Dopamine (DA) type 1 receptor (D1R) signaling in the striatum presumably regulates neuronal excitability and reward-related behaviors through PKA. However, whether and how D1Rs and PKA regulate neuronal excitability and behavior remain largely unknown. Here, we developed a phosphoproteomic analysis method to identify known and novel PKA substrates downstream of the D1R and obtained more than 100 candidate substrates, including Rap1 GEF (Rasgrp2). We found that PKA phosphorylation of Rasgrp2 activated its guanine nucleotide-exchange activity on Rap1. Cocaine exposure activated Rap1 in the nucleus accumbens in mice. The expression of constitutively active PKA or Rap1 in accumbal D1R-expressing medium spiny neurons (D1R-MSNs) enhanced neuronal firing rates and behavioral responses to cocaine exposure through MAPK. Knockout of Rap1 in the accumbal D1R-MSNs was sufficient to decrease these phenotypes. These findings demonstrate a novel DA-PKA-Rap1-MAPK intracellular signaling mechanism in D1R-MSNs that increases neuronal excitability to enhance reward-related behaviors.

cAMP responsive element binding protein 3-like 3 (CREB3L3), a transcription factor expressed in the liver and small intestine, governs fasting-response energy homeostasis. Tissue-specific CREB3L3 knockout mice have not been generated till date. To our knowledge, this is the first study using the one-step CRISPR/Cas9 system to generate CREB3L3 floxed mice and subsequently obtain liver- and small intestine-specific Creb3l3 knockout (LKO and IKO, respectively) mice. While LKO mice as well as global KO mice developed hypertriglyceridemia, LKO mice exhibited hypercholesterolemia in contrast to hypocholesterolemia in global KO mice. LKO mice demonstrated up-regulation of hepatic Srebf2 and its corresponding target genes. No phenotypic differences were observed between IKO and floxed mice. Severe liver injury was observed in LKO mice fed a methionine-choline deficient diet, a model for non-alcoholic steatohepatitis. These results provide new evidence regarding the hepatic CREB3L3 role in plasma triglyceride metabolism and hepatic and intestinal CREB3L3 contributions to cholesterol metabolism.

The hippocampus plays an important role in the formation of contextual memory between the environment and the rewarding effect of abused drugs. The dopaminergic neural transmission in the hippocampus seems to be critical for such memory. Using conditioned place preference in rats, we found that the protein level of the dopamine D(1) receptor and its prerequisite mRNA in the hippocampus increased in animals that showed a clear preference for the environment paired with cocaine. The increase was not a simple reflection of the repeated administration of cocaine. Instead, it is attributable to conditioning, because systematic contingency between drug administration and exposure to a particular environment was necessary for the increase. Furthermore, we found that the mRNA of the dopamine D(1) receptors increased in the granule cell layer of the dentate gyrus. These results suggest that the alteration of dopamine D(1) receptor in the hippocampus, especially in the dentate gyrus, is related to the induction of drug-induced contextual memory. The finding implicates the relevance of the dopaminergic signal transduction in the hippocampus to drug dependence.

Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.

The basic helix-loop-helix transcription factor Hand2 is essential for the proliferation and noradrenergic differentiation of sympathetic neuron precursors during development. Here we address the function of Hand2 in postmitotic, differentiated sympathetic neurons. Knockdown of endogenous Hand2 in cultured E12 chick sympathetic neurons by siRNA results in a significant (about 60%) decrease in the expression of the noradrenergic marker genes dopamine-beta-hydroxylase (DBH) and tyrosine hydroxylase (TH). In contrast, expression of the pan-neuronal genes TuJ1, HuC and SCG10 was not affected. To analyze the in vivo role of Hand2 in differentiated sympathetic neurons we used mice harboring a conditional Hand2-null allele and excised the gene by expression of Cre recombinase under control of the DBH promotor. Mouse embryos homozygous for Hand2 gene deletion showed decreased sympathetic neuron number and TH expression was strongly reduced in the residual neuron population. The in vitro Hand2 knockdown also enhances the CNTF-induced expression of the cholinergic marker genes vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT). Taken together, these findings demonstrate that the Hand2 transcription factor plays a key role in maintaining noradrenergic properties in differentiated neurons.

Parkinson's disease is characterized by loss of nigral dopaminergic neurons and presence of Lewy bodies, whose major component is alpha-synuclein. In the present study, we generated transgenic mice termed Syn130m that express truncated human alpha-synuclein (amino acid residue number: 1-130) in dopaminergic neurons. Notably, dopaminergic neurons were selectively diminished in the substantia nigra pars compacta of Syn130m, while transgenic mice that expressed comparable amount of full-length human alpha-synuclein did not develop such pathology. Therefore, the truncation of human alpha-synuclein seems to be primarily responsible for the loss of nigral dopaminergic neurons. The nigral pathology resulted in impairment of axon terminals in the striatum and concomitant decrease in striatal dopamine content. Behaviorally, spontaneous locomotor activities of Syn130m were reduced, but the abnormality was ameliorated by treatment with L-DOPA. The loss of nigral dopaminergic neurons was not progressive and seemed to occur during embryogenesis along with the onset of expression of the transgene. Our results indicate that truncated human alpha-synuclein is deleterious to the development and/or survival of nigral dopaminergic neurons.

Pelvic ganglia are derived from the sacral neural crest and contain both sympathetic and parasympathetic neurons. Various members of the neurotrophin and GDNF families of neurotrophic factors have been shown to play important roles in the development of a variety of peripheral sympathetic and parasympathetic neurons; however, to date, the role of these factors in the development of pelvic ganglia has been limited to postnatal and older ages. We examined the effects of NGF, NT-3, GDNF, neurturin and artemin on cell migration and neurite outgrowth from explants of the pelvic ganglia from embryonic and newborn mice grown on collagen gels, and correlated the responses with the immunohistochemical localization of the relevant receptors in fixed tissue.

The nigrostriatal pathway is of great importance for the execution of movements, especially in the context of Parkinson's disease. In research, analysis of this pathway often requires the application of severe animal experiments. Organotypic nigrostriatal slice cultures offer a resource-saving alternative to animal experiments for research on the nigrostriatal system.

Some diseases that are associated with dopamine deficiency are accompanied by psychiatric symptoms, including Parkinson's disease. However, the mechanism by which this occurs has not been clarified. Previous studies found that dopamine-deficient (DD) mice exhibited hyperactivity in a novel environment. This hyperactivity is improved by clozapine and donepezil, which are used to treat psychiatric symptoms associated with dopamine deficiency (PSDD). We considered that DD mice could be used to study PSDD. In the present study, we sought to identify the pharmacological mechanism of PSDD. We conducted locomotor activity tests by administering quetiapine and drugs that have specific actions on serotonin (5-hydroxytryptamine [5-HT]) receptors and muscarinic receptors. Changes in neuronal activity that were induced by drug administration in DD mice were evaluated by examining Fos immunoreactivity. Quetiapine suppressed hyperactivity in DD mice while the 5-HT1A receptor antagonist WAY100635 inhibited this effect. The number of Fos-positive neurons in the median raphe nucleus increased in DD mice that exhibited hyperactivity and was decreased by treatment with quetiapine and 5-HT1A receptor agonists. In conclusion, hyperactivity in DD mice was ameliorated by quetiapine, likely through 5-HT1A receptor activation. These findings suggest that 5-HT1A receptors may play a role in PSDD, and 5-HT1A receptor-targeting drugs may help improve PSDD.

Dopamine-deficient (DD) mice exhibit psychomotor hyperactivity that might be related to a decrease in muscarinic signaling. In the present study, muscarinic acetylcholine receptor M2 (CHRM2) density decreased in the cortex in DD mice. This is significant because cortical CHRM2 acts as an autoreceptor; therefore, changes in CHRM2 levels could alter acetylcholine in DD mice. We also found that the CHRM1/CHRM4 agonist xanomeline and CHRM2 agonist arecaidine propargyl ester tosylate inhibited hyperactivity in DD mice, suggesting that postsynaptic CHRM1 and CHRM2 and presynaptic CHRM2 may be involved in hyperactivity in DD 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.

Methylglyoxal (MG), one of the uremic toxins, is a highly reactive alpha-dicarbonyl compound. Recent clinical studies have demonstrated the close associations of cognitive impairment (CI) with plasma MG levels and presence of kidney dysfunction. Therefore, the present study aims to examine whether MG is a direct causative substance for CI development. Eight-week-old male Sprague-Dawley (SD) rats were divided into two groups: control (n = 9) and MG group (n = 10; 0.5% MG in drinking water), and fed a normal diet for 12 months. Cognitive function was evaluated by two behavioral tests (object exploration test and radial-arm maze test) in early (4-6 months of age) and late phase (7-12 months of age). Serum MG was significantly elevated in the MG group (495.8 ± 38.1 vs. 244.8 ± 28.2 nM; p < 0.001) at the end of study. The groups did not differ in cognitive function during the course of study. No time-course differences were found in oxidative stress markers between the two groups, while, antioxidants such as glutathione peroxidase and superoxide dismutase activities were significantly increased in the MG group compared to the control. Long-term MG administration to rats with normal kidney function did not cause CI. A counter-balanced activation of the systemic anti-oxidant system may offset the toxicity of MG in this model. Pathogenetic significance of MG for CI requires further investigation.

The development of gene therapy techniques to introduce transgenes that promote neuronal survival and protection provides effective therapeutic approaches for neurological and neurodegenerative diseases. Intramuscular injection of adenoviral and adeno-associated viral vectors, as well as lentiviral vectors pseudotyped with rabies virus glycoprotein (RV-G), permits gene delivery into motor neurons in animal models for motor neuron diseases. Recently, we developed a vector with highly efficient retrograde gene transfer (HiRet) by pseudotyping a human immunodeficiency virus type 1 (HIV-1)-based vector with fusion glycoprotein B type (FuG-B) or a variant of FuG-B (FuG-B2), in which the cytoplasmic domain of RV-G was replaced by the corresponding part of vesicular stomatitis virus glycoprotein (VSV-G). We have also developed another vector showing neuron-specific retrograde gene transfer (NeuRet) with fusion glycoprotein C type, in which the short C-terminal segment of the extracellular domain and transmembrane/cytoplasmic domains of RV-G was substituted with the corresponding regions of VSV-G. These two vectors afford the high efficiency of retrograde gene transfer into different neuronal populations in the brain. Here we investigated the efficiency of the HiRet (with FuG-B2) and NeuRet vectors for retrograde gene transfer into motor neurons in the spinal cord and hindbrain in mice after intramuscular injection and compared it with the efficiency of the RV-G pseudotype of the HIV-1-based vector. The main highlight of our results is that the HiRet vector shows the most efficient retrograde gene transfer into both spinal cord and hindbrain motor neurons, offering its promising use as a gene therapeutic approach for the treatment of motor neuron diseases.

Recently, by using a combination of two viral vectors, we developed a technique for pathway-selective and reversible synaptic transmission blockade, and successfully induced a behavioral deficit of dexterous hand movements in macaque monkeys by affecting a population of spinal interneurons. To explore the capacity of this technique to work in other pathways and species, and to obtain fundamental methodological information, we tried to block the crossed tecto-reticular pathway, which is known to control orienting responses to visual targets, in mice. A neuron-specific retrograde gene transfer vector with the gene encoding enhanced tetanus neurotoxin (eTeNT) tagged with enhanced green fluorescent protein (EGFP) under the control of a tetracycline responsive element was injected into the left medial pontine reticular formation. 7-17 days later, an adeno-associated viral vector with a highly efficient Tet-ON sequence, rtTAV16, was injected into the right superior colliculus. 5-9 weeks later, the daily administration of doxycycline (Dox) was initiated. Visual orienting responses toward the left side were impaired 1-4 days after Dox administration. Anti-GFP immunohistochemistry revealed that a number of neurons in the intermediate and deep layers of the right superior colliculus were positively stained, indicating eTeNT expression. After the termination of Dox administration, the anti-GFP staining returned to the baseline level within 28 days. A second round of Dox administration, starting from 28 days after the termination of the first Dox administration, resulted in the reappearance of the behavioral impairment. These findings showed that pathway-selective and reversible blockade of synaptic transmission also causes behavioral effects in rodents, and that the crossed tecto-reticular pathway clearly controls visual orienting behaviors.

Most neuronal types have a well-identified electrical phenotype. It is now admitted that a same phenotype can be produced using multiple biophysical solutions defined by ion channel expression levels. This argues that systems-level approaches are necessary to understand electrical phenotype genesis and stability. Midbrain dopaminergic (DA) neurons, although quite heterogeneous, exhibit a characteristic electrical phenotype. However, the quantitative genetic principles underlying this conserved phenotype remain unknown. Here we investigated the quantitative relationships between ion channels' gene expression levels in midbrain DA neurons using single-cell microfluidic qPCR. Using multivariate mutual information analysis to decipher high-dimensional statistical dependences, we unravel co-varying gene modules that link neurotransmitter identity and electrical phenotype. We also identify new segregating gene modules underlying the diversity of this neuronal population. We propose that the newly identified genetic coupling between neurotransmitter identity and ion channels may play a homeostatic role in maintaining the electrophysiological phenotype of midbrain DA neurons.

Increases in sodium concentrations ([Na+]) in body fluids elevate blood pressure (BP) by enhancing sympathetic nerve activity (SNA). However, the mechanisms by which information on increased [Na+] is translated to SNA have not yet been elucidated. We herein reveal that sympathetic activation leading to BP increases is not induced by mandatory high salt intakes or the intraperitoneal/intracerebroventricular infusions of hypertonic NaCl solutions in Nax-knockout mice in contrast to wild-type mice. We identify Nax channels expressed in specific glial cells in the organum vasculosum lamina terminalis (OVLT) as the sensors detecting increases in [Na+] in body fluids and show that OVLT neurons projecting to the paraventricular nucleus (PVN) are activated via acid-sensing ion channel 1a (ASIC1a) by H+ ions exported from Nax-positive glial cells. The present results provide an insight into the neurogenic mechanisms responsible for salt-induced BP elevations.

Pseudotyped lentiviral vectors give access to pathway-selective gene manipulation via retrograde transfer. Two types of such lentiviral vectors have been developed. One is the so-called NeuRet vector pseudotyped with fusion glycoprotein type E, which preferentially transduces neurons. The other is the so-called HiRet vector pseudotyped with fusion glycoprotein type B2, which permits gene transfer into both neurons and glial cells at the injection site. Although these vectors have been applied in many studies investigating neural network functions, it remains unclear which vector is more appropriate for retrograde gene delivery in the brain. To compare the gene transfer efficiency and inflammatory response of the NeuRet vs. HiRet vectors, each vector was injected into the striatum in macaque monkeys, common marmosets, and rats. It was revealed that retrograde gene delivery of the NeuRet vector was equal to or greater than that of the HiRet vector. Furthermore, inflammation characterized by microglial and lymphocytic infiltration occurred when the HiRet vector, but not the NeuRet vector, was injected into the primate brain. The present results indicate that the NeuRet vector is more suitable than the HiRet vector for retrograde gene transfer in the primate and rodent brains.

The cerebellum regulates complex animal behaviors, such as motor control and spatial recognition, through communication with many other brain regions. The major targets of the cerebellar projections are the thalamic regions including the ventroanterior nucleus (VA) and ventrolateral nucleus (VL). Another thalamic target is the central lateral nucleus (CL), which receives the innervations mainly from the dentate nucleus (DN) in the cerebellum. Although previous electrophysiological studies suggest the role of the CL as the relay of cerebellar functions, the kinds of behavioral functions mediated by cerebellothalamic tracts projecting to the CL remain unknown. Here, we used immunotoxin (IT) targeting technology combined with a neuron-specific retrograde labeling technique, and selectively eliminated the cerebellothalamic tracts of mice. We confirmed that the number of neurons in the DN was selectively decreased by the IT treatment. These IT-treated mice showed normal overground locomotion with no ataxic behavior. However, elimination of these neurons impaired motor coordination in the rotarod test and forelimb movement in the reaching test. These mice showed intact acquisition and flexible change of spatial information processing in the place discrimination, Morris water maze, and T-maze tests. Although the tract labeling indicated the existence of axonal collaterals of the DN-CL pathway to the rostral part of the VA/VL complex, excitatory lesion of the rostral VA/VL did not show any significant alterations in motor coordination or forelimb reaching, suggesting no requirement of axonal branches connecting to the VL/VA complex for motor skill function. Taken together, our data highlight that the cerebellothalamic tracts projecting to the CL play a key role in the control of motor skills, including motor coordination and forelimb reaching, but not spatial recognition and its flexibility.

Transcranial direct-current stimulation (tDCS) is a non-invasive procedure that achieves polarity-dependent modulation of neuronal membrane potentials. It has recently been used as a functional intervention technique for the treatment of psychiatric and neurological diseases; however, its neuronal mechanisms have not been fully investigated in vivo.

Transcription factor E3 (TFE3) is a transcription factor that binds to E-box motifs and promotes energy metabolism-related genes. We previously reported that TFE3 directly binds to the insulin receptor substrate-2 promoter in the liver, resulting in increased insulin response. However, the role of TFE3 in other tissues remains unclear. In this study, we generated adipose-specific TFE3 transgenic (aP2-TFE3 Tg) mice. These mice had a higher weight of white adipose tissue (WAT) and brown adipose tissue than wild-type (WT) mice under fasting conditions. Lipase activity in the WAT in these mice was lower than that in the WT mice. The mRNA level of adipose triglyceride lipase (ATGL), the rate-limiting enzyme for adipocyte lipolysis, was significantly decreased in aP2-TFE3 Tg mice. The expression of Foxo1, which directly activates ATGL expression, was also suppressed in transgenic mice. Promoter analysis confirmed that TFE3 suppressed promoter activities of the ATGL gene. In contrast, G0S2 and Perilipin1, which attenuate ATGL activity, were higher in transgenic mice than in WT mice. These results indicated that the decrease in lipase activity in adipose tissues was due to a decrease in ATGL expression and suppression of ATGL activity. We also showed that thermogenesis was suppressed in aP2-TFE3 Tg mice. The decrease in lipolysis in WAT of aP2-TFE3 Tg mice inhibited the supply of fatty acids to brown adipose tissue, resulting in the inhibition of the expression of thermogenesis-related genes such as UCP1. Our data provide new evidence that TFE3 regulates lipid metabolism by controlling the gene expression related to lipolysis and thermogenesis in adipose tissue.