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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
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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
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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). Intriguingly, the
larval zebrafish of an enhancer-trap line in which the
afferent pathway to the habenula from the putative
telencephalic nucleus was inactivated showed the
similar behavioral defect in escape behavior in the
active avoidance paradigm, implicating the habenula
in the establishment of the condition similar to the
learned helplessness.
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