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Genetic Dissection of the Zebrafish Habenula, a Possible Switching Board for Selection of Behavioral Strategy to Cope with Fear and Anxiety Hitoshi Okamoto, Masakazu Agetsuma,* Hidenori Aizawa{ RIKEN Brain Science Institute, Saitama 351-0198, Japan Received 24 February 2011; revised 2 May 2011; accepted 5 May 2011 ABSTRACT: 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 as an intersection of the neural circuits for controlling emotion and behavior. We have recently elucidated THE HABENULA AS A POTENTIAL INTERFACE OF FRONTOSTRIATAL AND SEPTOHIPPOCAMPAL SYSTEMS WITH THE BRAIN STEM MONOAMINAERGIC SYSTEMS The habenula and its afferent and efferent fiber tracts constitute the dorsal diencephalic conduction pathway, which conveys neural information from the limCorrespondence to: H. Okamoto ([email protected]). *Present address: Howard Hughes Medical Institute, Columbia University, Biological Sciences, 901 NWC Building, 550 West 120th Street, New York, NY 10027, USA. { Present address: Department of Molecular Neuroscience, Medical Research Institute and School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 Japan. ' 2011 Wiley Periodicals, Inc. Published online 12 May 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/dneu.20913 386 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. ' 2011 Wiley Periodicals, Inc. Develop Neurobiol 72: 386–394, 2012 Keywords: habenula; fear; anxiety; helplessness; left– right asymmetry bic forebrain to the interpeduncular nucleus (IPN) at the boundary between the midbrain and the hindbrain, which is further connected with the monoaminergic neurons such as the serotonin neurons in the raphe and the dopamine neurons in the ventral tegmental area (VTA) (Sutherland, 1982). This pathway is conserved throughout the vertebrate evolution (see Fig. 1). Efferent projection from the habenula to IPN is one of the most preserved tract in the forebrain from lamprey to human, and the output pathway from the habenula consists of fasciculated axons termed the fasciculus retroflexus [blue lines encircled by blue rings in Fig. 1(A,B)]. This fiber bundle contains not only the axons projecting to IPN but also the ones projecting to the nuclei abundant in monoaminergic neurons, i.e., dopaminergic neurons in the VTA and the substantia nigra pars compacta (SNc), serotonergic neurons in the raphe nuclei, and other brain stem nuclei such as the rostromedial tegmental nucleus (RMTg) and the nucleus incertus. RMTg was Genetic Dissection of the Zebrafish Habenula Figure 1 Comparison of the afferent and efferent neural pathways of the habenula in mammals and zebrafish. Schematic diagram of the sagittal sections of the rat (A) and zerbrafish (B) brain showing the phylogenetic conservation of the habenular pathways. The habenula in both species connects the basal forebrain nuclei (green circles) with the nuclei in the ventral midbrain and hindbrain (red circles). SNc, substantia nigra pars compacta; VTA, ventral tegmental area. recently identified as a nucleus which receives the afferent projection from the habenula and, in turn, sends the axons to VTA and SNc (Jhou et al., 2009b; Kaufling et al., 2009), and it is supposed that this nucleus mediates the inhibitory influence of the habenular activation on the dopaminergic activity (Jhou et al., 2009a). In the mammalian brain, the habenula is composed of two compartments [Fig. 2(A)]. The medial habenula receives the input from the septal nucleus, the bed nucleus of the anterior commissure (Herkenham and Nauta, 1977; Qin and Luo, 2009), and the diagonal band [Fig. 2(B)]. Since these nuclei get inputs both from the hippocampus and the amygdala, the contextual information of the outside world with the estimated saliency is fed into the medial habenula. The lateral habenula receives the inputs from the internal segment of the globus pallidus, a part of the cortico-basal ganglia loops which represent the activation status of the behavior programs encoded internally in the cortico-basal ganglia loops [Fig. 2(C)] (Herkenham and Nauta, 1977). Therefore, in the habenula, both the information representing the external situation with saliency value and the information representing the internal status of the behavioral program activated in response to the external information inputs can be juxtaposed [Fig. 2(A)]. The septo-hippocampal system is suspected to act as a comparator of conflicts in a given situation (Gray and 387 McNaughton, 2000). When it detects the conflicts, e.g., in the dilemma whether the animal should make an approach to or avoid from the goal or in the discrepancy between the expectation of reward and the obtained result, this system inhibits the ongoing behaviors and switches the behaviors of the individual animals into the stop-and-explore mode. The animals fall into the state of anxiety while the conflicts are not solved. The physical adjunction of these two habenular nuclei gives an anatomically favorable situation if the selected behavioral programs would be examined with regard to whether they are suitable for the given individual situation. Therefore, it is possible that the habenula plays an important role as part of or in association with the septo-hippocampal comparator system of conflicts which acts upstream of neural substrates for anxiogenesis. In fact, it was recently reported that the lateral habenula is activated in response to aversive stimulus as well as outcomes that seem inappropriate for the chosen behaviors, as opposed to the inactivation of the midbrain dopaminergic neurons (Matsumoto and Hiko- Figure 2 Schematic illustration of the afferent and efferent neural pathways of the medial and lateral habenula in mammals. Habenula could integrate the neural information from the major neural systems (A) including the limbic circuits connected with the medial habenula (B) and the cortico-basal ganglia-thalamic circuit connecting with the lateral habenula (C). Amg, amygdala; Cx, cortex; FR, fasciculus retroflexus; Fx, fornix; GP, globus pallidus; Hip, hippocampus; IPN, interpeduncular nucleus; LHyp, lateral hypothalamus; LHb, lateral habenula; MHb, medial habenula; RN, raphe nucleus; Sp, septum; SM, stria medullaris; ST, stria terminalis; Str, striatum; Th, thalamus; VTA, ventral tegmental area. Developmental Neurobiology 388 Okamoto et al. saka, 2007, 2009). The habenular lesions in rats prevented change in the response strategy under stressful conditions that was more appropriate for a given environmental contingency (Thornton and Evans, 1982). Namely, in forced swimming test, the animals with bilateral lesions of the habenula cannot utilize the extrinsic cue for escape even if it was provided. In rats, the lateral habenula receives inputs from the medial habenula asymmetrically (Kim and Chang, 2005). It is not known how such asymmetric connection between two components of the habenula is related to the hypothetical function of the habebula as a part of the conflict detecting system. In zebrafish, such asymmetric connection between the dorsal habenula (dHb) and ventral habenula (vHb) has not been observed. The medial habenula projects to the IPN, whereas the lateral habenula projects directly to the median and dorsal raphe, substantia nigra (SN), and VTA (Herkenham and Nauta, 1979). Stimulation of the lateral habenula has an inhibitory effect on both the dopaminergic and serotonergic neurons (Wang and Aghajanian, 1977; Christoph et al., 1986; Matsumoto and Hikosaka, 2007). In addition, destroying the medial and lateral habenular pathways leads to increased monoamine metabolism (Nishikawa and Scatton, 1985; Nishikawa et al., 1986) accompanied by increased locomotion (Lecourtier et al., 2004), reduction of REM sleep (Valjakka et al., 1998), and impaired responses to stress (Amat et al., 2001). These studies suggested that the lateral habenula plays a pivotal role in controlling motor and cognitive behaviors by influencing the activity of dopamine and serotonin neurons. In addition, as the habenula gets direct inputs both from the dopaminergic neurons in the VTA which respond to the rewarding situation (Phillipson and Griffith, 1980) and from the lateral hypothalamus neurons and the serotonergic neurons in the raphe which respond to the adverse situation (see Fig. 2) (Herkenham and Nauta, 1977), the habenula or the habenulo-interpeduncular system itself might act as the integrator to evaluate the positive and negative aspects of given situation as a potential interface of frontostriatal and septohippocampal systems with the brain stem monoaminaergic systems. ZEBRAFISH AS A MODEL ANIMAL TO STUDY THE FUNCTION OF THE HABENULA Zebrafish are genetically accessible and amenable to imaging neural activities due to their transparency during development. Resolving the neuroanatomy of zebrafish habenulae should therefore inform the functional dissecDevelopmental Neurobiology tion of neural circuits regulating monoaminergic systems. Fish and amphibian habenulae can be subdivided into dHb and vHb based on differences in cytoarchitecture (Braford and Northcutt, 1983; Kemali and Làzàr, 1985). The zebrafish dHb projects to the IPN (Aizawa et al., 2005; Gamse et al., 2005) and is thus analogous to the medial habenula of mammals [Fig. 3(A,B)]. Axonal tracing in live and fixed fish showed projection of zebrafish ventral habenular axons to the ventral part of the median raphe, but not to the IPN where the dHb projected (Amo et al., 2010). The vHb expressed protocadherin 10a, a specific marker of the rat lateral habenula, while the dHb showed no such expression. Gene expression analyses revealed that the ventromedially positioned vHb in the adult originated from the region of primordium lateral to the dHb 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. THE LEFT–RIGHT ASYMMETRY OF THE HABENULO-INTERPEDUNCLAR PROJECTION In zebrafish, the dHb shows conspicuous left–right differences, which recent genetic analyses revealed to emerge in the habenular subnuclei and their projections to the IPN [Fig. 3(A,B)] (Aizawa et al., 2007; Carl et al., 2007; Inbal et al., 2007; Kuan et al., 2007; Snelson et al., 2008; Regan et al., 2009). Based on expression of GFP in the brn3a-GFP transgenic fish and other molecular markers, the dHb in zebrafish can be subdivided into medial (dHbM) and lateral subnuclei (dHbL) (Aizawa et al., 2005), although further subdivision may also be possible by using more marker genes. A prominent left–right difference in the size ratio of these subnuclei accounted for asymmetry in neural connectivity, which is termed \laterotopy" (Aizawa et al., 2005). That is, axons from the left habenula project predominantly to the dorsal and intermediate IPN (d/iIPN), whereas the right habenula predominantly innervates the ventral IPN (vIPN). The larger dHbM on the right innervates the ventral and intermediate IPN (v/iIPN) together with axons from the smaller dHbM on the left. Conversely, the larger dHbL on the left is the predominant source of axons that innervate the dIPN with axons from the smaller dHbL on the right. As a result of this characteristic subnuclear organization, in adult fish, axons Genetic Dissection of the Zebrafish Habenula 389 Figure 3 The asymmetry in the habenulo-interpeduncular projection in zebrafish. A,B: The schematic illustrations of the habenulo-interpeduncular pathways in adult zebrafish (A, dorsal oblique view; B, sagittal view) Cbl, cerebellum; dIPN, dorsal interpeduncular nucleus; GC, griseum centrale; IL, inferior lobe of the hypothalamus; lHb, left habenula; MLF, medial longitudinal fasciclus; MR, median raphe; OB, olfactory bulb; TeO, optic tectum; PO, pineal organ; PP, parapineal organ; rHb, right habenula, Tel, telencephalon; vIPN, ventral interpeduncular nucleus. C: Schematic illustration of the asymmetric neurogenesis in the left and right dorsal habenula of zebrafish. The asymmetric Nodal pathway activation in the left habenula was detected by expression of GFP-transgene (lefty1:GFP). from the left habenula project predominantly to the d/ iIPN whereas the right habenula predominantly innervates the ventral IPN. In the v/iIPN, habenular axons surround IPN cell bodies that are predominantly located at the midline central core of the nucleus. TEMPORALLY REGULATED ASYMMETRIC NEUROGENESIS CAUSES LEFT–RIGHT DIFFERENCE IN THE ZEBRAFISH HABENULAR STRUCTURES Nodal belongs to the TGF-b superfamily, and its signaling cascade plays an important role in determining the left–right axis in viscera [Fig. 3(C)] (Hamada et al., 2002). Unilateral and transient Nodal activation in the dorsal diencephalon was believed to correlate with the orientation of habenular asymmetry, however left–right differences still remained after bilateral Nodal activation or even in the absence of Nodal signaling (Concha et al., 2000; Aizawa et al., 2005; Carl et al., 2007; Roussigné et al., 2009). This suggests the presence of other mechanisms for the establishment of habenular asymmetry. Since the establishment of habenular asymmetry is based on the differences in the number of neurons that belong to each subnucleus between the two sides of the Developmental Neurobiology 390 Okamoto et al. Figure 4 Specific silencing of the neural transmission from the lateral subnuclei of the dorsal habenula makes adult zebrafish prone to freeze upon presentation of the conditioned fear stimulus. A,B: The expression of DsRed2 and GFP in the lateral (dHbL) and medial (dHbM) subnuclei of the dorsal habenula (A) and in the dorsal (dIPN) and the ventral (vIPN) half of the interpeduncular nucleus (B) in the adult Tg(narp:GAL4VP16; UAS:DsRed2; brn3a-hsp70:GFP) zebrafish. C: The apparatus for fear conditioning two zebrafish independently. D,E: The trajectories of the wild-type (D) and manipulated (E) zebrafish after presentation of the conditioned stimulus. brain, the issue of habenular asymmetry is based on how these subnuclei are asymmetrically generated during development. This led us to examine whether the complementary enlargement or reduction of the dHbM and dHbL might derive from asymmetric neurogenesis during development (Aizawa et al., 2007). In fact, neurons of the medial and lateral subnuclei are born at different stages during development. The birthdate analyses by BrdU-pulse labeling of the Tg(brn3ahsp70:GFP) fish indicated that neural precursors for the dHbL are born at earlier stages than those for the dHbM. In addition, more neural precursors are born on the left side than on the right side mostly likely due to the right side-dominance in the activity of the Notch signaling in the habenula. Because of these two mechanisms, the left dHbL ends up significantly larger than the right dHbL [Fig. 3(C)]. Since both the neural precursors for the dHbL and the dHbM are derived from the common stem cells, more neurogenesis in the left habenula induces quicker depletion of these stem cells than in the right habenula. This in turn causes more neurogenesis at later stages of the embryonic development from the remaining stem cells on the right side to give rise to more dHbM neurons on the right side than on the left side and ultimately leads to establishment of Developmental Neurobiology the left–right asymmetric subnuclear organization of the dorsal habenula (Aizawa et al., 2007). HABENULA AS THE EXPERIENCEDEPENDENT MODULATORS OF BEHAVIORAL STRATEGY TO COPE WITH STRESS As compared to the recent advance in the understanding of the function of the lateral habenula, the medial habenula remains functionally ambiguous due mainly to a lack of suitable technology for manipulating the medial habenular neurons reproducibly and with subdivision-specific precision. To further investigate the physiological meaning of this prominent asymmetric axonal projection pattern, we have established the transgenic zebrafish line expressing Gal4-VP16 specifically in the dHbL by using the BAC clone of the zebrafish neural activityregulated petaxin (Narp) gene which is specifically expressed in the subsets of the habenula and whose coding regions had been replaced with the gal4-vp16 gene (Agetsuma et al., 2010). By crossing such lines with other transgenic lines carrying the tetanus toxin Genetic Dissection of the Zebrafish Habenula gene or the nitroreductase gene under control of the target site of Gal4-VP16, we have succeeded in establishing the lines in which the neural signal transmission by way of the dHbL is selectively impaired either constitutively or conditionally [Fig. 4(A,B)]. After establishment of the fear conditioning in which the electrical shock was given to fish as unconditioned stimulus paired with presentation of the red light as conditioned stimulus [Fig. 4(C)], the manipulated fish showed extremely enhanced levels of freezing response to presentation of the conditioned stimulus, while the normal conditioned fish shows simply agitated behavior by increasing the frequency of turning [Fig. 4(D,E)]. This result suggests that the tract connecting the left-dominant dHbL with dIPN may normally function to suppress the choice of freezing as a response to fear after establishment of fear conditioning. This modulation of fear response by this neural tract is dependent of experience of fish. In the first conditioning trial, both the normal and manipulated fish showed freezing behavior at the similar frequency after the electrical shock, but the normal fish ceased to show freezing as they experience more and more conditioning trials, while the manipulated fish continued to freeze (Agetsuma et al., 2010). Narp is specifically enriched in the habenula (Reti et al., 2002), and encodes a secreted protein that is released at synaptic sites and affects trafficking of AMPA receptors by binding to the extracellular surface of AMPA receptors (O’Brien et al., 1999). Narp knockout mice show defects in reward devaluation (Johnson et al., 2007) and extinction of morphine place preference (Crombag et al., 2009), even though they are normal in many learning tasks (Johnson et al., 2007). Therefore, the highly enriched expression of Narp in the habenula may be at least partially responsible for the habenular functions as the modulators of behavioral strategy changes to cope with stress. RECIPROCAL CONNECTION OF THE IPN WITH THE RAPHE AND THE NUCLEI IN THE DORSAL TEGMENTAL REGION MAY BE CRITICAL FOR BEHAVIORAL CHOICE IN FEAR We showed that the majority of labeled axons from the dIPN projected bilaterally to the dorsal directions, through the region putatively corresponding to the dorsal raphe (DR) [Fig. 3(B)] (Agetsuma et al., 2010). They further extended laterally around the medial longitudinal fascicle [MLF, Fig. 3(B)]. This trajectory then turned caudally and elongated through the longitudinally extended region termed the gri- 391 seum centrale (GC) (Wullimann et al., 1996) underlying the rhombencephalic ventricle [Fig. 3(B)]. The GC is the periventricular structure that most likely includes the regions corresponding to the periaqueductal gray (PAG), the dorsal tegmental nucleus, and the nucleus incertus (NI) in the mammalian brain. As the IPN, the PAG, and the NI are implicated in control of behaviors under fear or stress conditions (Groenewegen et al., 1986; Fanselow, 1994; Bandler et al., 2000; Goto et al., 2001; Banerjee et al., 2010). This observation supports our hypothesis that the dHbL-d/iIPN pathway modulates fear behaviors. In mammals, the IPN together with the DR, and the NI is hypothesized to comprise the brain stem network involved in controlling behavioral activation (Shibata and Suzuki, 1984; Groenewegen et al., 1986; Goto et al., 2001; Maier and Watkins, 2005; Forster et al., 2006), making it highly likely that our findings in zebrafish could be extended to mammals. In mammals, different parts of PAG differentially regulate coping strategies against stress (Bandler et al., 2000). Both the rostral and caudal parts of the lateral or dorso-lateral PAG are responsible for active coping of stress, with the rostral parts evoking confrontational defensive posture and the caudal parts evoking flight escape behaviors. In contrast, the ventrolateral PAG is responsible for a passive coping reaction with freezing. Our observation with the zebrafish with specific defects in the dHbL-d/iIPN transmission suggests that the dHbL-d/iIPN pathway may modulate PAG for proper selection of the strategies to cope with stress, and silencing of this pathway biases the animals predisposed to a passive coping reaction, i.e., freezing (Agetsuma et al., 2010). In contrast to dIPN, the vIPN has the reciprocal connection with the median raphe (MR) [Fig. 3(C)] (Agetsuma et al., 2010). Therefore, these results revealed the existence of subdivided, parallel pathways, namely the dHbL-d/iIPN-GC pathway and the dHbM-v/iIPN-MR pathway. Considering the roles of the median raphe neurons in adjustment of adaptive behaviors and of PAG in instinctive defense behaviors such as fight, flight, and freeze, it is worth studying whether the specific connections of the dIPN and vIPN may further contribute to refinement of behavioral strategies to cope with stress varying from innate fight or flight to more adaptive goal-directed behavior for escape. It is known that human activates different systems of the brain, such as the central amygdala and the PAG for panic behavior vs. the lateral amygdala and the prefrontal cortex for adaptive behavior for escape, depending on the imminence of the threat (Mobbs et al., 2007). However, which part of the brain is reDevelopmental Neurobiology 392 Okamoto et al. sponsible for choice among these alternatives. Our results suggest the potential roles of habenula as a switching board for selection of behavioral strategy to cope with stressful condition appropriate for environmental contingencies. Recently, certain populations of the telencephalic neurons including those in the olfactory bulb were discovered to directly project to the right dHbM neurons in zebrafish (Miyasaka et al., 2009). Application of the fear substance derived from the con-specific skin extract causes intense freezing as we observed in the fish with silencing of the dHbL-d/iIPN pathway (Jesuthasan and Mathuru, 2008). If the skin extractevoked innate fear response is mediated by this direct connection of the olfactory system with the right dHbM, our results may suggest that the dHbL-d/iIPN and dHbM-v/iIPN pathways may have the opposing roles in control of freezing behaviors. THE HABENULAR ASYMMETRY AND THE BEHAVIORAL LATERALITY Adult zebrafish are known to use preferentially the right eye when they are approaching novel objects (Miklósi and Andrew, 1999). In the mutant zebrafish in which the direction of the left–right asymmetry in the habenula was inverted, the left eye instead of the right eye was preferentially used for novelty recognition (Barth et al., 2005). It is intriguing whether these observations are related to a possible difference in the properties of the left and right habenulo-IPN pathways in modulation of fear response strategies. Visual images of novel objects captured by the right eye may be less likely to evoke flight response than those captured by the left eye. And the difference in the ratio of the dHbL vs. dHbM may be responsible for asymmetric setting of threshold for induction of flight behavior. THE HABENULA AND MENTAL DISORDERS AFFECTING FEAR AND ANXIETY Habenula is metabolically hyperactive in congenitally helpless rats (Shumake et al., 2003). Habenula ablation completely blocks development of learned helplessness (Amat et al., 2001). These observations together with ours have implicated disorder of habenular functions in etiology of chronic depression or post-traumatic stress disorder (PTSD). It was recently shown that the larval zebrafish fail to learn to escape but rather reduce mobility in the conditioned learning Developmental Neurobiology for the active avoidance paradigm if the larval fish were pretreated with electrical shock under inescapable condition (Lee et al., 2010). 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