Nobuyuki Kai

Diversity Revealed by a Novel Family of Cadherins Expressed in Neurons at a Synaptic Complex

In mammals, neurons are highly differentiated and play distinctive functions even in the same brain region. We found a novel cadherin-related neuronal receptor (Cnr) gene family by studying Fyn-binding activity in mouse brain. CNR1 protein is located in the synaptic junction and forms a complex with Fyn. Sequence analysis of eight Cnr products of approximately 20 genes indicates that these comprise a novel cadherin family of the cadherin superfamily. The expression patterns of each member of this novel family were grossly similar to each other but restricted to subpopulations of neurons of the same type. The diversity of the Cnr family genes suggests that there are molecular mechanisms that govern highly differentiated neural networks in the mammalian CNS.

Decline of striatal dopamine release in parkin-deficient mice shown by ex vivo autoradiography.

Parkin is the causal gene of autosomal recessive juvenile parkinsonism (AR‐JP). Dopamine (DA) metabolism has been linked to Parkinsons disease (PD). To understand the pathogenesis of AR‐JP, we generated parkin‐deficient mice to assess the status of DA signaling pathway and examine DA release and DA receptor by ex vivo autoradiography. Ex vivo autoradiography using [11C]raclopride showed a clear decrease in endogenous DA release after methamphetamine challenge in parkin‐deficient mice. Furthermore, parkin deficiency was associated with considerable upregulation of DA (D1 and D2) receptor binding in vivo in the striatum and increased DA levels in the midbrain. Our results suggest that dopaminergic neurons could behave abnormally before neuronal death.

Aberrant responses to acoustic stimuli in mice deficient for neural recognition molecule NB-2

NB‐2, a member of the contactin subgroup in the immunoglobulin superfamily, is expressed specifically in the postnatal nervous system, reaching a maximum level at 3 weeks postnatal. NB‐2 displays neurite outgrowth‐promoting activity in vitro. To assess its function in the nervous system, we generated mutant mice in which a part of the NB‐2 gene was ablated and replaced with the tau‐LacZ gene. The general appearance of NB‐2‐deficient mice and their gross anatomical features were normal. The LacZ expression patterns in heterozygous mice revealed that NB‐2 is preferentially expressed in the central auditory pathways. In the audiogenic seizure test, NB‐2‐deficient mice exhibited a lower incidence of wild running, but a higher mortality rate than the wild‐type littermates. c‐Fos immunohistochemistry demonstrated that neural excitability induced by the audiogenic seizure test in the NB‐2‐deficient mice was prominently attenuated in both the dorsal and external cortices of the inferior colliculus, where enhanced neural excitability was observed in the wild‐type mice. In response to pure‐tone stimulation after priming, NB‐2‐deficient mice exhibited a diffuse and low level of c‐Fos expression in the central nucleus of the inferior colliculus, which was distinctly different from the band‐like c‐Fos expression corresponding to the tonotopic map in the wild‐type littermates. Taken together, these results suggest that a lack of NB‐2 causes impairment of the neuronal activity in the auditory system.

Subthalamic Neurons Coordinate Basal Ganglia Function through Differential Neural Pathways

The subthalamic nucleus (STN) is a key component of basal ganglia circuitry that mediates a variety of motor functions. The STN neurons send glutamatergic projections to the output structures of basal ganglia, including the substantia nigra pars reticulata (SNr) and the entopeduncular nucleus, and also innervate the globus pallidus (GP). However, the mechanism by which the STN regulates motor functions in the neural circuitry is not fully understood. Here we performed conditional ablation of the STN neurons by using immunotoxin-mediated cell targeting. We then analyzed dopamine (DA)-mediated motor behavior and firing activity of the SNr and GP neurons. Ablation of the STN neurons increased spontaneous movement and reduced hyperactivity in response to DA stimulation. Ablation of these neurons modulated the pattern and rate of spontaneous firing of the SNr neurons, although it did not substantially affect spontaneous firing of the GP neurons. The ablation attenuated DA-induced suppression of the firing rate of the SNr neurons and inhibited DA-induced elevation of the rate of the GP neurons. In addition, pharmacological blockade of GP activation in response to DA stimulation inhibited the suppression of SNr activity and the resultant motor activation. These results suggest that the STN neurons suppress spontaneous behavior through their direct projection to the output neurons and that, in response to DA, they contribute to expression of behavior by acting on the output neurons mainly through the GP-mediated pathways. We conclude that the STN coordinates motor behavior through differential neural pathways depending on the state of DA transmission.

Enhanced flexibility of place discrimination learning by targeting striatal cholinergic interneurons

Behavioural flexibility is mediated through the neural circuitry linking the prefrontal cortex and basal ganglia. Here we conduct selective elimination of striatal cholinergic interneurons in transgenic rats by immunotoxin-mediated cell targeting. Elimination of cholinergic interneurons from the dorsomedial striatum (DMS), but not from the dorsolateral striatum, results in enhanced reversal and extinction learning, sparing the acquisition of place discrimination. This enhancement is prevented by infusion of a non-selective muscarinic acetylcholine receptor agonist into the DMS either in the acquisition, reversal or extinction phase. In addition, gene-specific silencing of M4 muscarinic receptor by lentiviral expression of short hairpin RNA (shRNA) mimics the place reversal learning promoted by cholinergic elimination, whereas shRNA-mediated gene silencing of M1 muscarinic receptor shows the normal performance of reversal learning. Our data indicate that DMS cholinergic interneurons inhibit behavioural flexibility, mainly through the M4 muscarinic receptor, suggesting that this role is engaged to the stabilization of acquired reward contingency and the suppression of response switch to changed contingency.

Striatal Indirect Pathway Contributes to Selection Accuracy of Learned Motor Actions

The dorsal striatum, which contains the dorsolateral striatum (DLS) and dorsomedial striatum (DMS), integrates the acquisition and implementation of instrumental learning in cooperation with the nucleus accumbens (NAc). The dorsal striatum regulates the basal ganglia circuitry through direct and indirect pathways. The mechanism by which these pathways mediate the learning processes of instrumental actions remains unclear. We investigated how the striatal indirect (striatopallidal) pathway arising from the DLS contributes to the performance of conditional discrimination. Immunotoxin targeting of the striatal neuronal type containing dopamine D2 receptor in the DLS of transgenic rats resulted in selective, efficient elimination of the striatopallidal pathway. This elimination impaired the accuracy of response selection in a two-choice reaction time task dependent on different auditory stimuli. The impaired response selection was elicited early in the test sessions and was gradually restored as the sessions continued. The restoration from the deficits in auditory discrimination was prevented by excitotoxic lesion of the NAc but not by that of the DMS. In addition, lesion of the DLS mimicked the behavioral consequence of the striatopallidal removal at the early stage of test sessions of discriminative performance. Our results demonstrate that the DLS-derived striatopallidal pathway plays an essential role in the execution of conditional discrimination, showing its contribution to the control of selection accuracy of learned motor responses. The results also suggest the presence of a mechanism that compensates for the learning deficits during the repetitive sessions, at least partly, demanding accumbal function.

Striatal direct pathway modulates response time in execution of visual discrimination

The dorsal striatum in the basal ganglia circuitry is a principal structure that mediates the acquisition and performance of instrumental learning. The projections from the dorsal striatum are composed of two subpopulations of medium spiny neurons that constitute the direct and indirect pathways. The mechanism by which these striatal projections control the learning processes of instrumental actions remains unknown. We addressed the behavioral role of the striatal direct (striatonigral) pathway in the performance of visual discrimination. Immunotoxin targeting of the striatal neuronal type containing dopamine D1 receptor in mice resulted in a moderate level of elimination of the striatonigral pathway. Targeting of the neural pathway from the whole region of the dorsal striatum lengthened the response time but did not affect the accuracy of response selection in a two‐choice reaction time task dependent on light stimulus. This lengthened motor response was induced early in the test sessions and was gradually restored to normal levels during repetitive sessions. In addition, subregion‐specific pathway targeting revealed that the delay in learned motor response was generated by the elimination of the striatonigral pathway arising from the dorsomedial striatum but not from the dorsolateral striatum. Our findings indicate that the striatonigral pathway, in particular from the dorsomedial striatum, contributes to the regulation of response time in the execution of visual discrimination. The restoration of motor response deficits during repetitive sessions suggests the presence of a mechanism by which the response facilitation is acquired through continuation of learning despite the removal of the striatonigral pathway.

Up-regulation of dopamine D1 receptor in the hippocampus after establishment of conditioned place preference by cocaine.

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.

Laser capture microdissection and cDNA array analysis for identification of mouse KIAA/FLJ genes differentially expressed in the embryonic dorsal spinal cord

During early development, centrally projecting dorsal root ganglion (DRG) neurons extend their axons toward the dorsal spinal cord. We previously reported that this projection is achieved by dorsal spinal cord-derived chemoattraction. However, the molecular nature of the chemotrophic cue is not yet fully understood. To identify novel genes differentially expressed in the dorsal spinal cord in the embryonic day 10.5 mouse, we used the Kazusa cDNA array system comprising approximately 1700 mouse KIAA/FLJ (mKIAA/mFLJ) cDNA clones and laser capture microdissection (LCM) in combination with PCR-based cDNA amplification. We observed that a certain population of genes showed significantly increased expression in the dorsal spinal cord. In situ hybridization analysis verified the expression of mRNAs of 6 genes (Hip1r, Nav2, Fstl5, Cacna1h, Bcr, and Bmper) in the cells that constitute the dorsal spinal cord. The dorsal spinal cord-specific genes identified in this study provide a basis for studying the molecular nature of the neural development including the axonal guidance of DRG neurons. These results also demonstrate that the combined use of LCM coupled with the Kazusa cDNA array technology will be useful for the identification of large proteins expressed in the restricted small regions of embryos.

Differential roles of dopamine D1 and D2 receptor-containing neurons of the nucleus accumbens shell in behavioral sensitization

The nucleus accumbens (Nac) mediates the reinforcing and motor stimulating properties of psychostimulants. It receives dopaminergic afferents from the ventral midbrain and is divided into two distinct subregions: shell and core. Each of these contains two subtypes of medium spiny neurons, which express either dopamine D1 (D1R) or D2 (D2R) receptors. However, functional dissociation between the two subtypes in psychostimulant response remains to be elucidated. We performed selective ablation of each subtype in the Nac shell in mice, using immunotoxin‐mediated cell targeting, and examined the behavioral sensitization evoked by repeated administration of methamphetamine. The D1R cell‐ablated mice exhibited delayed induction of sensitized locomotion compared to control mice, whereas the D2R cell‐ablated mice showed a mildly enhanced rate of induction of sensitization. In vivo microdialysis revealed a marked blockade of the increase in extracellular dopamine in the Nac of the D1R cell‐ablated animals in response to methamphetamine, indicating that the observed delay in behavioral sensitization in these mice involves an impairment in accumbal dopamine release. Our results reveal differential roles of D1R‐ and D2R‐containing accumbal shell neurons in the development of behavioral sensitization to psychostimulants.