Kazuo Funabiki

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.

TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade

An essential step in intricate visual processing is the segregation of visual signals into ON and OFF pathways by retinal bipolar cells (BCs). Glutamate released from photoreceptors modulates the photoresponse of ON BCs via metabotropic glutamate receptor 6 (mGluR6) and G protein (Go) that regulates a cation channel. However, the cation channel has not yet been unequivocally identified. Here, we report a mouse TRPM1 long form (TRPM1-L) as the cation channel. We found that TRPM1-L localization is developmentally restricted to the dendritic tips of ON BCs in colocalization with mGluR6. TRPM1 null mutant mice completely lose the photoresponse of ON BCs but not that of OFF BCs. In the TRPM1-L-expressing cells, TRPM1-L functions as a constitutively active nonselective cation channel and its activity is negatively regulated by Go in the mGluR6 cascade. These results demonstrate that TRPM1-L is a component of the ON BC transduction channel downstream of mGluR6 in ON BCs.

Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation

Exquisitely precise synapse formation is crucial for the mammalian CNS to function correctly. Retinal photoreceptors transfer information to bipolar and horizontal cells at a specialized synapse, the ribbon synapse. We identified pikachurin, an extracellular matrix–like retinal protein, and observed that it localized to the synaptic cleft in the photoreceptor ribbon synapse. Pikachurin null-mutant mice showed improper apposition of the bipolar cell dendritic tips to the photoreceptor ribbon synapses, resulting in alterations in synaptic signal transmission and visual function. Pikachurin colocalized with both dystrophin and dystroglycan at the ribbon synapses. Furthermore, we observed direct biochemical interactions between pikachurin and dystroglycan. Together, our results identify pikachurin as a dystroglycan-interacting protein and demonstrate that it has an essential role in the precise interactions between the photoreceptor ribbon synapse and the bipolar dendrites. This may also advance our understanding of the molecular mechanisms underlying the retinal electrophysiological abnormalities observed in muscular dystrophy patients.

The role of GABAergic inputs for coincidence detection in the neurones of nucleus laminaris of the chick

1 Synaptic inputs to nucleus laminaris (NL) neurones were studied in a brainstem slice preparation of chick embryos (E15‐20) using the whole‐cell patch clamp technique. NL neurones are third order auditory neurones and are proposed to behave as coincidence detectors concerned with interaural timing discrimination. 2 Under voltage clamp conditions, electrical stimuli applied to either ventral or dorsal dendritic layers evoked EPSCs. These fast currents decayed with a time constant of 1.1 ms near the resting potential, reversed close to 0 mV, and were blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 20 μM) or 6,7‐dinitro‐quinoxaline‐2,3‐dione (DNQX, 20 μM). Coincident or near coincident stimulation of the ventral and the dorsal dendritic layers increased the probability of action potential generation (response probability). 3 In the presence of CNQX (40 μM) other postsynaptic currents (PSCs) were observed, which reversed close to the equilibrium potential for chloride (ECl), and were reversibly blocked by bicuculline (20 μM) and, therefore, were mediated by GABAA receptors. Spontaneous GABAergic PSCs were inward going near the resting membrane potential immediately after starting whole‐cell recording with a low Cl− (5 mM, ECl= ‐90 mV) pipette medium, but became outward‐going with time. This indicates that GABAergic inputs may generate depolarizing potentials in intact NL neurones. 4 Local GABA (10 μM) application reduced both the EPSP and EPSC amplitude and shortened the EPSP decay time constant (from 5.3 to 2.1 ms), while the EPSC decay time constant was not affected (from 1.3 to 1.2 ms). These GABA effects were mostly due to the shunting conductance of the postsynaptic GABAA receptors. 5 Depolarizing current injections combined with electrical stimuli to a unilateral axon bundle simulated bilateral synaptic inputs. Response probability increased with decreased interstimulus intervals, while local GABA (10 μM) application to the soma narrowed the time dependence of the response probability. 6 These results suggest that GABAergic inputs to NL neurones may serve to improve coincidence detection of the bilateral excitatory inputs through an increase in membrane conductance.

Retarded vestibular compensation in mutant mice deficient in δ2 glutamate receptor subunit

The δ2 subunit of ionotropic glutamate receptors is expressed only in the cerebellar Purkinje cell. In mutant mice deficient in the δ2 protein, cerebellar long-term depression and motor coordination are impaired. We examined behavioural plasticity in these mutant mice after unilateral vestibular destruction. After intratympanic injection of sodium arsanilate, the mice showed roll head tilt and their righting response under a rotation load was impaired. These symptoms improved with time. However, compensation of the righting response was retarded in the mutant mice. These results suggest that motor learning of the δ2 mutant mice is disturbed, and that the static and dynamic components of vestibular compensation may be controlled by different neuronal mechanisms.

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.

Dynamic properties, interactions and adaptive modifications of vestibulo-ocular reflex and optokinetic response in mice

Dynamic properties of horizontal vestibulo-ocular reflex (VOR) and optokinetic response (OKR) were studied in mice. The VOR was examined in the dark (VORD), in the light (VORL) and in the condition in which most of the visual field moves synchronously with the head motion (VORF). A mouse and/or a surrounding screen with vertical stripes was rotated sinusoidally, and the gain and phase of eye movements were measured in wide dynamic stimulation ranges. The working conditions of VOR and OKR were supplementary; OKR worked at low speeds of head turn and VOR at high speeds. Examination of VORL and VORF revealed non-linear interaction of VOR and OKR. The continuous sinusoidal head oscillation coupled with the in-phase or the out-of-phase oscillation of the surrounding screen, decreased or increased the VORD gain, and increased or decreased the VORD phase lead, respectively. Continuous oscillation of the surrounding screen increased the OKR gain and decreased the phase delay. These changes of VOR and OKR work to reduce the retinal slip. The present study provides fundamental information concerning the dynamic properties of VOR and OKR and the nature of their adaptive modifications in mice, which have been extensively used in genetic manipulation recently.

Conditioned eyeblink learning is formed and stored without cerebellar granule cell transmission

Classical conditioning of the eyeblink reflex is elicited by paired presentation of a conditioned stimulus and an unconditioned stimulus and represents a basic form of cerebellum-dependent motor learning. Purkinje cells and the deep nuclei receive convergent information of conditioned stimulus and unconditioned stimulus through the mossy fiber and climbing fiber projections, respectively. To explore the relative importance of these neural circuits and the underlying mechanism in associative eyeblink learning, we adopted a novel gene-manipulating technique, termed reversible neurotransmission blocking (RNB). In this technology, cerebellar granule cells specifically expressed neurotransmission-blocking tetanus toxin in a doxycycline (DOX)-dependent manner. Extracellular recording of Purkinje cells in awake RNB mice revealed that DOX treatment and withdrawal reversibly turned off and on simple spikes elicited by granule cell inputs, respectively, without interference with complex spikes evoked by climbing fiber inputs. Blockade of granule cell inputs to Purkinje cells abolished eyeblink conditioned responses (CRs) in a DOX-dependent manner. Importantly, when granule cell inputs recovered by removal of DOX, normal CRs were immediately produced in the DOX-treated, CR-negative RNB mice from the beginning of reconditioning. This learning process in RNB mice during DOX treatment was completely abolished by bilateral lesion of the interpositus nucleus before eyeblink conditioning. These results indicate that the convergent information at the interpositus nucleus is critical for acquisition and storage of learning in intimate association with the Purkinje cell circuit for expression of CRs in eyeblink conditioning.

Voltage-gated ionic currents and their roles in timing coding in auditory neurons of the nucleus magnocellularis of the chick

Avian cochlear neurons of the nucleus magnocellularis (NMC) are known to encode temporal information of sound. The neuron generated only a single action potential at a stable timing even though suprathreshold currents of long duration (> 100 ms) was injected. The threshold for the action potential was -42 mV. In voltage-clamp experiments, a TTX-sensitive Na current was activated at membrane potentials more positive than -50 mV. A low voltage activated (LVA) Ca current and a high voltage activated (HVA) Ca current were observed. The LVA Ca current was activated from -65 mV and showed a voltage dependent inactivation. The HVA Ca current was activated from -40 mV and did not show any inactivation. The LVA Ca current and the HVA Ca current were sensitive to Ni2+ (0.1 mM) and Nifedipine (10-20 mM), respectively. NMC neurons showed a TEA-sensitive K current and a 4-AP-sensitive K current. With 4-AP (0.5 mM) in a bathing medium, the threshold of action potential was decreased to -49 mV and the timing of action potential generation showed a wider distribution than that of control. Ni2+ (0.1 mM) reversed effects of 4-AP on the threshold and the variability of action potential onsets. It is concluded that a 4-AP-sensitive current counteracts the LVA Ca current that facilitates Na spike generation, and sets a threshold to a higher level for generating a single action potential at a precise timing following synaptic inputs from the auditory nerve.

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.