Margherita Zaupa

Seyedeh Maryam

Alavi Naini

Maroun Abi Younes

Erika Bullier

Erik R Duboué

Hervé Le Corronc

Hédi Soula

Sebastien Wolf

Raphael Candelier

Pascal Legendre

Marnie E Halpern

Jean-Marie Mangin

Elim Hong
email: elim.hong@inserm.fr

Maryam Alavi Naini

Affiliations

Trans-inhibition of axon terminals underlies competition in the habenulo-interpeduncular pathway

Keywords: Habenula, interpeduncular nucleus, nicotine, GABA B receptor, calcium imaging, electrophysiological recording, zebrafish

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The bilaterally paired habenular nuclei (Hb) are part of the highly conserved dorsal diencephalic conduction system connecting the limbic forebrain with monoaminergic brainstem areas [START_REF] Mclaughlin | The medial habenula and interpeduncular nucleus circuitry is critical in addiction, anxiety, and mood regulation[END_REF][START_REF] Fakhoury | The dorsal diencephalic conduction system in reward processing: Spotlight on the anatomy and functions of the habenular complex[END_REF] . Anatomically, they consist of medial (MHb) and lateral (LHb) nuclei in mammals that are homologous to the dorsal (dHb) and ventral (vHb) nuclei of teleosts. The LHb/vHb send projections through the fasciculus retroflexus (FR) directly to serotonergic raphe neurons and dopaminergic neurons in the ventral tegmental area. Axons from MHb/dHb neurons course through the FR and terminate at an unpaired midbrain target, the interpeduncular nucleus (IPN). The Hb-IPN pathway is critical for understanding addiction and mood disorders due to its involvement in regulating behaviors such as stress, anxiety and fear learning [START_REF] Mclaughlin | The medial habenula and interpeduncular nucleus circuitry is critical in addiction, anxiety, and mood regulation[END_REF][START_REF] Yamaguchi | Distinct roles of segregated transmission of the septo-habenular pathway in anxiety and fear[END_REF][START_REF] Seigneur | Cbln2 and Cbln4 are expressed in distinct medial habenula-interpeduncular projections and contribute to different behavioral outputs[END_REF][START_REF] Agetsuma | The habenula is crucial for experience-dependent modification of fear responses in zebrafish[END_REF][START_REF] Duboué | Left Habenular Activity Attenuates Fear Responses in Larval Zebrafish[END_REF] .

The MHb/dHb contains glutamatergic neurons are divided into those that corelease acetylcholine (cholinergic) or neuropeptides, including substance P (noncholinergic) or somatostatin [START_REF] Cuello | Substance P containing and cholinergic projections from the habenula[END_REF][START_REF] Kadar | Age-J o u r n a l P r e -p r o o f related changes in the cholinergic components within the central nervous system of cw1 female mice. I. Structural analysis[END_REF][START_REF] Hong | Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway[END_REF] . In rodents, cholinergic and non-cholinergic neuronal populations project to central and peripheral IPN domains, respectively [START_REF] Cuello | Substance P containing and cholinergic projections from the habenula[END_REF] . Gene expression analyses, fluorescent dye tracing and transgenic reporters have demonstrated left-right differences between the bilateral dHb of larval zebrafish [START_REF] Agetsuma | The habenula is crucial for experience-dependent modification of fear responses in zebrafish[END_REF][START_REF] Aizawa | Laterotopic representation of left-right information onto the dorso-ventral axis of a zebrafish midbrain target nucleus[END_REF][START_REF] Gamse | Directional asymmetry of the zebrafish epithalamus guides dorsoventral innervation of the midbrain target[END_REF] .

For example, cholinergic neurons are located mainly in the right dHb and project to the ventral IPN (vIPN), whereas non-cholinergic neurons are more abundant in the left dHb and innervate the dorsal IPN (dIPN) [START_REF] Hong | Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway[END_REF][START_REF] Decarvalho | Neurtransmitter map of the asymmetric dorsal habenular nuclei of Zebrafish[END_REF] (Figure 1A). In rodents, cholinergic and non-cholinergic MHb neurons receive afferent input from neurons located in the triangular septum (TS) and the bed of the anterior commissure (BAC), respectively [START_REF] Otsu | Functional Principles of Posterior Septal Inputs to the Medial Habenula[END_REF] .

Toxin-induced ablation of TS-cholinergic or BAC-non-cholinergic MHb projections selectively impaired anxiety or fear learning behaviors [START_REF] Yamaguchi | Distinct roles of segregated transmission of the septo-habenular pathway in anxiety and fear[END_REF] . Conditional genetic deletion J o u r n a l P r e -p r o o f of Cerebellin (Cbln) genes in the MHb, Cbln4 in cholinergic or Cbln2 in noncholinergic neurons, affects freezing behavior or spatial learning, respectively [START_REF] Seigneur | Cbln2 and Cbln4 are expressed in distinct medial habenula-interpeduncular projections and contribute to different behavioral outputs[END_REF] .

The dHb responds differently to external factors such as light, odor and aversive stimuli in zebrafish larvae [START_REF] Duboué | Left Habenular Activity Attenuates Fear Responses in Larval Zebrafish[END_REF][START_REF] Zhang | Left Habenula Mediates Light-Preference Behavior in Zebrafish via an Asymmetrical Visual Pathway[END_REF][START_REF] Dreosti | J o u r n a l P r e -p r o o f Left-right asymmetry is required for the habenulae to respond to both visual and olfactory stimuli[END_REF] . Inhibition of neural transmission in the noncholinergic (dHbL) versus cholinergic (dHbM) pathways in adult zebrafish is associated with winner or loser states in a social conflict paradigm [START_REF] Chou | Social conflict resolution regulated by two dorsal habenular subregions in zebrafish[END_REF] . Studies in rodents and zebrafish suggest that the cholinergic and non-cholinergic MHb/dHb neuronal populations form functionally distinct, parallel circuits. However, it is unknown whether the two pathways interact or directly influence the activity of one another.

To understand functional connectivity of the dHb-IPN pathway, we took advantage of the small size of the larval zebrafish brain to monitor neural activity in whole explant preparations. Calcium imaging revealed that dHb terminals at the IPN can be resolved into distinct domains based on their signaling dynamics. We discovered an inverse relationship between cholinergic and non-cholinergic neurons only at their axon terminals. Results from pharmacology, imaging and electrophysiology experiments show that synchronized activity of cholinergic dHb neurons results in GABA release from the IPN, leading to presynaptic inhibition of non-cholinergic axon terminals. We propose a hardwired mode of competition between two neuronal populations, whereby synchronized activation of one group inhibits the activity of the other.

Results

Cholinergic and non-cholinergic dHb terminals exhibit negatively correlated calcium events

J o u r n a l P r e -p r o o f

The bilateral dHb on the dorsal surface of the diencephalon form connections with the IPN deeply embedded in the ventral midbrain, making it challenging to simultaneously monitor and manipulate both pre-and post-synaptic neuronal populations. To identify the hardwired mode of action of the dHb-IPN pathway, we analyzed spontaneous activity in both regions using brain explants from larval zebrafish.

The entire brain was removed from

TgBAC(gng8:GAL4ff) c426 ;Tg(UAS:GCaMP7a) larvae expressing GCaMP in dHb neuronal cell bodies and their terminals at the IPN [START_REF] Duboué | Left Habenular Activity Attenuates Fear Responses in Larval Zebrafish[END_REF] . We found that whereas most dHb neurons exhibit spontaneous activity (Video S1, Figure S1A), a large cluster of neurons on the right together with a small cluster on the left exhibited correlated calcium bursts (Figure 1B-E, Video S1). The neuronal clusters with highly correlated calcium bursts were consistently identified by k-means and hierarchical clustering methods (Figure 1B-E, Figure S1). Their location (Figure 1B, Figure S1, green and blue) corresponds to that previously described for cholinergic and somatostatinergic neuronal clusters in the dHb (Figure 1A) [START_REF] Hong | Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway[END_REF][START_REF] Decarvalho | Neurtransmitter map of the asymmetric dorsal habenular nuclei of Zebrafish[END_REF] .

We examined whether similar calcium signals were present in dHb axon terminals at the IPN. As the left dHb contains cholinergic and non-cholinergic neurons, which form connections with the dorsal and ventral IPN (Figure 1A Notably, terminals at the dIPN and raphe exhibited negatively correlated calcium signals (Figure 1L), which we refer to as negatively correlated events.

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Calcium inhibition only occurred in terminals at the dIPN when high amplitude bursts were observed at the vvIPN and raphe (Figure 1N) but not when calcium bursts were restricted to the dvIPN (Figure 1O). This suggests that dHb neurons that target the dvIPN differ from those innervating the vvIPN and raphe. Moreover, prior to or following a negatively correlated event, terminals at the dIPN showed higher GCaMP signals compared to those observed in the vIPN (Figure 1K,N; Figure S2A,B). The higher signals were not merely due to greater terminal density at the dIPN relative to the vIPN because we found no difference in the ratio of axon terminals in the two regions when labeled with GFP (Figure S2A'-B).

Quantification of calcium signaling kinetics revealed that the time to reach maximum amplitude (peak time) occurred earlier in terminals at the vvIPN compared to the raphe (1.13 ± 0.42 s vs. 1.59 ± 0.72 s), and the duration at half peak time (half duration) of the calcium burst was shorter (2.72 ± 0.56 s vs. 3.64 ± 1.55 s) (Figure S3A-C). Peak inhibition in terminals at the dIPN occurred later (4.33 ± 1.4 s) and lasted longer (9.2 ± 2.9 s) than the calcium bursts (Figure S3D,E). Activation at the vvIPN was followed by a period of inhibition not observed in terminals at the raphe.

Therefore, to resolve the relationship between the negatively correlated events, we measured activity in terminals at the dIPN and at the raphe as a proxy for the vvIPN.

We found a 931 ± 50 ms delay between the onset of the calcium burst and inhibition (Figure 2A), suggesting that calcium activity at the vvIPN triggers calcium inhibition in the dIPN.

To identify the neurotransmitter phenotype of neurons that exhibit calcium bursts at the vvIPN and raphe, we assessed Vesicular Acetylcholine Transporter (VAChT) expression by immunolabeling in Tg(nptx2:Gal4-VP16) rw0143a ;Tg(UAS:GFP) c354 larvae. We previously found that this transgenic line J o u r n a l P r e -p r o o f mainly labels non-cholinergic neurons in the left dHb and their projections to the dIPN [START_REF] Decarvalho | Neurtransmitter map of the asymmetric dorsal habenular nuclei of Zebrafish[END_REF] . We confirmed that GFP expression does not colocalize with VAChT protein (Figure 2B) whereas dHb terminals at the vvIPN and raphe are positive for VAChT staining in TgBAC(gng8:GAL4ff) c426 ;Tg(UAS:GFP) c354 larvae (Figure 2B). We therefore conclude that calcium bursts at these regions are due to the synchronized activation of dHb cholinergic neurons (Figure 2C). Negatively correlated events are already apparent in 3-day-old larvae (Figure S4) upon emergence of spontaneous activity in dHb neurons (data not shown), suggesting that it is a hardwired mode of activity, coincident with establishment of the dHb-IPN pathway.

To determine whether external stimuli provoke negatively correlated events in vivo, we applied a mild electric shock to larvae. Indeed, we observed negatively correlated events between dHb terminals at the dIPN and vIPN/raphe (Figure 2D).

However, due to tissues surrounding the brain, the resolution of calcium activity was inferior in larvae compared with the brain explants. Nevertheless, this result indicates that negatively correlated events can be induced in vivo, suggesting it is a physiologically relevant mode of neuronal activation.

Nicotinic acetylcholine receptors do not mediate negatively correlated events

The MHb/dHb-IPN pathway shows the highest expression and greatest variety of nicotinic acetylcholine receptor (nAChR) subunits in the brain [START_REF] Antolin-Fontes | The habenulo-interpeduncular pathway in nicotine aversion and withdrawal[END_REF] . Since calcium inhibition in non-cholinergic dHb terminals is preceded by a calcium burst in cholinergic terminals, we investigated whether inhibition depends on the activation of nAChRs [START_REF] Hong | Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway[END_REF] . Perfusion of nicotine resulted in prolonged calcium inhibition of dHb terminals at the dIPN, which corresponded with an extended series of calcium bursts in dHb terminals at the raphe (Figure 3A,A', 260 ± 176 s). To determine the J o u r n a l P r e -p r o o f contribution of nAChRs in the generation of negatively correlated events, we administered mecamylamine and d-tubocurarine, effective blockers of cholinergic transmission in the larval IPN [START_REF] Hong | Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway[END_REF] . nAChRs antagonists did not attenuate, but rather increased the frequency of negatively correlated events (Figure 3B-C'). Thus, activation of nAChRs is not required for negatively correlated events between cholinergic and non-cholinergic dHb terminals at the IPN.

dHb terminal calcium bursts correlate with GABAergic currents in IPN neurons

To understand the basis for negatively correlated events, we combined calcium imaging of dHb terminals with patch-clamp recordings of neurons at the interface between the dIPN and vIPN. Recording at -60 mV (equilibrium potential of Cl -ions) revealed that calcium bursts correlated with a barrage of postsynaptic inward currents consisting of fast and slow events (Figure 4A,B), which is due to release of glutamate and acetylcholine [START_REF] Hong | Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway[END_REF] . Recording at 0 mV (reversal potential of cationic ions) revealed that calcium bursts also correlated with a barrage of outward currents In zebrafish, there are two GABA B 1 receptor genes, GABA B receptor 1a (gabbr1a) and 1b (gabbr1b) [START_REF] Cocco | Characterization of the γ-aminobutyric acid signaling system in the zebrafish (Danio rerio Hamilton) central nervous system by reverse transcription-quantitative polymerase chain reaction[END_REF] . We found that gabbr1a, but not gabbr1b, is prominently expressed in the dHb in a larger domain of the left nucleus compared with the right, in the location of non-cholinergic neurons (Figure 5D,E). Strikingly, gabbr1a transcripts localized to the dHb nuclei and were not detected in other brain regions including the IPN (Figure 5D). Together, these results suggest that calcium inhibition is due to the activation of presynaptic GABA B receptors in non-cholinergic terminals at the dIPN.

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Retrograde signaling from GABAergic IPN neurons to non-cholinergic habenular inputs

To determine that inhibition of dHb terminals at the dIPN results from GABA released upon IPN activation, we carried out electrical stimulation of the IPN with calcium imaging (Figure 6A, Video S4). Activation of the IPN effectively triggered negatively correlated events consisting of the vIPN/raphe calcium burst (likely due to the activation of the dHb terminals at the vIPN) and dIPN calcium inhibition (Figure 6B).

We next perfused a GABA B antagonist, cgp55845, during negatively correlated events induced by electrical stimulation and found a 90 ± 13 % decrease in calcium inhibition at the dIPN (-0.15 ± 0.07 vs. -0.02 ± 0.02) without any change in calcium bursts at the raphe (0.22 ± 0.08 vs 0.2 ± 0.06) (Figure 6B-E).

To verify the presence of GABAergic neurons in the IPN, we imaged the TgBAC(gng8:GAL4ff) c426 ;Tg(UAS:GFP) c426 ;Tg(gad1b:|R|-GFP) larvae in which dHb terminals are labeled with GFP and GABAergic neurons by RFP [START_REF] Satou | Transgenic tools to characterize neuronal properties of discrete populations of zebrafish neurons[END_REF] . We found that nearly half of IPN neurons were labeled by gad1b:RFP (48 ± 2%, n = 3 larvae, N = 479 cells, Figure 6F), with more located in the dIPN than vIPN (79 ± 11% vs. 21 ± 11%, n = 3 larvae, N = 232 gad1b:RFP + cells). These results suggest that a higher concentration of GABA is released from neurons in the dIPN.

Discussion

The dHb of zebrafish larvae respond to external factors including light, odor and aversive stimuli [START_REF] Duboué | Left Habenular Activity Attenuates Fear Responses in Larval Zebrafish[END_REF][START_REF] Zhang | Left Habenula Mediates Light-Preference Behavior in Zebrafish via an Asymmetrical Visual Pathway[END_REF][START_REF] Dreosti | J o u r n a l P r e -p r o o f Left-right asymmetry is required for the habenulae to respond to both visual and olfactory stimuli[END_REF] . A prerequisite to investigating the innate hardwired activity of the dHb-IPN pathway was eliminating the influence of external factors. We achieved this by performing calcium imaging using a whole brain explant preparation, in which central neural circuits including the dHb-IPN pathway remain intact. We found a J o u r n a l P r e -p r o o f stereotyped mode of spontaneous activity in dHb soma and axon terminals at the IPN.

Through pharmacological and electrophysiological manipulations, we determined that synchronized activation of dHb cholinergic neurons inhibit non-cholinergic neurons by an atypical mechanism of target-mediated, trans-inhibition at the level of axon terminals.

Negatively correlated events at axon terminals of cholinergic and noncholinergic dHb neurons

Clustering algorithms are commonly used to segregate neurons into different functional groups during calcium imaging of neuronal soma [START_REF] Panier | Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy[END_REF][START_REF] Dunn | Brain-wide mapping of neural activity controlling zebrafish exploratory locomotion[END_REF][START_REF] Jetti | Spontaneous activity governs olfactory representations in spatially organized habenular microcircuits[END_REF] . The k value and threshold for hierarchical clustering were determined from a previous study of the zebrafish dHb [START_REF] Jetti | Spontaneous activity governs olfactory representations in spatially organized habenular microcircuits[END_REF] and manual inspection of the activity of individual neurons assigned to different clusters. We obtained similar but not identical results from k-means and hierarchical clustering methods. Nevertheless, the minor difference resulting from the clustering algorithms does not alter the conclusion showing that most dHb neurons on the right and a small cluster on the left show high levels of synchronized spontaneous activity. The location of these neurons corresponds to different neurotransmitter phenotypes: cholinergic and somatostatin [START_REF] Decarvalho | Neurtransmitter map of the asymmetric dorsal habenular nuclei of Zebrafish[END_REF] , suggesting that dHb neurons expressing the same neurotransmitter function together.

In analyzing group dynamics of axon terminals, we found it important to also carry out manual inspection of raw data. For example, only calcium peaks at the vvIPN and not the dvIPN correlated with dIPN inhibition. VAChT antibody labeling in transgenic lines confirmed that cholinergic dHb terminals innervate only the vvIPN, indicating that the dvIPN is innervated by different types of neurons. In addition, a J o u r n a l P r e -p r o o f small domain in the caudal vIPN clustered together with the terminals at the dIPN.

While there are currently no available molecular markers to differentiate between the dHb axon terminals at the cvIPN and dIPN, spatial segregation between the two areas suggests that they are targeted by different dHb neurons. In conclusion, cholinergic neurons exhibit synchronized activity and project to the vvIPN/raphe, which displays negatively correlated events with axon terminals at the dIPN (Figure 2C).

Nicotine promotes negatively correlated events in the dHb-IPN pathway

We found that nicotine induces prolonged duration of the negatively correlated event.

We previously showed that a cocktail of nAChR antagonists inhibits approximately 80% of cholinergic currents in IPN neurons [START_REF] Hong | Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway[END_REF] . However, application of antagonists failed to attenuate but, rather, increased the frequency of negatively correlated events. The cholinergic system can increase the excitability of GABAergic interneurons in the neocortex and the lateral habenula [START_REF] Picciotto | Acetylcholine as a Neuromodulator: Cholinergic Signaling Shapes Nervous System Function and Behavior[END_REF][START_REF] Jo | Cholinergic modulation of purinergic and GABAergic co-transmission at in vitro hypothalamic synapses[END_REF] . Likewise, our data suggest that GABAergic neurons are activated by nAChRs in the IPN. We hypothesize that blocking nAChRs throughout the whole brain inhibits GABAergic signaling, resulting in a global increase in synchronized network activity.

While nAChR activation can generate a negatively correlated event, the release of GABA from IPN neurons to induce inhibition is predominantly triggered by glutamatergic rather than cholinergic transmission from the dHb terminals at the IPN during spontaneous activity,

Localization of the GABA B receptor to non-cholinergic terminals at the dIPN mediates presynaptic inhibition

J o u r n a l P r e -p r o o f activates GABAergic neurons in the IPN resulting in the 3) release of GABA that through retrograde signaling 4) activates inhibitory presynaptic GABA B receptors on non-cholinergic dHb terminals at the dIPN, leading to 5) inhibition of glutamatergic transmission (Figure 6G).

How does activation of vvIPN neurons induce GABA release by dIPN neurons? Single cell labeling experiments showed the diverse morphology of IPN neurons including those that appear to be interneurons extending their processes between the dorsal and vIPN [START_REF] Bianco | Brain asymmetry is encoded at the level of axon terminal morphology[END_REF] . We hypothesize that communication between vvIPN and dIPN neurons could be mediated by such interneurons connecting the dorsal and ventral IPN. Another possibility is that more vvIPN neurons are GABAergic than visualized by the transgenic line used in this study, akin to what has been shown for the adult IPN [START_REF] Mueller | The distribution of GAD67-mRNA in the adult zebrafish (teleost) forebrain reveals a prosomeric pattern and suggests previously unidentified homologies to tetrapods[END_REF] . Thus, GABAergic vvIPN neurons might form direct synaptic connections, or through non-synaptic volume transmission, release GABA to inhibit non-cholinergic terminal activity at the dIPN.

As the MHb/dHb-IPN pathway is evolutionarily conserved in all vertebrates, including rodents and zebrafish [START_REF] Hong | Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway[END_REF][START_REF] Ren | Habenula " Cholinergic" Neurons Corelease Glutamate and Acetylcholine and Activate Postsynaptic Neurons via Distinct Transmission Modes[END_REF] , it is likely that this mode of hardwired activity is not unique to zebrafish. Indeed, a recent study on mice reports that presynaptic J o u r n a l P r e -p r o o f

Model for competitive selection between dHb circuits on behavior

It is crucial for an animal to identify the most salient stimulus amongst many concurrent external stimuli and select a single behavioral response. One example is the flight, freeze or fight response [START_REF] Eilam | Die hard: A blend of freezing and fleeing as a dynamic J o u r n a l P r e -p r o o f defense -Implications for the control of defensive behavior[END_REF] . During early stages of development, hardwired circuits to execute specific behavioral repertoires, such as escape (flight), are essential for survival [START_REF] Hale | Neural circuits that drive startle behavior, with a focus on the Mauthner cells and spiral fiber neurons of fishes[END_REF] . The process of transforming multiple sensory inputs into a single behavioral outcome is termed competitive selection and includes selective attention and decision-making [START_REF] Mysore | Mechanisms of competitive selection: A canonical neural circuit framework[END_REF] . Computational formulations such as winner-take-all models have been proposed [START_REF] Yuille | A Winner-Take-All Mechanism Based on Presynaptic Inhibition Feedback[END_REF][START_REF] Maass | On the computational power of winner-take-all[END_REF] and recent studies have begun to elucidate the mechanisms for behavioral selection in competing circuits [START_REF] Fadok | A competitive inhibitory circuit for selection of active and passive fear responses[END_REF][START_REF] Shang | Divergent midbrain circuits orchestrate escape and freezing responses to looming stimuli in mice[END_REF][START_REF] Hong | Antagonistic control of social versus repetitive self-grooming behaviors by separable amygdala neuronal subsets[END_REF][START_REF] Koyama | A circuit motif in the zebrafish hindbrain for a two alternative behavioral choice to turn left or right[END_REF] .

Higher baseline GCaMP fluorescence in the terminals at the dIPN relative to the vIPN suggests that non-cholinergic neurons display high frequency oscillation [START_REF] Li | Measuring sharp waves and oscillatory population activity with the genetically J o u r n a l P r e -p r o o f encoded calcium indicator GCaMP6f[END_REF] .

We propose that the synchronized burst in cholinergic terminals at the vIPN transiently inhibits this constitutive activity that occur in non-cholinergic neurons, ensuring only one pathway is active at any given time.

What could be the advantage of having local inhibition at the terminal level?

Different external stimuli preferentially activate left or right dHb neurons [START_REF] Duboué | Left Habenular Activity Attenuates Fear Responses in Larval Zebrafish[END_REF][START_REF] Zhang | Left Habenula Mediates Light-Preference Behavior in Zebrafish via an Asymmetrical Visual Pathway[END_REF][START_REF] Dreosti | J o u r n a l P r e -p r o o f Left-right asymmetry is required for the habenulae to respond to both visual and olfactory stimuli[END_REF] .

However, the ratio of asymmetric dHb activation depends on the intensity of the stimulus for both light and electric shock (personal observations), suggesting that the left and right dHb can be co-activated by the same stimulus. We suspect that this setup of local inhibition would ensure that 1) only one circuit is active at a time and 2) upon exposure to multiple stimuli a hierarchy is established between the stimuli.

J o u r n a l P r e -p r o o f

Aversive behavior refers to emotional states with negative valence, such as fear or anxiety. Fear is defined as a response to a factual known threat while anxiety to an unknown, poorly defined threat. Brain areas that contribute to fear and anxiety exhibit great overlap including the amygdala, medial prefrontal cortex and the hippocampus [START_REF] Tovote | Neuronal circuits for fear and anxiety[END_REF] . While recent optogenetic and in vivo studies have begun unraveling the microcircuits that participate in these behaviors [START_REF] Fadok | A competitive inhibitory circuit for selection of active and passive fear responses[END_REF][START_REF] Tovote | Midbrain circuits for defensive behaviour[END_REF] , how they interact with other areas of the brain are unclear. The highly conserved MHb/dHb-IPN pathway consists of two circuits composed of different neuronal populations, which mediate fear-or anxiety-related behaviors [START_REF] Yamaguchi | Distinct roles of segregated transmission of the septo-habenular pathway in anxiety and fear[END_REF][START_REF] Seigneur | Cbln2 and Cbln4 are expressed in distinct medial habenula-interpeduncular projections and contribute to different behavioral outputs[END_REF][START_REF] Agetsuma | The habenula is crucial for experience-dependent modification of fear responses in zebrafish[END_REF][START_REF] Chou | Social conflict resolution regulated by two dorsal habenular subregions in zebrafish[END_REF] . We propose that the trans-inhibition mechanism between cholinergic and non-cholinergic dHb-IPN circuits underlies competitive selection of aversive behavioral responses.

A brief electric shock induces fast swim followed by freezing behavior before returning to baseline locomotion in larval zebrafish [START_REF] Duboué | Left Habenular Activity Attenuates Fear Responses in Larval Zebrafish[END_REF] . Here, we demonstrate that it can also promote negatively correlated events in the dHb terminals at the IPN. We hypothesize that activation of the cholinergic pathway by electric shock results in a stereotypical fast swim (flight) response, which is followed by inhibition of the noncholinergic pathway allowing the animal to freeze. Prior to puberty, a flight/freeze response would be more beneficial than a fight response. In juveniles/adults, this dual circuit likely becomes more complex as different neuropeptides become expressed in the dHb-IPN pathway and with the maturation of behavioral repertoires from prior experience. For example, adult zebrafish exhibit a fight or flight response, manifested by a "winner" or "loser" phenotype in a social conflict paradigm, which is correlated with potentiation in non-cholinergic and cholinergic neuronal terminals, respectively [START_REF] Chou | Social conflict resolution regulated by two dorsal habenular subregions in zebrafish[END_REF] . We hypothesize that the "loser"/flight behavior corresponds to the default response to an aversive stimulus upon activation of the cholinergic pathway.

J o u r n a l P r e -p r o o f

The "winner"/fight behavior could result from preferential hyper-activation of the noncholinergic pathway, corresponding to a heightened level of anxiety.

In conclusion, investigating neuronal population dynamics within an intact pathway has revealed an atypical mode of trans-inhibition between two excitatory neuronal populations at their axon terminals. This hardwired mode of competition could underlie the mechanism for competitive selection between these neural circuits and provide a physiological framework to explore the relationship between anxiety and fear. (G) Model illustrating the mechanism underlying negatively correlated events in dHb terminals at the IPN (see discussion).

STAR Methods

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Elim Hong ( elim.hong@inserm.fr ).

Material availability

All plasmids generated in this study are available upon request to the Lead Contact.

Data and code availability

 All data reported in this paper will be shared by the Lead Contact upon request.

 This paper does not report original code.

 Any additional information required to reanalyze the data reported in this paper is available from the Lead Contact upon request.

J o u r n a l P r e -p r o o f μM, Acros Organics), tetrodotoxin (3 μM), baclofen (2 μM) and cgp55845 (1 μM) were used. The drugs were purchased from Tocris Bioscience unless otherwise noted.

Calcium Imaging

Calcium imaging was performed on a spinning disk microscope (Zeiss Axio Examiner.Z1) using a 40X water immersion objective (NA =0.95) or Leica SP5 laser scanning confocal microscope using a 25X water immersion objective (NA = 0.95).

Images were acquired at a rate between 2.2-6.7Hz, depending on the experiment.

Nicotine was perfused for 5 minutes followed by E3 medium to wash out the drug.

For experiments with mecamylamine hydrochloride and D-tubocurarine chloride, as a short-term application did not show any changes in calcium signals, we carried out the following experiment to assay for long-term change: baseline calcium signals were recorded for 15 minutes before pipetting the drug directly to the explant. The calcium signals were recorded for an additional 45 minutes after drug application.

For the cgp55845 and baclofen experiments, 10-15 minutes baseline activity was recorded followed by drug application using the perfusion system and imaged for another 15 minutes.

Patch-clamp recordings and simultaneous Ca 2+ imaging

Explants from TgBAC(gng8:GAL4ff) c426 ;Tg(UAS:GCaMP7a) larvae were placed in a recording chamber and constantly perfused with Ringer's solution at a rate of 1-2 mL/min. The IPN was made accessible for patch-clamp recordings and Ca 2+ imaging by orienting the explants with the ventral surface upward. dHb terminals and the IPN were located and imaged using a 63x water immersion objective mounted on an

RNA in situ Hybridization

PCR fragments for gabbr1a (ENSDARG00000018967) (553-bp) and gabbr1b

(ENSDARG00000016667) (539-bp) were subcloned into pCRII-TOPO vector using the TOPO®TA cloning Kit (Invitrogen). Reverse primer sequences were from [START_REF] Cocco | Characterization of the γ-aminobutyric acid signaling system in the zebrafish (Danio rerio Hamilton) central nervous system by reverse transcription-quantitative polymerase chain reaction[END_REF] . The following forward primers were used: gabbr1a (ACGTATGGTTCCTCATCGGC) gabbr1b (AGACAAGAGAAAGCACCTGGA). Restriction enzymes and RNA polymerase used to synthesize the antisense RNA probes are as follows: gabbr1a

(KpnI/T7), gabbr1b (KpnI/T7). Colorimetric in situ hybridization (ISH) assay was performed as described in [START_REF] Thisse | In situ hybridization on whole-mount zebrafish embryos and young larvae[END_REF] . Following ISH, the larvae were mounted in 100%

glycerol and imaged on a Nikon Eclipse E800 microscope.

J o u r n a l P r e -p r o o f

Electrical shock assay

Custom built electrical stimuli setup consisted of copper mesh electrodes that were mounted 6 cm apart on opposing sides and connected to a DS3 isolated constant current stimulator (Digitimer, Ltd.). Three electrical field stimulations (6mA, 20V, 500msec) were triggered every five minutes by MetaMorph (Molecular Devices). The were paralyzed in 2% pancuranium bromide and mounted in the middle of 90mm diameter petri dishes filled with 30 ml E3 medium to ensure consistent stimulation. Shock assay was performed in 6-7 dpf agarose-restricted larvae, paralyzed with 2% pancuranium bromide.

Electrical stimulation

The IPN neurons were stimulated using a low-resistance (<1MΩ) glass pipette filled with Ringer solution containing an AgCl electrode. Train stimulations (20µs duration 50Hz stimulations lasting 400ms) were triggered every 1 minute with an isolated current stimulator (DS3; Digitimer Ltd.) using a Master-8 pulse generator (A.M.P.I). Prior to each experiment, a dose-dependent curve was performed to select the optimal setting that induced negatively correlated events in the terminals at the IPN. Stimuli ranged between 100 µA -1.2 mA.

QUANTIFICATION AND STATISTICAL ANALYSIS

Calcium data analysis

Average fluorescence intensity of habenular soma was calculated by manually drawing regions of interest (ROI) in FIJI as described in [START_REF] Duboué | Left Habenular Activity Attenuates Fear Responses in Larval Zebrafish[END_REF] . To carry out a non-biased analysis of the dHb-IPN pathway and dHb terminals at the IPN, 2x2 μm 2 grid ROIs were drawn over the entire recording using FIJI macro. Average fluorescence value during a calcium burst. Peak of calcium inhibition was defined by the lowest value during a calcium inhibition event. Half duration was calculated as the width of the calcium signal at the half value of a peak during a calcium burst or inhibition. All graphs and plots were made on MATLAB using custom written scripts.

Statistical analysis

Statistical tests were carried out using a Wilcoxon signed rank test for averaged paired samples, Kruskal-Wallis test followed by a Dunn's test for comparison between more than 2 groups, Mann-Whitney test was used with independent samples and a linear mixed model with fixed effect was used to compare multiple values from repeated events. All statistical tests were performed on R or GraphPad Prism (GraphPad Software, Inc.). p <0.05 was considered as statistically significant.

  )[START_REF] Hong | Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway[END_REF][START_REF] Decarvalho | Neurtransmitter map of the asymmetric dorsal habenular nuclei of Zebrafish[END_REF] , we carried out simultaneous calcium imaging of neurons in the left dHb and axon terminals at the IPN (Video S2). We used k-means clustering to identify domains in an unbiased manner since results were comparable with hierarchical clustering. Distinct domains of spontaneous activity defined stereotypic and correlated patterns of dHb neurons. The soma of the left dHb segregated into two (dorsal and ventral) and dHb axon terminals fell into three domains at the dorsal IPN (dIPN), ventral IPN (vIPN) and raphe nucleus (raphe) (Figure1F). The activation pattern of ventral dHb J o u r n a l P r e -p r o o f soma clustered together with that of the raphe area, suggesting that these regions are functionally connected (Figure1F, green). Surprisingly, we found that calcium signals in the dIPN were negatively correlated with those in the ventral dHb soma and raphe (Figure1F-I, red). When an increased calcium signal (burst) occurred in the soma of the ventral dHb and terminals at the raphe, a transient decrease from baseline calcium signaling (inhibition) was observed at the dIPN (Figure1H,I).Calcium inhibition was limited to terminals at the dIPN and was not observed in the soma of dHb neurons (Figure1F). Indeed, analysis of different dHb focal planes in multiple explants failed to reveal neuronal cell bodies exhibiting negatively correlated calcium signals during spontaneous activity (Figure1C-E, FigureS1). The results indicate that calcium signals in dHb soma are not always reflective of the calcium dynamics at their terminals.Calcium signaling data also revealed a local negative interaction between dHb terminals at the IPN. We analyzed activity in dHb terminals throughout the IPN (Video S3) and by applying k-means clustering and manual inspection of the calcium signals, we identified five spatially segregated domains of axon terminals with distinct patterns of activity: 1) dIPN showing mainly calcium inhibition with few calcium bursts (red); 2) dorsal half of the vIPN (dvIPN) displaying frequent calcium bursts of low and high amplitude, with the high amplitude bursts followed by calcium inhibition (grey); 3) ventral half of the vIPN (vvIPN), showing high amplitude calcium bursts followed by inhibition (blue); 4) caudal area of the vIPN (cvIPN) (magenta), which clustered together with the dIPN exhibiting calcium inhibition; and 5) raphe area exhibiting calcium bursts without inhibition (green) (Figure1J-P).

(

  Figure4A',B') indicative of GABAergic inhibitory postsynaptic currents. The cholinergic/glutamatergic postsynaptic barrage was relatively short (half duration = 262 ± 124 ms) and synchronized with the onset of the calcium burst (delay = 67 ± 69 ms). In contrast, the barrage of GABAergic synaptic currents had a delayed onset (delay = 162 ± 80 ms) and lasted longer (half duration = 387 ± 143 ms). These results suggest that calcium bursts correspond to the release of glutamate and acetylcholine from dHb terminals at the IPN, which is followed by local release of GABA from IPN neurons.J o u r n a l P r e -p r o o f 10Presynaptic GABA B receptors in dHb axon terminals mediate calcium inhibitionGABA has an inhibitory presynaptic action on transmitter release through metabotropic GABA B receptors[START_REF] Dutar | A physiological role for GABAB receptors in the central nervous system[END_REF] . We asked whether GABA released by the IPN could activate presynaptic metabotropic GABA B receptors, thereby repressing dHb terminal activity. Application of cgp55845, a GABA B receptor antagonist, resulted in a 74 ± 9 % reduction in the amplitude of calcium inhibition (-0.16 ± 0.03 vs. -0.04 ± 0.01) without affecting the amplitude of calcium bursts (0.22 ± 0.09 vs. 0.26 ± 0.13) (Figure5A,A'). To test the role of GABA B receptors directly, we applied tetrodotoxin to inhibit the propagation of action potentials and eliminate all negatively correlated events. We then administered baclofen, a GABA B receptor agonist, and observed a prolonged decrease in the calcium baseline, similar in amplitude to that observed during inhibition of dHb terminals at the dIPN (Figure5B,B'). Calcium imaging of dHb terminals together with patch-clamp recordings of IPN neurons confirmed that prolonged inhibition corresponds with a decrease in the frequency of fast postsynaptic inward currents recorded in IPN neurons (Figure5C,C').

Figure 1 .

 1 Figure 1. Identification of functional domains in dHb-IPN pathway during

Figure 2 .

 2 Figure 2. Synchronized cholinergic terminal activity at vIPN/raphe precedes

Figure 3 .

 3 Figure 3. Nicotine induces prolonged negatively correlated event

Figure 4 .

 4 Figure 4. Calcium bursts correspond to acetylcholine/glutamate and

Figure 5 .

 5 Figure 5. Calcium inhibition occurs via presynaptic GABA B receptors

J o u r

  n a l P r e -p r o o f epifluorescence microscope BX51W1 (Olympus) equipped with a 470 nm LED fluorescent excitation light. Patch electrodes were filled with an intracellular solution containing 130 mM CsMeSO4, 4 mM MgCl2, 4 mM Na-ATP, 0.3 mM Na-GTP, 10 mM Hepes, and 10 mM EGTA, 5 mM QX-314 adjusted to pH 7.2, 290 mOsm, for resistances between 5 and 8 MΩ. This intracellular solution allowed discrimination of cationic excitatory currents and chloride inhibitory currents based on their distinct reversal potentials (E cation = 0 mV ; E Cl = -60 mV). By recording the cells at -60 mV, EPSCs appeared as inward currents, whereas IPSCs were barely detectable.Conversely, IPSCs appeared as outward currents when recording the cells at a holding potential of -0 mV, while EPSCs became undetectable. Whole-cell recordings were obtained using a Multiclamp 700B amplifier connected to a digidata 1440 acquistion system monitored using pClamp 10 software (Molecular Devices).Analyses were performed using Clampfit 10 (Molecular Devices). For all experiments, data were filtered at 4 kHz during recording and the traces were digitized at 20 kHz.Simultaneous time-lapse imaging of GCaMP fluorescence in dHb terminals was performed using either an Orca Flash4.0 (Hamamatsu, Japan) or Orca 03G (Hamamatsu, Japan) camera and recorded using the HC Image Live software (Hamamatsu, Japan) at an acquisition frequency of 5 Hz or 10Hz with an exposure time of 200 ms and 100 ms, respectively. Time-lapse and patch-clamp acquisition were synchronized via a TTL signal sent to the camera via the pClamp 10 software.Time-lapse frames were encoded at a 256x256 pixel resolution using a 16-bit grey scale. Time-lapse images were analyzed using the ImageJ software (National Institute of Health, USA) and fluorescence signals were plotted using Clampfit 10.J o u r n a l P r e -p r o o fImmunohistochemistry TgBAC(gng8:GAL4ff) c426 ;Tg(UAS:GFP) c354 6 dpf larvae were fixed in BT fix (4% PFA, 0.15mM CaCl 2 , 4% sucrose in 1x PBS) overnight in 4°C for rabbit anti-VAChT (1/200, Synaptic systems) labeling. Larvae were embedded in 4% low melt agarose and sectioned to 50μm using a vibratome (Leica, Inc). Floating sections were incubated in primary antibody in incubating solution (0.8% Triton-X in 1x PBS) for 3 days on a shaker in 4°C. The sections were washed and incubated in secondary anti-rabbit Alexa Fluor 594 (1:1000) with Hoechst 33342 (1:2000) overnight at 4°C on a shaker. TgBAC(gng8:GAL4ff) c426 ;Tg(UAS:GFP c354 ;Tg(gad1b:|R|-GFP) larvae were fixed overnight in 4% PFA, sectioned to 50μm and incubated with Hoechst 33342(1:2000) overnight at 4°C on a shaker. All sections were then mounted on slides using Mowiol (Sigma). They were imaged using the Leica TCS SP5 AOBS upright microscope using a 63x (N.A. = 1.4) objective.

J o u r n a l P r e -p r o o f 29 intensity

 29 for each ROI was then extracted per frame and analyzed on MATLAB using custom written scripts. Amplitude of calcium signal was calculated according to the formula: ∆F/F = (F i -F mean )/F mean , whereby, F i is the mean intensity in a single ROI at a single time point while F mean is the mean intensity in a single ROI throughout the entire recording time. Multiple clustering algorithms were tested including functional clustering algorithm[START_REF] Feldt | Functional clustering algorithm for the analysis of dynamic network data[END_REF] and spectral clustering on calcium traces and correlation coefficient values. After comparing the clusters generated using various algorithms, we found k-means and hierarchical clustering based on pairwise linear correlation matrix were the most representative of calcium signals in the spontaneous activity recordings. K-means and hierarchical clustering based on pairwise linear correlation matrix were carried out on MATLAB using kmeans and linkage functions ('euclidean' distance and 'complete' method), respectively. Different k values were tested and k = 5 was selected based on manual inspection of calcium signals in the time-lapse recordings and also using a heatmap. The correlation matrix displayed in figures was generated by calculating the correlation coefficients between the average calcium signal traces for each cluster. The threshold for hierarchical clustering was determined by manual inspection of the calcium signals in the recording and heatmap. Once specific IPN domains were identified by k-means, larger ROIs were manually drawn containing the different IPN domains. Analysis of calcium burst frequency using 'findpeaks' function was performed in MATLAB (Mathworks). Peak of calcium burst, calcium inhibition and half duration were identified using findpeaks and half prominence functions in MATLAB and verified manually. Calcium burst peak was defined by the highest J o u r n a l P r e -p r o o f

  All statistical details including statistical tests, exact values of n, value representation (mean ± SD) are indicated in the figure legends.  Synchronized Hb cholinergic activity inhibits non-cholinergic terminals  Presynaptic inhibition of non-cholinergic terminals is mediated by GABA B receptors  GABA B R activation by retrograde signaling from the interpeduncular nucleus eToc (350 characters) Zaupa et al., show that calcium signals in habenular soma are not reflective of activity in axon terminals at the interpeduncular nucleus. They uncover an atypical mode of lateral inhibition between two habenular circuits where the activation of cholinergic neurons inhibits non-cholinergic activity by retrograde signaling from their target site. J o u r n a l P r e -p r o o f -/ nptx + vvIPN: VAChT + raphe: VAChT + dvIPN: VAChT -cvIPN: ? J o u r n a l P r e -p r o o f r n a l P r e -p r o o f

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  We found that gabbr1a is expressed asymmetrically in a large area in the left and smaller area in the right dHb, indicating that the GABA B receptor is present in noncholinergic neurons that innervate the dIPN in larvae. Moreover, gad1b-positive neurons predominate in the dIPN, suggesting that GABA released from dIPN neurons activates presynaptic GABA B receptors on non-cholinergic terminals at the dIPN. Based on calcium imaging, pharmacology, electrical stimulation and molecular studies, we propose a model whereby 1) activation of cholinergic dHb neurons 2)

Acknowledgements

We thank Hitoshi Okamoto for insightful feedback on this project; Marco Diana and Nathalie Leresche for advice on pharmacology experiments; Eric Schwartz and Soumaiya Imarraine for helpful comments on the manuscript; Jean-Pierre Coutanceau for aid in zebrafish husbandry; and the IBPS aquatic animal facility and imaging facility. Hitoshi Okamoto, Koichi Kawakami and Shin-ichi Higashijima provided the Tg(nptx2:Gal4-VP16) rw0143a , Tg(UAS:GCaMP7a) and Tg(gad1b:|R|-GFP) lines. This study was supported by a graduate fellowship from the Alexis and Anne Marie Habib Foundation (to M.A.Y), NIH grant R37HD091280 (to M.E.H.) and the ATIP-Avenir young investigator grant (INSERM/CNRS) and Emergence grant (Sorbonne Université) (to E.H). experiments. M.Z., S.M.A.N., S.W., H.S., J-M.M. and E.H. analyzed data. E.H. wrote the manuscript with input from all authors.

Author Contributions

METHOD DETAILS Explant Dissection

TgBAC(gng8:GAL4ff) c426 ;Tg(UAS:GCaMP7a) 3, 6 and 7 day-post-fertilization (dpf) larvae were used for experiments. Larvae were anesthetized in Tricaine (0.01%) and brains dissected using fine forceps in ice cold Ringer's solution (134mM NaCl, 2.1 KCl, 1.2mM MgCl 2 , 10 mM HEPES, 10mM Glucose, 2.1 CaCl 2 , pH = 7.6) and mounted in 1.2% low melting agarose for calcium imaging and electrophysiology experiments.

Pharmacology

Drugs were applied to the explants either directly using a pipette or by using a perfusion peristaltic pump (Ismatec) at a rate of 5-6 ml/min. Mecamylamine hydrochloride (100 μM), (+)-D-tubocurarine chloride (10 μM), nicotine ditartrate (10