Ajay S. Mathuru

The Habenula Prevents Helpless Behavior in Larval Zebrafish

Animals quickly learn to avoid predictable danger. However, if pre-exposed to a strong stressor, they do not display avoidance even if this causes continued contact with painful stimuli [1, 2]. In rodents, lesioning the habenula, an epithalamic structure that regulates the monoaminergic system, has been reported to reduce avoidance deficits caused by inescapable shock [3]. This is consistent with findings that inability to overcome a stressor is accompanied by an increase in serotonin levels [4]. However, other studies conclude that habenula lesions cause avoidance deficits [5, 6]. These contradictory results may be caused by lesions affecting unintended regions [6]. To clarify the role of the habenula, we used larval zebrafish, whose transparency and amenability to genetic manipulation enables more precise disruption of cells. We show that larval zebrafish learn to avoid a light that has been paired with a mild shock but fail to do so when pre-exposed to inescapable shock. Photobleaching of habenula afferents expressing the photosensitizer KillerRed causes a similar failure in avoidance. Expression of tetanus toxin in dorsal habenula neurons is sufficient to prevent avoidance. We suggest that this region may signal the ability to control a stressor, and that its disruption could contribute to anxiety disorders.

The Alarm Response in Zebrafish: Innate Fear in a Vertebrate Genetic Model

The alarm response is an antipredator behavior displayed by many fish species and was first described 70 years ago. It is triggered through the olfactory system by substances released from injured skin and is characterized by dramatic, measurable changes in locomotion as well as physiology. We propose that this is an ideal time to revisit this response and to utilize it as an assay for understanding how neural circuits mediate innate fear. A suitable organism for these studies is the zebrafish, a genetic model with a rapidly expanding toolkit for molecular manipulation of the nervous system. Individual neurons mediating the response, ranging from receptor neurons to those in higher brain centers, should first be identified. New tools, specifically transgenic lines that allow spatial and temporal control of neural activity, provide a way to define and test the role of specific neurons, while genetic screens provide a route to identifying individual molecules essential for a normal response. Optical recording, which has proven successful in studies of information processing in the bulb, will provide valuable insights into neural circuitry function during the alarm response. When carried out on mutants, physiological analysis can provide insight into aspects of signal processing that are essential for normal behavior. The alarm response thus provides a paradigm to examine innate fear in a vertebrate system, enabling analysis at multiple levels from genes to the entire neural circuit. Additionally, the context dependency of the response can be utilized to investigate attention and decision making.

The medial habenula as a regulator of anxiety in adult zebrafish.

The habenula consists of a set of nuclei located in the epithalamus. It regulates the release of multiple neuromodulators including serotonin and dopamine, and consists of two major subdivisions—medial and lateral. In all vertebrates, the medial habenula projects to the interpeduncular nucleus (IPN), a midline structure with poorly defined functions (Morley, 1986). Both the medial habenula and the IPN are rich in nicotinic receptors (nAChR). Activity in this pathway, triggered by opioids and nicotine, leads to a rise in dopamine in the nucleus accumbens (Glick et al., 2006; McCallum et al., 2012) and thus underlies the rewarding aspect of substance abuse. Strong activation of nicotinic receptors in the medial habenula or IPN, however, is sufficient to mediate the aversion to high concentration of nicotine (Fowler et al., 2011; Frahm et al., 2011). In contrast, absence of activity in this pathway is critical for the effects of withdrawal (Salas et al., 2009; Baldwin et al., 2011). Hence, depending on the level of activity, the medial habenula-IPN pathway can trigger reward, aversion or the physical and emotional changes that are characteristic of withdrawal.

The Right Dorsal Habenula Limits Attraction to an Odor in Zebrafish

BACKGROUND The habenula consists of an evolutionarily conserved set of nuclei that control neuromodulator release. In lower vertebrates, the dorsal habenula receives innervation from sensory regions, but the significance of this is unclear. Here, we address the role of the habenula in olfaction by imaging neural activity in larval zebrafish expressing GCaMP3 throughout the habenula and by carrying out behavioral assays. RESULTS Activity in several hundred neurons throughout the habenula was recorded using wide-field fluorescence microscopy, fast focusing, and deconvolution. This enabled the creation of 4D maps of odor-evoked activity. Odors activated the habenula in two broad spatiotemporal patterns. Increasing concentrations of a putative social cue (a bile salt) evoked a corresponding increase in neuronal activity in the right dorsal habenula. In behavioral assays, fish were attracted to intermediate concentration of this cue but avoided higher concentration. Increasing cholinergic activity through nicotine exposure rendered the intermediate concentration aversive in a habenula-dependent manner. Pharmacologically blocking nicotinic receptors or lesioning the right dorsal habenula attenuated avoidance. CONCLUSIONS These data provide physiological and functional evidence that the habenula functions as a higher center in zebrafish olfaction and suggest that activity in the right dorsal subdomain gates innate attraction to specific odors.

Disruption of Esrom and Ryk identifies the roof plate boundary as an intermediate target for commissure formation

Growth cones are guided to their final destination by intermediate targets. Here, we identify intermediate targets and signaling components acting on zebrafish habenula commissural axons. Live imaging establishes that axons pause at the medial habenula before and after crossing the roof plate. esrom mutants axons fail to advance beyond the ipsilateral medial habenula. Tsc2 function is reduced in mutant axons, indicating cell autonomous defects in signaling. Consistent with signaling properties changing outside the roof plate, EphB is surface localized on axon segments within a zone demarcated by the medial habenula. wnt4a is expressed in the medial habenula and morpholino knockdown causes loss of the commissure. Electroporation of truncated Ryk causes axons to reenter the midline after reaching the contralateral habenula. These data identify Esrom as a mediator of growth cone navigation at an intermediate target and underscore the importance of midline boundaries as signaling centers for commissure formation.

A Microfluidic Device to Sort Cells Based on Dynamic Response to a Stimulus

Single cell techniques permit the analysis of cellular properties that are obscured by studying the average behavior of cell populations. One way to determine how gene expression contributes to phenotypic differences among cells is to combine functional analysis with transcriptional profiling of single cells. Here we describe a microfluidic device for monitoring the responses of single cells to a ligand and then collecting cells of interest for transcriptional profiling or other assays. As a test, cells from the olfactory epithelium of zebrafish were screened by calcium imaging to identify sensory neurons that were responsive to the odorant L-lysine. Single cells were subsequently recovered for transcriptional profiling by qRT-PCR. Responsive cells all expressed TRPC2 but not OMP, consistent with known properties of amino-acid sensitive olfactory neurons. The device can be adapted for other areas in biology where there is a need to sort and analyze cells based on their signaling responses.

Modeling Alzheimer’s and Other Age Related Human Diseases in Embryonic Systems

Modeling human disease in animals is an important strategy to discover potential methods of intervention. We suggest that there is much to be gained by employing a multi-model approach that takes advantage of different animal systems used in the laboratory simultaneously. We use the example of modeling Alzheimer’s disease in Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio to illustrate how such an approach can be employed to investigate the pathophysiology of the disease.

Correction to: Lie-X : Depth Image Based Articulated Object Pose Estimation, Tracking, and Action Recognition on Lie Groups

The original author list did not accurately reflect the contributions of the following colleagues.

Familiarity with companions aids recovery from fear in zebrafish

Interaction with social partners during or after a stressful episode aids recovery in humans and other mammals. We asked if a comparable phenomenon exists in zebrafish (Danio rerio) that live in shoals in the wild. In the first experiment, we observed that most quantifiable parameters of swimming behavior were similar when zerbafish swam alone or with companions. However, after exposure to an alarm substance (Schreckstoff), individuals recovering alone continued to display behaviors associated with fear after removal of the stimulus, while those recovering with companions did not. In the next two experiments, we examined the role of familiarity of companions. Subjects spent more time in the vicinity of familiar companions in a two-choice assay. While both familiar and unfamiliar companions reduced behavioral signs of distress, familiar companions additionally modulated cortisol and endogenous isotocin in subjects. Shortly after being united with familiar companions, isotocin spiked followed by a dampening of circulating cortisol levels. These results suggest that zebrafish experience fear attenuation in the presence of others and familiar companions are more effective at buffering the stress associated with escaping predation. Changes in behavior, circulating cortisol and isotocin levels due to social partners are reminiscent of changes due to amelioration of fear in some mammalian species in the presence of companions. The two phenomena may be related.

Conspecific injury raises an alarm in medaka

In the late 1930s, Karl von Frisch reported that semiochemicals released upon injury, act as alarm substances (Schreckstoff) in fish. In Ostariophysi species, club cells in the epidermis are believed to contain cues related to alarm substance; however, the function of club cells, primarily as reservoirs of alarm substance has been debated. Here, I describe an alarm response in the Japanese rice fish Oryzias latipes (medaka), a member of the order Beloniformes. The response to alarm substance (Schreckreaction) in medaka is characterized by bouts of immobility and an increase in cortisol levels within minutes of exposure to conspecific skin extract. Histological analysis, however, suggests that club cells are either rare or absent in the medaka epidermis. In addition to describing an uncharacterized behavior in a vertebrate popular for genetic and developmental studies, these results support the hypothesis that the primary function of epidermal club cells may be unrelated to a role as alarm substance cells. The existence of similar behavioral responses in two evolutionarily distant but well established laboratory models, the zebrafish and the medaka, offers the possibility of comparative analyses of neural circuits encoding innate fear.