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 medial habenula regulates the expression of fear in zebrafish. Following silencing with tetanus toxin or lesioning with nitroreductase (Agetsuma et al., 2010; Lee et al., 2010), enhanced freezing was seen in aversive conditioning paradigms in both adult and larvae. The freezing response in habenula-lesioned fish was experience-dependent. In the case of adults, both control and lesioned fish froze when first exposed to an electric shock paired with a red light (the conditioning stimulus, CS) in a Pavlovian conditioning paradigm. As more shocks were delivered, freezing decreased in control fish, but not in habenula-lesioned fish (Agetsuma et al., 2010). The experiment with larval fish involved instrumental conditioning: fish had to swim away from the side of the tank containing a red light, which had been paired with a mild electric shock. In this case, freezing appeared gradually in lesioned fish in the later half of the conditioning session, but never in control fish (Lee et al., 2010). One interpretation that has been suggested for these observations is that silencing the medial habenula biases fish to a passive coping strategy of freezing, due to modulation in the activity of the downstream griseum centrale [homologous to periaqueductal gray (PAG) in mammals] and nucleus incertus (Okamoto et al., 2012). As different regions of the PAG have been implicated in selection of different actions for coping against stress, a role for the medial habenula in such selection via PAG is a distinct possibility. An additional possibility is that medial habenula silencing increases anxiety in animals. A consequence of elevated generalized anxiety in an animal is predisposition toward larger fear responses (Davis et al., 2010). The swimming behavior of an isolated fish when introduced into a novel tank can be used as a measure of baseline generalized anxiety (Levin et al., 2007): anxious fish spendmore time near the bottom in a novel tank. Therefore, by using this assay, it is possible to quantify if adult fish where the medial habenula has been silenced display a similar, a lower, or a higher level of baseline anxiety when compared to control fish. We compared the behavior of adult fish that express the light chain of tetanus toxin (TeTXlc) in the medial habenula (Lee et al., 2010), under the control of the GAL4s1019t driver (Scott et al., 2007), with fish that did not express this transgene. TeTXlc expressing fish spent more time in the bottom half of the tank compared to controls (Figures 1A,B), consistent with the possibility that these fish may indeed have a higher baseline generalized anxiety. Mild stressors can elicit disproportionate reactions in anxious animals. In rats, for example, strong illumination, which is anxiogenic, causes an abnormally large startle to a tone (Walker and Davis, 1997). We examined the response of TeTXlc-expressing fish to naturalistic stimuli that are expected to be mildly stressful. Low concentration of the alarm substance (Jesuthasan andMathuru, 2008) normally triggers a response that naïve, control fish recover from within seconds (0.1 unit; Mathuru et al., 2012). We followed this with an overhead shadow, which is thought to mimic the threat of a predator (Luca and Gerlai, 2012). These stimuli provide an opportunity to examine the role of medial habenula when the subject is challenged with stimuli that mimic complex natural stressors as opposed to its role in conditioning experiments that use unnatural repetitive stimuli. In such conditions, control fish displayed behavior indicative of mild fear—marginal increase in episodes of darting [Wilcoxon Signed Rank Test (n = 7), p = 0.1; Figure 1F], while other measures remained unchanged. TeTxlcexpressing fish, in contrast, spent more time spent in the bottom quarter of the tank (Mann–Whitney U-test, n1, n2, 7, 9,U = 7, p = 0.005; Figure 1C), displayed