorporated into the local bulbar circuit. The spine size of mGluR2-expressing new GCs in the ablated area was larger than that of those in the intact area. In contrast, mGluR2-negative new GCs did not show ablated area-specific spine enlargement. These results indicate that local OB areas have a mechanism to coordinate the loss and incorporation of GC subsets by compensatory incorporation of new GC subsets, which involves subset-specific cellular incorporation and subset-specific regulation of spine size.