Shigetada Nakanishi

Distinct Roles of Synaptic Transmission in Direct and Indirect Striatal Pathways to Reward and Aversive Behavior

In the basal ganglia, convergent input and dopaminergic modulation of the direct striatonigral and the indirect striatopallidal pathways are critical in rewarding and aversive learning and drug addiction. To explore how the basal ganglia information is processed and integrated through these two pathways, we developed a reversible neurotransmission blocking technique, in which transmission of each pathway was selectively blocked by specific expression of transmission-blocking tetanus toxin in a doxycycline-dependent manner. The results indicated that the coordinated modulation of these two pathways was necessary for dopamine-mediated acute psychostimulant actions. This modulation, however, shifted to the predominant roles of the direct pathway in reward learning and cocaine sensitization and the indirect pathway in aversive behavior. These two pathways thus have distinct roles: the direct pathway critical for distinguishing associative rewarding stimuli from nonassociative ones and the indirect pathway for rapid memory formation to avoid aversive stimuli.

Distinct Roles of Segregated Transmission of the Septo-Habenular Pathway in Anxiety and Fear

The posterior septum consisting of the triangular septum (TS) and the bed nucleus of the anterior commissure (BAC) is predominantly linked with the medial habenula (MHb) and has been implicated in the control of anxiety and fear responses. However, its anatomical and functional linkage has largely remained elusive. We established a transgenic mouse model in which the TS and BAC projection neurons were visualized by GFP fluorescence and selectively eliminated by immunotoxin-mediated cell targeting. The linkage between the TS/BAC and the MHb constitutes two parallel pathways composed of the TS-ventral MHb, the core part of the interpeduncular nucleus (IPN), and the BAC-dorsal MHb, the peripheral part of the IPN. Ablation of the TS and BAC projection neurons selectively impaired anxiety and enhanced fear responses and learning, respectively. Inputs from the TS and BAC to the MHb are thus segregated by two parallel pathways and play specialized roles in controlling emotional behaviors.

Membrane potential-regulated Ca2+ signalling in development and maturation of mammalian cerebellar granule cells

In many developing neuronal cell types, the resting membrane potential is relatively depolarized, then gradually hyperpolarizes during the early postnatal period. The regulatory roles of membrane potential changes in neuronal development and maturation have been extensively studied in developing cerebellar granule cells, using primary culture under depolarizing and non‐depolarizing conditions in combination with in vivo analysis. Depolarization enhances calcium entry via voltage‐sensitive Ca2+ channels (VSCCs) and activates Ca2+–calmodulin‐dependent protein kinase (CaMK) and calcineurin phophatase (CaN). The activation of CaN induces many genes encoding extracellular and intracellular signalling molecules implicated in granule cell development. The inactivation of CaN in turn up‐regulates many other genes characteristic of mature granule cells, including NR2C NMDA receptor and GABAAα1 and α6 receptors. The induction of NR2C also requires CaMK‐up‐regulated brain‐derived neurotrophic factor (BDNF), indicating a convergence of signalling mechanism of the CaMK and CaN cascades. The inactivation of CaN maintains the phosphorylated and sumoylated form of a transcriptional myocyte enhances factor 2A (MEF2A) regulator. This form of MEF2A acts as a transcriptional repressor and is essential for the dendritic morphogenesis of differentiated granule cells. Collectively, the membrane potential change and the resulting Ca2+ signalling play a pivotal role in development and maturation of neuronal cells.

Aversive behavior induced by optogenetic inactivation of ventral tegmental area dopamine neurons is mediated by dopamine D2 receptors in the nucleus accumbens

Significance Dopamine (DA) neurons in the ventral tegmental area (VTA) react to aversive stimuli mostly by transient silencing. It remains unclear whether this reaction directly induces aversive responses in behaving mice. We examined this question by optogenetically controlling DA neurons in the VTA and found that the inactivation of DA neurons resulted in aversive response and learning. The nucleus accumbens (NAc), the major output nuclei of VTA DA neurons, was considered to be responsible for this response, so we examined which of the fundamental pathways in the NAc was critical to this behavior by using knockdown of D1 or D2 receptor, and found that the D2 receptor-specific pathway was crucial for this behavior. Dopamine (DA) transmission from the ventral tegmental area (VTA) is critical for controlling both rewarding and aversive behaviors. The transient silencing of DA neurons is one of the responses to aversive stimuli, but its consequences and neural mechanisms regarding aversive responses and learning have largely remained elusive. Here, we report that optogenetic inactivation of VTA DA neurons promptly down-regulated DA levels and induced up-regulation of the neural activity in the nucleus accumbens (NAc) as evaluated by Fos expression. This optogenetic suppression of DA neuron firing immediately evoked aversive responses to the previously preferred dark room and led to aversive learning toward the optogenetically conditioned place. Importantly, this place aversion was abolished by knockdown of dopamine D2 receptors but not by that of D1 receptors in the NAc. Silencing of DA neurons in the VTA was thus indispensable for inducing aversive responses and learning through dopamine D2 receptors in the NAc.

Pathway-specific control of reward learning and its flexibility via selective dopamine receptors in the nucleus accumbens

In the basal ganglia, inputs from the nucleus accumbens (NAc) are transmitted through both direct and indirect pathways and control reward-based learning. In the NAc, dopamine (DA) serves as a key neurotransmitter, modulating these two parallel pathways. This study explored how reward learning and its flexibility are controlled in a pathway-specific and DA receptor-dependent manner. We used two techniques (i) reversible neurotransmission blocking (RNB), in which transmission of the direct (d-RNB) or the indirect pathway (I-RNB) in the NAc on both sides of the hemispheres was selectively blocked by transmission-blocking tetanus toxin; and (ii) asymmetric RNB, in which transmission of the direct (d-aRNB) or the indirect pathway (I-aRNB) was unilaterally blocked by RNB techniques and the intact side of the NAc was infused with DA agonists or antagonists. Reward-based learning was assessed by measuring goal-directed learning ability based on visual cue tasks (VCTs) or response-direction tasks (RDTs). Learning flexibility was then tested by switching from a previously learned VCT to a new VCT or RDT. d-RNB mice and D1 receptor antagonist-treated d-aRNB mice showed severe impairments in learning acquisition but normal flexibility to switch from a previously learned strategy. In contrast, I-RNB mice and D2 receptor agonist-treated I-aRNB mice showed normal learning acquisition but severe impairments not only in the flexibility to the learning switch but also in the subsequent acquisition of learning a new strategy. D1 and D2 receptors thus play distinct but cooperative roles in reward learning and its flexibility in a pathway-specific manner.

Pathway-specific modulation of nucleus accumbens in reward and aversive behavior via selective transmitter receptors

The basal ganglia–thalamocortical circuitry plays a central role in selecting actions that achieve reward-seeking outcomes and avoid aversive ones. Inputs of the nucleus accumbens (NAc) in this circuitry are transmitted through two parallel pathways: the striatonigral direct pathway and the striatopallidal indirect pathway. In the NAc, dopaminergic (DA) modulation of the direct and the indirect pathways is critical in reward-based and aversive learning and cocaine addiction. To explore how DA modulation regulates the associative learning behavior, we developed an asymmetric reversible neurotransmission-blocking technique in which transmission of each pathway was unilaterally blocked by transmission-blocking tetanus toxin and the transmission on the intact side was pharmacologically manipulated by local infusion of a receptor-specific agonist or antagonist. This approach revealed that the activation of D1 receptors and the inactivation of D2 receptors postsynaptically control reward learning/cocaine addiction and aversive learning in a direct pathway-specific and indirect pathway–specific manner, respectively. Furthermore, this study demonstrated that aversive learning is elicited by elaborate actions of NMDA receptors, adenosine A2a receptors, and endocannabinoid CB1 receptors, which serve as key neurotransmitter receptors in inducing long-term potentiation in the indirect pathway. Thus, reward and aversive learning is regulated by pathway-specific neural plasticity via selective transmitter receptors in the NAc circuit.

Intrinsic ON Responses of the Retinal OFF Pathway Are Suppressed by the ON Pathway

Parallel ON and OFF pathways conduct visual signals from bipolar cells in the retina to higher centers in the brain. ON responses are thought to originate by exclusive use of metabotropic glutamate receptor 6 (mGluR6) expressed in retinal ON bipolar cells. Paradoxically, we find ON responses in retinal ganglion cells of mGluR6-null mice, but they occur at long latency. The long-latency ON responses are not blocked by metabotropic glutamate or cholinergic receptor antagonists and are not produced by activation of receptive field surrounds. We show that these longer-latency ON responses are initiated in the OFF pathways. Our results expose a previously unrecognized intrinsic property of OFF retinal pathways that generates responses to light onset. In mGluR6-null mice, long-latency ON responses are observed in the visual cortex, indicating that they can be conducted reliably to higher visual areas. In wild-type (WT) mice, APB (dl-2-amino-4-phosphonobutyric acid), an mGluR6 agonist, blocks normal, short-latency ON responses but unmasks longer-latency ones. We find that these potentially confusing ON responses in the OFF pathway are actively suppressed in WT mice via two pharmacologically separable retinal circuits that are activated by the ON system in the retina. Consequently, we propose that a major function of the signaling of the ON pathway to the OFF pathway is suppression of these mistimed, and therefore inappropriate, light-evoked responses.

Distinct dopaminergic control of the direct and indirect pathways in reward-based and avoidance learning behaviors.

The nucleus accumbens (NAc) plays a pivotal role in reward and aversive learning and learning flexibility. Outputs of the NAc are transmitted through two parallel routes termed the direct and indirect pathways and controlled by the dopamine (DA) neurotransmitter. To explore how reward-based and avoidance learning is controlled in the NAc of the mouse, we developed the reversible neurotransmission-blocking (RNB) technique, in which transmission of each pathway could be selectively and reversibly blocked by the pathway-specific expression of transmission-blocking tetanus toxin and the asymmetric RNB technique, in which one side of the NAc was blocked by the RNB technique and the other intact side was pharmacologically manipulated by a transmitter agonist or antagonist. Our studies demonstrated that the activation of D1 receptors in the direct pathway and the inactivation of D2 receptors in the indirect pathway are key determinants that distinctly control reward-based and avoidance learning, respectively. The D2 receptor inactivation is also critical for flexibility of reward learning. Furthermore, reward and aversive learning is regulated by a set of common downstream receptors and signaling cascades, all of which are involved in the induction of long-term potentiation at cortico-accumbens synapses of the two pathways. In this article, we review our studies that specify the regulatory mechanisms of each pathway in learning behavior and propose a mechanistic model to explain how dynamic DA modulation promotes selection of actions that achieve reward-seeking outcomes and avoid aversive ones. The biological significance of the network organization consisting of two parallel transmission pathways is also discussed from the point of effective and prompt selection of neural outcomes in the neural network.

The effect of mGluR2 activation on signal transduction pathways and neuronal cell survival.

In earlier studies, we found profound alterations in specific signal transduction pathways such as mitogen-activated protein kinase signal pathway that mirrored neuronal cell death in Alzheimer disease (AD). To further delineate the mechanism(s) involved in such aberrant signaling, we subsequently showed that mGluR2 is increased in pyramidal neurons in the hippocampus of AD and often co-localizes with neurofibrillary pathology. Based on these data, we suggested that selective neuronal degeneration in AD may arise through the differential expression and activation of specific receptor populations, such as, mGluR2. In this study, to examine the mechanistic relevance of the above-mentioned in vivo findings, we used cell culture models to show that the activation of mGluR2 leads to the activation of extracellular signal-related kinase (ERK) pathways. Importantly, attesting to the in vivo significance of our findings, this pro-survival signaling pathway is also found to be ectopically activated in AD. We also found that the activation of mGluR2 increases the phosphorylation of tau and that the specific activation of mGluR2 reduces oxidative stress mediated cytotoxicity in neuronal cells. Taken together our findings strongly suggest that mGluR2 may participate in mediating the survival of neurons in the face of selective neuronal dysfunction and degeneration in AD. Additionally, our findings lend support to the notion that tau phosphorylation is a neuroprotective antioxidant response to cellular insults.

Circuit-dependent striatal PKA and ERK signaling underlies rapid behavioral shift in mating reaction of male mice

Significance Selection of actions that allow the seeking of rewards and avoidance of uncomfortable environments is a fundamental animal behavior. Here, we report an in vivo method, in which the activities of PKA and ERK were optically recorded by microendoscopy of Förster resonance energy transfer responses of biosensors in distinct D1 and D2 dopamine receptor-expressing neurons of the dorsal striatum. The PKA and ERK were coordinately but reciprocally regulated not only by rewarding and aversive stimuli but also between the two parallel projection neurons. Importantly, the cell type-specific regulation of PKA and ERK was causally linked to active and indifferent mating reactions of male mice. The dynamic modulation of PKA and ERK in the striatum underlies the selection of alternative actions. The selection of reward-seeking and aversive behaviors is controlled by two distinct D1 and D2 receptor-expressing striatal medium spiny neurons, namely the direct pathway MSNs (dMSNs) and the indirect pathway MSNs (iMSNs), but the dynamic modulation of signaling cascades of dMSNs and iMSNs in behaving animals remains largely elusive. We developed an in vivo methodology to monitor Förster resonance energy transfer (FRET) of the activities of PKA and ERK in either dMSNs or iMSNs by microendoscopy in freely moving mice. PKA and ERK were coordinately but oppositely regulated between dMSNs and iMSNs by rewarding cocaine administration and aversive electric shocks. Notably, the activities of PKA and ERK rapidly shifted when male mice became active or indifferent toward female mice during mating behavior. Importantly, manipulation of PKA cascades by the Designer Receptor recapitulated active and indifferent mating behaviors, indicating a causal linkage of a dynamic activity shift of PKA and ERK between dMSNs and iMSNs in action selection.