Dopaminergic modulation of olfactory‐evoked motor output in sea lampreys (Petromyzon marinus L.)

Abstract Detection of chemical cues is important to guide locomotion in association with feeding and sexual behavior. Two neural pathways responsible for odor‐evoked locomotion have been characterized in the sea lamprey (Petromyzon marinus L.), a basal vertebrate. There is a medial pathway originating in the medial olfactory bulb (OB) and a lateral pathway originating from the rest of the OB. These olfactomotor pathways are present throughout the life cycle of lampreys, but olfactory‐driven behaviors differ according to the developmental stage. Among possible mechanisms, dopaminergic (DA) modulation in the OB might explain the behavioral changes. Here, we examined DA modulation of olfactory transmission in lampreys. Immunofluorescence against DA revealed immunoreactivity in the OB that was denser in the medial part (medOB), where processes were observed close to primary olfactory afferents and projection neurons. Dopaminergic neurons labeled by tracer injections in the medOB were located in the OB, the posterior tuberculum, and the dorsal hypothalamic nucleus, suggesting the presence of both intrinsic and extrinsic DA innervation. Electrical stimulation of the olfactory nerve in an in vitro whole‐brain preparation elicited synaptic responses in reticulospinal cells that were modulated by DA. Local injection of DA agonists in the medOB decreased the reticulospinal cell responses whereas the D2 receptor antagonist raclopride increased the response amplitude. These observations suggest that DA in the medOB could modulate odor‐evoked locomotion. Altogether, these results show the presence of a DA innervation within the medOB that may play a role in modulating olfactory inputs to the motor command system of lampreys.


| INTRODUCTION
Lampreys represent the oldest extant group of vertebrates and their behavior is strongly influenced by olfactory inputs. Sea lampreys (Petromyzon marinus L.) rely heavily on the detection of chemical cues for feeding (Kleerekoper & Mogensen, 1963) and sexual behaviors (Buchinger, Siefkes, Zielinski, Brant, & Li, 2015). As odorant perfusion on the olfactory epithelium of lampreys from prolarval (Zielinski, Fredricks, Mcdonald, & Zaidi, 2005) to spawning (Li, Sørensen, & Gallaher, 1995) stages activates sensory neurons, olfaction is thought to induce motor behavior throughout life. During the transformation from larva to young adult, the peripheral olfactory apparatus becomes well developed with a lamellar olfactory epithelium and an anatomically distinct accessory olfactory organ (Ren et al., 2009). Moreover, compared to other vertebrate species, lampreys have a large proportion of their brain dedicated to processing olfactory inputs, the size of the olfactory bulbs (OB) even exceeding that of the cerebral hemispheres (Nieuwenhuys, 1977). Furthermore, secondary olfactory neurons have extensive projections throughout the prosencephalon (Northcutt & Puzdrowski, 1988).
Our research group has previously identified a neural substrate responsible for generating locomotion in response to olfactory inputs (Derjean et al., 2010). Odorant detection is carried out by olfactory sensory neurons that project to the medial part of the OB (medOB; Green et al., 2017). There, a unique population of projection neurons located inside a single glomerulus (medOB glomerulus) conveys the inputs to the posterior tuberculum (PT; Daghfous et al., 2018;Derjean et al., 2010;Green, Basilious, Dubuc, & Zielinski, 2013;Pérez-Fernández, Stephenson-Jones, Suryanarayana, Robertson, & Grillner, 2014). The PT then sends descending dopaminergic and glutamatergic inputs to the mesencephalic locomotor region (MLR; Ryczko et al., 2013Ryczko et al., , 2016Ryczko et al., , 2017, a structure known to play a crucial role in the control of locomotion in all vertebrate species tested so far. The MLR then activates reticulospinal (RS) cells (Sirota, Viana Di Prisco, & Dubuc, 2000), which provide the main descending inputs to the central pattern generators for locomotion in the spinal cord.
Recent findings in our lab have revealed that olfactory projections from the rest of the OB (main OB: MOB) can also activate the PT, but via projections to the lateral pallium (Daghfous et al., 2018). The wellcharacterized, locomotion-controlling neural circuits that are part of an axis from the PT to the spinal cord can thus be activated by both a medial (medOB-PT) and a lateral (MOB-lateral pallium-PT) olfactomotor pathways to generate locomotor responses to olfactory cues. These two parallel olfactomotor pathways convey information from the olfactory sensory organ in the periphery, which is activated by various naturally occurring food-related or reproductive olfactory cues such as amino acids, bile acids, and pheromones (Green et al., 2017). Thus, they may be equally involved in food-seeking and mate-finding.
Multiple odorants that can trigger locomotion elicit responses in the medOB (Green et al., 2017). Moreover, the medial olfactomotor pathway is functional throughout life, but odor-driven behaviors differ among larval, parasitic (Kleerekoper & Mogensen, 1963;Silva, Servia, Vieira-Lanero, & Cobo, 2013), and spawning (Johnson, Yun, Buchinger, & Li, 2012) developmental stages. Hence, the activity in this neural circuit must be adjusted to various conditions, external and internal. Modulatory mechanisms upstream of the PT could efficiently regulate motor responses to odorants. The medOB would thus be a good target for modulation of the medial olfactomotor pathway.

| MATERIALS AND METHODS
Experiments were performed on 72 larvae, 11 newly transformed adults, and 23 spawning-phase adult sea lampreys (Petromyzon marinus) of both sexes. Larval and newly transformed specimens were collected from the Pike River (Pike River, QC, Canada) and the Morpion Stream
The brain tissue at the site of tracer injection was lesioned beforehand with an entomological needle to precisely cut the axons, allowing them to pick up the tracer. Biocytin crystals (Sigma-Aldrich, St. Louis, MO) were inserted in the lesioned area to dissolve. The brains were then kept overnight under Ringer's solution perfusion at 8 C to allow transport of the tracer to the cell body. To label projection neurons of the medOB, biocytin was injected in the PT. The roof of the caudal part of the third ventricle was cut open along the midline to gain access to the PT. Biocytin injections were also performed in the medOB to retrogradely label neurons projecting to this area.

| Tyrosine hydroxylase immunofluorescence
The brains were fixed by immersion in 4% paraformaldehyde in phosphate-buffered saline (PBS; 0.1 M, pH 7.4 with 0.9% NaCl) for 24 hours at 4 C, then rinsed in PBS and incubated in a 20% sucrose solution in PBS overnight for cryoprotection. The tissue was frozen in 2-methylbutane at −50 C and cut transversally with a cryostat. The sections (25 μm thickness) were collected on ColorFrost Plus microscope slides (Thermo Fisher Scientific) and allowed to dry on a warming plate at 37 C for a minimum of 12 hours.
The sections were rinsed (three 10-min immersions) in PBS, immerged for 1 hour in a permeabilizing solution (normal goat serum 10%, Triton X-100 0.3%, in PBS), and incubated overnight at 4 C with a primary antibody targeting TH (rabbit anti-TH, Millipore, Cat# AB152, RRID:AB_390204) diluted 1:400 in the permeabilizing solution. The next day, the sections were rinsed and incubated 1 hour at room temperature with a goat anti-rabbit antibody conjugated to Alexa Fluor 594 (Molecular Probes, Cat# A-11012, RRID: AB_141359) diluted 1:400 in the permeabilizing solution. The sections were then rinsed, mounted with Vectashield ® (with or without DAPI), and stored in the dark at 4 C.
The fixed brains were then incubated in a 20% sucrose solution in Tris-buffered saline with low sodium metabisulfite overnight for cryoprotection, frozen in 2-methylbutane at −50 C, and cut transversally with a cryostat. The sections (25 μm thickness) were collected on ColorFrost Plus microscope slides (Thermo Fisher Scientific) and dried on a warming plate at 37 C for a minimum of 12 hours.
The sections were first rinsed in Tris-buffered saline with high sodium metabisulfite (TBS-m: Tris 0.05 M with 1.0% sodium metabisulfite, pH 7.4) and incubated in a reducing solution (sodium borohydride 0.2% in Tris-buffered saline 0.05 M with 0.9% NaCl, pH 7.4) for 45 min to decrease autofluorescence induced by the glutaraldehyde fixation. The glass slides were rinsed again and immerged in a permeabilizing solution (normal goat serum 10%, Triton X-100 0.3%, in TBS-m) for 60 min before overnight incubation at 4 C with a monoclonal mouse anti-DA antibody (Millipore, Cat# MAB5300, RRID:AB_94817) diluted 1:300 in the permeabilizing solution. The next day, the sections were rinsed and incubated 1 hour with a goat anti-mouse antibody conjugated to Alexa Fluor 594 (Jackson ImmunoResearch Labs, Cat# 115-585-146, RRID:AB_2338881) diluted 1:200 in the permeabilizing solution at room temperature. Sections were then rinsed, mounted with Vectashield ® (with or without DAPI) and stored in the dark at 4 C.

| Antibody characterization
The antibodies used in this study are listed in Table 1. The rabbit anti-TH antibody has been used reliably on lamprey tissue in independent studies examining the presence of DA neurons (Barreiro-Iglesias, Villar-Cervino, Villar-Cheda, Robertson et al., 2012). Additionally, our research group has previously used this antibody in lampreys and salamanders (Ryczko et al., 2013(Ryczko et al., , 2016. The pattern of labeling of the mouse antibody targeting DA in our material corresponded closely to that reported with other DA antibodies in the lamprey (Abalo, Villar-Cheda, Anadon, & Rodicio, 2005; Barreiro-Iglesias, Villar-Cervino, Pierre et al., 1997). Cross-reactivity of the mouse anti-DA antibody was determined by the manufacturer (Millipore) using an ELISA test with the following compounds: DA-glutaraldehyde (G)-bovine serum albumin (BSA) 1; Tyrosine-G-BSA 1:36,000; L-DOPA-G-BSA 1:10,000; Noradrenaline-G-BSA 1:>50,000; Adrenaline-G-BSA 1:>50,000. The specificity of the fluorescent secondary antibodies was verified by omitting the primary antibody from the procedures. In every case, no labeling was obtained under these conditions.

| Additional labeling
Streptavidin conjugated to Alexa Fluor 488 or 350 (diluted 1:200, S11223 or S11249, Thermo Fisher Scientific) was added to the secondary antibody solution to visualize biocytin. The primary olfactory afferents were stained with Griffonia simplicifolia isolectin B4 (GSIB4), which binds to galactosyl residues present on axons of olfactory sensory neurons, as previously done by others (Tobet, Chickering, & Sower, 1996) and us (Daghfous et al., 2018). This labeling was carried out after the immunofluorescence protocol by incubating the slides with GSIB4 conjugated to Alexa Fluor 488 (I21411, Life Technologies) diluted 1:100 in the appropriate rinsing solution (TBS-m or PBS) for 60 min at room temperature. The sections were then rinsed and fixed in 4% paraformaldehyde in PBS for 1 hour at room temperature before they were rinsed again and mounted.

| Fluorescence microscopy
The sections were observed and photographed on an E600 epifluorescence microscope equipped with a DMX1200 digital camera

| Electrophysiological experiments
Whole brain preparations from larval specimens were isolated in vitro as described above and pinned down in an experimental chamber (total volume = 50 mL) continuously perfused with a Ringer's solution at a rate of 4 mL/min and maintained between 8 and 13 C.  Tocris Bioscience, Bristol, UK). All drugs were dissolved in Ringer's solution and kept at −20 C (or 4 C for less than 7 days) until application.

| Data analysis
Electrophysiological data were analyzed with Spike2 software (Cambridge Electronic Design, Version 5.19) and a homemade script for excitatory postsynaptic potentials (EPSPs). Statistical analyses were carried out on Sigmaplot (Systat Software Inc., Version 11.0).
One-way ANOVA for repeated measures or Friedman ANOVA on ranks for repeated measures were used and followed by multiple comparison procedures (Holm-Sidak or Tukey) to test equality of means in the different treatments (Control -Drug -Washout). For all statistical analyses, a significance level of .05 was adopted. Results are presented as the mean ± SD.

| Dopamine immunofluorescence in the olfactory bulb
An immunofluorescence procedure was performed in 12 larval, six newly transformed adult, and 11 spawning-phase adult lampreys of both sexes to analyze the distribution of DA and TH immunoreactivity in the OB. The most notable feature brought to light was the presence of two distinct types of processes that were differentially distributed in the OB ( Figure 1).
The processes from a first type were varicose and strongly labeled.
They were readily seen under epifluorescence microscopy, even at low magnification ( Figure 1). These strongly labeled processes were preferentially located medially and caudally within the OB, although scarce and isolated processes were found in all bulbar regions In the OB of newly transformed and spawning-phase adults, DA immunofluorescence yielded additional labeling. Notably in adult lampreys, a second type of DA+ processes were detected (Figures 1 and   5). These processes were in sharp contrast with those previously described: they were weakly labeled, did not display varicosities, and were spread homogeneously across the granular layer. Isolated processes also occasionally reached the glomerular layer (Figure 5c2,d2).
In addition, processes with same characteristics were often seen originating from local cell bodies, which were also exclusively observed in newly transformed and spawning phase adults. These somata had a similar, weak labeling intensity, and were small-sized (10-15 μm), round or ovoid and often bipolar ( Figure 6). From the most caudal to the most rostral levels, they were homogeneously they showed a more intense labeling, making them easier to visualize.
They exhibited similar size, shape, and distribution when compared to the DA+ cell bodies described above, the only difference being that a greater number of TH somata could be visualized. Moreover, despite the absence of DA+ somata and weakly labeled processes in larval specimens, both were observed consistently in larvae following TH immunofluorescence.

| Dopaminergic afferents to the medial olfactory bulb
In the adult OB, the weakly labeled DA+ processes could stem from local neurons, as they displayed the same labeling intensity than the DA+ cell bodies and their associated processes in the granular layer.
However, no labeled cell bodies were found to be as intensely labeled as the strongly labeled processes. It is possible that an extrinsic source

| The effect of dopamine on reticulospinal cell responses induced by olfactory inputs
Our anatomical results altogether suggest that DA+ processes from extrinsic sources (DHN and PT) innervate the medOB in addition to We also tested the effects of DA on suprathreshold responses to get a better indication on whether DA also affects motor behavior. activity. Therefore, the local effects of DA in the medOB may significantly affect motor output.

| The role of D1 and D2 receptors in modulating olfactomotor transmission
To further characterize the action of DA on olfactomotor transmission, ligands selective for D1 or D2 receptor were pressure-injected in the medOB (Figure 11). A local microinjection of a D1 receptor agonist, dihydrexidine (0.1 mM), caused a significant decrease of RS cell responses to ON stimulation (Figure 11c). Depression of both amplitude (to 71.5 ± 36.0%; F = 11.180, df = 2, p < .001, n = 6 cells in six larvae) and area (to 64.7 ± 87.0%; χ 2 = 6.222, df = 2, p = .045, n = 6 cells in six larvae) was observed and no significant differences were detected between control and washout of dihydrexidine Dopaminergic processes have been observed previously in the lamprey OB, from larval to adult stages, both in the river lamprey (Lampetra fluviatilis L.) (Baumgarten, 1972;Pérez-Fernández et al., 2014;Pierre et al., 1994Pierre et al., , 1997Pierre-Simons, Reperant, Mahouche, & Ward, 2002;Pombal et al., 1997) and in P. marinus (Abalo et al., 2005;Barreiro-Iglesias et al., 2009Fernandez-Lopez et al., 2017;Yáñez, Molist, Rodríguez-Moldes, & Anadón, 1992). Compared to previous studies, a major difference in DA immunofluorescence observed in the OB was the presence of two distinct populations of DA+ processes with different developmental patterns and anatomical distributions. The processes from a first type were strongly labeled and denser in the medOB. The processes from a second type contrast sharply: they were weakly labeled and only observed in newly transformed and spawning-phase adults. They seemed to arise from a local population of similarly labeled DA+ neurons, which are also detected only in adult animals.
Our results suggest that the strongly labeled processes of the first type do not originate from local cell bodies. Although it is possible that such strongly labeled processes arose from weakly labeled DA+ somata in the OB, it is more likely that they stemmed from one of the numerous DA cell groups in the diencephalon (Abalo et al., 2005). In our material, these processes appeared to reach the OB from its caudal aspect passing through the septum (see Figure 2), close to axons from medOB projection neurons exiting the OB (see Figure 4). In larval lampreys, the strongly labeled DA+ processes were detected close to and inside the medOB despite the absence of (or failure to detect) local DA+ neurons in the OB. During prolarval development of P. marinus, the earliest detection of DA+ processes reaching the telencephalon coincides with that of DA+ neurons in the PT (Abalo et al., 2005). Most importantly, we provide evidence that DA+ neurons in the PT and DHN project to the medOB. However, PT injections did not allow us to observe anterogradely labeled DA+ processes in the OB, which suggests otherwise. This may be due to a limited number of DA neurons projecting to the medOB from the PT. Although the DA innervation of the OB is traditionally considered to be exclusively local (Smeets & Gonzalez, 2000), extrinsic projections to the OB were notably observed in rats, where a minor portion of SNc neurons send a direct DA projection to the OB (Höglinger et al., 2015). Furthermore, projections from the SNc and the VTA to the OB were observed in the sheep (Lévy, Meurisse, Ferreira, Thibault, & Tillet, 1999) and PT-OB projections were detected in two species of shark (Yáñez, F I G U R E 1 1 Effects of D1 and D2 receptor agonists and antagonists injection in the medOB on RS cell responses. The physiological effects of selective dopamine (DA) receptor ligands injection in the medial olfactory bulb (medOB) on reticulospinal (RS) cells synaptic responses to olfactory nerve stimulation was studied in isolated larval brains (a). The data were compiled in a boxplot (b) representing the relative response amplitude following local drug injections in comparison with control responses from every recording. In addition, the mean response amplitude of individual RS cells before, during and after injection of selective DA receptor agonists or antagonists was plotted as different line graphs (c1, d1, e1, and f1). Evoked responses from representative recordings are exhibited as six superimposed traces and their mean (thick black trace) during each treatment (c2-c4; d2-d4; e2-e4; f2-f4). (c) D1 receptor agonist dihydrexidine (0.1 mM) decreased the mean response amplitude to 71.5 ± 36.0% of control (n = 6 cells in six larvae). projections modulating OB sensory processing is common in vertebrates, including noradrenergic fibers from the locus coeruleus (Shipley, Halloran, & de la Torre, 1985) and serotoninergic fibers from the raphe nuclei (Broadwell & Jacobowitz, 1976). Hence, in the medOB of lampreys, the DA innervation might originate from extrinsic DA afferents in addition to the intrinsic innervation.

| Dopaminergic cell bodies in the lamprey olfactory bulb
Our study shows that DA+ somata were present in the granular layer of the OB in adult lampreys, but were not detected in larvae. The absence of DA+ cells in the OB of larval specimens was also reported previously (Abalo et al., 2005;Pierre-Simons et al., 2002;Yáñez et al., 1992), but studies on both P. marinus and L. fluviatilis have described DA+ neurons in adult specimens (Barreiro-Iglesias et al., 2009;Fernandez-Lopez et al., 2017;Pierre et al., 1997;Pombal et al., 1997).
The phenotype of these cells was confirmed to be DA (TH+/DOPA decarboxylase+/DA+/dopamine β-hydroxylase-) in an immunoreactivity study (Pierre et al., 1997). Moreover, the same authors did not find somata containing dopamine-β-hydroxylase or phenylethanolamine-N-methyltransferase in the OB, suggesting the absence of other catecholaminergic (noradrenergic or adrenergic) neurons.

| Dopaminergic modulation of the medial olfactory bulb
A single population of projection neurons located inside the medOB receives sensory inputs exclusively from chemosensory cells in the accessory olfactory organ (Green et al., 2017). The medOB projection neurons then project directly to the PT to drive swimming activity through activation of brainstem RS cells (Daghfous et al., 2018;Derjean et al., 2010). Direct medOB projections to the MLR were also observed (Daghfous et al., 2018). We now show that the injection of DA agonists in the medOB reduces the activation of RS cells in response to ON stimulation. The effects were even more powerful when gabazine, a GABA A receptor antagonist, was injected beforehand in the OB (see Daghfous et al., 2018). The spiking responses in RS cells were then totally suppressed. This suggests that activation of DA receptors in the medOB can lead to a substantially reduced motor output in response to olfactory inputs. This could explain the changes in motor responses to chemical cues that occur during the life cycle of the animal. For example, only during the spawning phase will lampreys respond to migratory pheromones released by larvae (Vrieze & Sørensen, 2001 (Ryczko et al., 2013).
Moreover, D1 receptors have been detected in the OB (Pérez-Fernández, 2013). Dihydrexidine, a D1 receptor agonist, reduced olfactomotor response amplitude (28.5% decrease), but was less efficient than DA (36.6% decrease) or quinpirole (51.3% decrease), although it was reported that dihydrexidine exhibits more than 10-fold higher affinity and potency at the D1 receptor than DA (Rosell et al., 2015). Altogether, these physiological results demonstrate that DA exerts a strong modulatory effect on olfactomotor processing in the medOB via D2 and possibly D1 receptors.
Based on previous findings and the present results, we hypothesize that projection neurons are the main site of action of DA involved in the modulation of olfactomotor activity.
Although it could be expected that olfactory glomeruli are devoid of cell bodies, the medOB glomerulus is definitely an exception and does contain the somata of PT-targeting projection neurons in its neuropil (Daghfous et al., 2018;Green et al., 2013;present results). Hence, D2 receptors might be present on the soma or dendrites of medOB projection neurons and directly modulate their activity and output to the PT. Moreover, their activity might also be modulated downstream, as their axons are observed in close proximity to DA+ processes caudal to the medOB (see Figure 4). Indeed, axo-axonic contacts established by DA fibers have been detected in the striatum of lizards (Henselmans & Wouterlood, 1994) and rats (Bouyer, Park, Joh, & Pickel, 1984;Freund, Powell, & Smith, 1984;Pickel & Chan, 1990), in the median eminence of sheep (Kuljis & Advis, 1989), and the cortex of monkeys (Sesack, Snyder, & Lewis, 1995). Furthermore, in the ventral pallidum of rats, D2 receptors are detected mainly on axons or terminals of non-DA neurons, suggesting an effect on presynaptic release (Mengual & Pickel, 2002 Another effect of DA may be the fine-tuning of medOB activity in response to odorants. The activation of the medial olfactomotor pathway could produce appetitive goal-directed locomotion, as the medOB is activated by amino acids, bile acids, and pheromones (Green et al., 2017), all of which can elicit goal-directed swimming (Bjerselius et al., 2000;Johnson, Yun, Thompson, Brant, & Li, 2009;Kleerekoper & Mogensen, 1963;Li et al., 2002). In the natural environment, these chemical cues are encountered in a wide range of concentrations and DA modulation in the medOB might contribute to olfactory processing during tracking behaviors. In rodents, DA modulation of glomerular activation was proposed to increase the range of odorant levels processed by the OB (Ennis, Hamilton, & Hayar, 2007).
Dopaminergic inhibition in the medOB could thus dynamically adapt olfactory sensitivity, which would allow the animal to follow an olfactory target more efficiently. Such mechanism may increase the range of odorant concentrations inducing an appropriate locomotor response, which is to swim toward their source.
In addition to a local DA source of inputs in the medOB, the two diencephalic DA cell populations projecting to the medOB identified here could provide means for adapting olfactomotor behaviors in different contexts. First, one source of DA innervation originates from the PT, a DA nucleus homologous to the SNc/VTA (Baumgarten, 1972;Pombal et al., 1997). As medOB projection neurons reach directly the PT to drive locomotion (Daghfous et al., 2018;Derjean et al., 2010), there are reciprocal connections between the medOB and the PT, which would thus allow PT neurons to control the inputs they receive. Interestingly, a reciprocal connection also exists between the PT and the tectum (Pérez-Fernández et al., 2014). It was recently found that DA neurons in the PT are activated by visual stimuli, coding saliency (Pérez-Fernández, Kardamakis, Suzuki, Robertson, & Grillner, 2017). In this study, authors reported that the PT modulates visuomotor transformations mediated in the tectum by modifying tectal neuron responsiveness to visual stimuli via direct DA projections. Similarly, the DA projections of the PT to the medOB could modulate olfactory processing so that odorants generate a motor output less effectively. This mechanism would allow for flexibility in the motor output evoked by the medial olfactomotor pathway.
Additionally, the DHN contained CSF-contacting DA+ cell bodies projecting to the medOB. Hypothalamic projections to the olfactory system are common in vertebrates and exert modulatory effects to control odor-driven behaviors (Gascuel et al., 2012). In lamprey, TH+ cells of the DHN are in contact with the CSF and give rise to long extrahypothalamic pathways reaching telencephalic structures (Pierre et al., 1994). Therefore, the CSF-contacting neurons may modulate the activity of the medOB to adjust the behavioral output according to the functional state of the hypothalamus or according to the rate of diverse hormones or other chemical substances in the CSF.