Hidenori Aizawa

The habenula is crucial for experience-dependent modification of fear responses in zebrafish

The zebrafish dorsal habenula (dHb) shows conspicuous asymmetry in its connection with the interpeduncular nucleus (IPN) and is equivalent to the mammalian medial habenula. Genetic inactivation of the lateral subnucleus of dHb (dHbL) biased fish towards freezing rather than the normal flight response to a conditioned fear stimulus, suggesting that the dHbL-IPN pathway is important for controlling experience-dependent modification of fear responses.

Microcircuitry coordination of cortical motor information in self-initiation of voluntary movements

Motor cortex neurons are activated at different times during self-initiated voluntary movement. However, the manner in which excitatory and inhibitory neurons in distinct cortical layers help to organize voluntary movement is poorly understood. We carried out juxtacellular and multiunit recordings from actively behaving rats and found temporally and functionally distinct activations of excitatory pyramidal cells and inhibitory fast-spiking interneurons. Across cortical layers, pyramidal cells were activated diversely for sequential motor phases (for example, preparation, initiation and execution). In contrast, fast-spiking interneurons, including parvalbumin-positive basket cells, were recruited predominantly for motor execution, with pyramidal cells producing a command-like activity. Thus, fast-spiking interneurons may underlie command shaping by balanced inhibition or recurrent inhibition, rather than command gating by temporally alternating excitation and inhibition. Furthermore, initiation-associated pyramidal cells excited similar and different functional classes of neurons through putative monosynaptic connections. This suggests that these cells may temporally integrate information to initiate and coordinate voluntary movement.

Molecular Characterization of the Subnuclei in Rat Habenula

The mammalian habenula is involved in regulating the activities of serotonergic and dopaminergic neurons. It consists of the medial and lateral habenulae, with each subregion having distinct neural connectivity. Despite the functional significance, manipulating neural activity in a subset of habenular pathways remains difficult because of the poor availability of molecular markers that delineate the subnuclear structures. Thus, we examined the molecular nature of neurons in the habenular subnuclei by analyzing the gene expressions of neurotransmitter markers. The results showed that different subregions of the medial habenula (MHb) use different combinations of neurotransmitter systems and could be categorized as either exclusively glutamatergic (superior part of MHb), both substance P‐ergic and glutamatergic (dorsal region of central part of MHb), or both cholinergic and glutamatergic (inferior part, ventral region of central part, and lateral part of MHb). The superior part of the MHb strongly expressed interleukin‐18 and was innervated by noradrenergic fibers. In contrast, the inferior part, ventral region of the central part, and lateral part of the MHb were peculiar in that acetylcholine and glutamate were cotransmitted from the axonal terminals. In contrast, neurons in the lateral habenula (LHb) were almost uniformly glutamatergic. Finally, the expressions of Htr2c and Drd2 seemed complementary in the medial LHb division, whereas they coincided in the lateral division, suggesting that the medial and lateral divisions of LHb show strong heterogeneity with respect to monoamine receptor expression. These analyses clarify molecular differences between subnuclei in the mammalian habenula that support their respective functional implications. J. Comp. Neurol. 520:4051–4066, 2012.

Identification of the Zebrafish Ventral Habenula As a Homolog of the Mammalian Lateral Habenula

The mammalian habenula consists of the medial and lateral habenulae. Recent behavioral and electrophysiological studies suggested that the lateral habenula plays a pivotal role in controlling motor and cognitive behaviors by influencing the activity of dopaminergic and serotonergic neurons. Despite the functional significance, manipulating neural activity in this pathway remains difficult because of the absence of a genetically accessible animal model such as zebrafish. To address the level of lateral habenula conservation in zebrafish, we applied the tract-tracing technique to GFP (green fluorescent protein)-expressing transgenic zebrafish to identify habenular neurons that project to the raphe nuclei, a major target of the mammalian lateral habenula. Axonal tracing in live and fixed fish showed projection of zebrafish ventral habenula axons to the ventral part of the median raphe, but not to the interpeduncular nucleus where the dorsal habenula projected. The ventral habenula expressed protocadherin 10a, a specific marker of the rat lateral habenula, whereas the dorsal habenula showed no such expression. Gene expression analyses revealed that the ventromedially positioned ventral habenula in the adult originated from the region of primordium lateral to the dorsal habenula during development. This suggested that zebrafish habenulae emerge during development with mediolateral orientation similar to that of the mammalian medial and lateral habenulae. These findings indicated that the lateral habenular pathways are evolutionarily conserved pathways and might control adaptive behaviors in vertebrates through the regulation of monoaminergic activities.

Genetic dissection of the zebrafish habenula, a possible switching board for selection of behavioral strategy to cope with fear and anxiety

The habenula is a part of an evolutionarily highly conserved conduction pathway within the limbic system that connects telencephalic nuclei to the brain stem nuclei such as interpeduncular nucleus (IPN), the ventral tegmental area (VTA), and the raphe. In mammals, the medial habenula receives inputs from the septohippocampal system, and relaying such information to the IPN. In contrast, the lateral habenula receives inputs from the ventral pallidum, a part of the basal ganglia. The physical adjunction of these two habenular nuclei suggests that the habenula may act asan intersection of the neural circuits for controlling emotion and behavior. We have recently elucidated that zebrafish has the equivalent structure as the mammalian habenula. The transgenic zebrafish, in which the neural signal transmission from the lateral subnucleus of the dorsal habenula to the dorsal IPN was selectively impaired, showed extremely enhanced levels of freezing response to presentation of the conditioned aversive stimulus. Our observation supports that the habenula may act as the multimodal switching board for controlling emotional behaviors and/or memory in experience dependent manners.

Reward-Modulated Motor Information in Identified Striatum Neurons

It is widely accepted that dorsal striatum neurons participate in either the direct pathway (expressing dopamine D1 receptors) or the indirect pathway (expressing D2 receptors), controlling voluntary movements in an antagonistically balancing manner. The D1- and D2-expressing neurons are activated and inactivated, respectively, by dopamine released from substantia nigra neurons encoding reward expectation. However, little is known about the functional representation of motor information and its reward modulation in individual striatal neurons constituting the two pathways. In this study, we juxtacellularly recorded the spike activity of single neurons in the dorsolateral striatum of rats performing voluntary forelimb movement in a reward-predictable condition. Some of these neurons were identified morphologically by a combination of juxtacellular visualization and in situ hybridization for D1 mRNA. We found that the striatal neurons exhibited distinct functional activations before and during the forelimb movement, regardless of the expression of D1 mRNA. They were often positively, but rarely negatively, modulated by expecting a reward for the correct motor response. The positive reward modulation was independent of behavioral differences in motor performance. In contrast, regular-spiking and fast-spiking neurons in any layers of the motor cortex displayed only minor and unbiased reward modulation of their functional activation in relation to the execution of forelimb movement. Our results suggest that the direct and indirect pathway neurons cooperatively rather than antagonistically contribute to spatiotemporal control of voluntary movements, and that motor information is subcortically integrated with reward information through dopaminergic and other signals in the skeletomotor loop of the basal ganglia.

Genetic Single-Cell Mosaic Analysis Implicates ephrinB2 Reverse Signaling in Projections from the Posterior Tectum to the Hindbrain in Zebrafish

The optic tectum is a visual center in vertebrates. It receives topographically ordered visual inputs from the retina in the superficial layers and then sends motor outputs from the deeper layers to the premotor reticulospinal system in the hindbrain. Although the topographic patterns of the retinotectal projection are well known, it is not yet well understood how tectal efferents in the tectobulbar tract project to the hindbrain. The retinotectal and the tectobulbar projections were visualized in a zebrafish stable transgenic line Tg(brn3a-hsp70:GFP). Using a single-neuron labeling system in combination with the cre/loxP and Gal4/UAS systems, we showed that the tectal neurons that projected to rhombomeres 2 and 6 were distributed with distinctive patterns along the anterior–posterior axis. Furthermore, we found that ephrinB2a was critically involved in increasing the probability of neurons projecting to rhombomere 2 through a reverse signaling mechanism. These results may provide a neuroanatomical and molecular basis for the motor command map in the tectum.

The Habenulo-Raphe Serotonergic Circuit Encodes an Aversive Expectation Value Essential for Adaptive Active Avoidance of Danger

Anticipation of danger at first elicits panic in animals, but later it helps them to avoid the real threat adaptively. In zebrafish, as fish experience more and more danger, neurons in the ventral habenula (vHb) showed tonic increase in the activity to the presented cue and activated serotonergic neurons in the median raphe (MR). This neuronal activity could represent the expectation of a dangerous outcome and be used for comparison with a real outcome when the fish is learning how to escape from a dangerous to a safer environment. Indeed, inhibiting synaptic transmission from vHb to MR impaired adaptive avoidance learning, while panic behavior induced by classical fear conditioning remained intact. Furthermore, artificially triggering this negative outcome expectation signal by optogenetic stimulation of vHb neurons evoked place avoidance behavior. Thus, vHb-MR circuit is essential for representing the level of expected danger and behavioral programming to adaptively avoid potential hazard.

Development of the mouse amygdala as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation

The amygdala is located in the caudal part of the ventral telencephalon. It is composed of many subdivisions and is involved in the control of emotion. It is important to know the mechanisms of amygdalar development in order to analyze the pathogenesis of emotional disorders, but they are still not adequately understood. In the present study the migration, differentiation, and distribution of amygdalar neurons in the mouse embryo were investigated by means of in utero electroporation. Ventricular zone cells in restricted regions, that is, the caudal ganglionic eminence (CGE), the ventral pallium, the lateral pallium, and the diencephalon, were labeled with an expression vector of the enhanced green fluorescent protein (EGFP) gene. Labeling at embryonic day (E)10 revealed that the central nucleus originates from the neuroepithelium in the ganglionic eminence and the labeling at E11 and E12 revealed that the basolateral complex originates from the neuroepithelium of the ventral and lateral pallia. The introduction of the EGFP gene into the neuroepithelium of the third ventricle at E11 showed that the medial nucleus originates, at least in part, from the neuroepithelium of the diencephalon and migrates over the diencephalo–telencephalic boundary. The radial glial arrangement corresponded well with the initial migration of amygdalar neurons, and the radial processes later formed the boundary demarcating the basolateral complex. These findings indicate that the neurons originating from the temporally and spatially restricted neuroepithelium in both the telencephalon and diencephalon migrate and differentiate to form the mosaic of amygdalar subdivisions. J. Comp. Neurol. 513:113–128, 2009.

Glial Dysfunction in the Mouse Habenula Causes Depressive-Like Behaviors and Sleep Disturbance

The lateral habenula (LHb) regulates the activity of monoaminergic neurons in the brainstem. This area has recently attracted a surge of interest in psychiatry because studies have reported the pathological activation of the habenula in patients with major depression and in animal models. The LHb plays a significant role in the pathophysiology of depression; however, how habenular neurons are activated to cause various depression symptoms, such as reduced motivation and sleep disturbance, remain unclear. We hypothesized that dysfunctional astrocytes may cause LHb hyperactivity due to the defective uptake activity of extracellular glutamate, which induces depressive-like behaviors. We examined the activity of neurons in habenular pathways and performed behavioral and sleep analyses in mice with pharmacological and genetic inhibition of the activity of the glial glutamate transporter GLT-1 in the LHb. The habenula-specific inhibition of GLT-1 increased the neuronal firing rate and the level of c-Fos expression in the LHb. Mice with reduced GLT-1 activity in the habenula exhibited a depressive-like phenotype in the tail suspension and novelty-suppressed feeding tests. These animals also displayed increased susceptibility to chronic stress, displaying more frequent avoidant behavior without affecting locomotor activity in the open-field test. Intriguingly, the mice showed disinhibition of rapid eye movement sleep, which is a characteristic sleep pattern in patients with depression. These results provide evidence that disrupting glutamate clearance in habenular astrocytes increases neuronal excitability and depressive-like phenotypes in behaviors and sleep.