The role of nonapeptides in electrocommunication behaviour in gymnotiform weakly electric fish
By Ali Mokdad
Department of Biology McGill University, Montreal
August 2016
A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science in Biology.
© Ali Mokdad 2016 1
Table of Contents
Table of Contents
Abstract ...... 1
Résumé ...... 3
Acknowledgments ...... and Contribution...... 5
General Introduction ...... 7
Review - The role of nonapeptides ...... in social ...... communication: A case...... 8 for gymnotiform weakly electric fish
Introduction ...... 11
Nonapeptide ...... systems in teleost fish ...... 11
Communication behaviour ...... 13
Vocal communication behaviour ...... 18
Electrocommunication behaviour ...... 18
Segregated neural pathways control ...... electrocommunication behaviour...... 20 ......
Connections between the electromotor system and POA-AH 22
Nonapeptide effects on electrocommunication behaviou r...... 25
Nonapeptides and wave-type fish ...... 28
Nonapeptides and pulse-type fish ...... 28
Conclusion ...... 31
Investigating the roles...... of isotocin in electrocommuni...... cation behaviour...... 37 in a weakly electric fish, Apteronotus leptorhynchus
Introduction ...... 40
Nonapeptide ...... systems and social behaviour...... 40
...... 40 2
Nonapeptides and descending motor pathways in teleosts
Electrocommunication behaviour ...... 41
Nonapeptide control of electromotor ...... circuits ...... 42
Goal ...... 44
Materials ...... and Methods ...... 46
Animal care and housing ...... 46
Recording and stimulation ...... 46
Experimental design ...... 47
Chirp assay ...... 48
Chirp and JAR ...... analysis ...... 48
Statistical analysis ...... 49
Results ...... 50
Effect ...... of isotocin on baseline...... EODf ...... 50
Effect of isotocin on JAR excursion ...... 50
Chirp analysis ...... 51
Effect of isotocin ...... on type-I and type-II chirp...... production ...... 51
Discussion ...... 52
Summary and Final ...... Conclusion ...... 53
Figures and Tables ...... 57
References ...... 60
...... 75
3
Abstract
The nonapeptide hormones, arginine vasotocin (AVT) and isotocin (IT), play important roles in mediating a wide range of social behaviour in fish. Gymnotiform weakly electric fish use their electric organ discharge (EOD) to produce an expansive array of social electric signals. These behaviours share relatively simple neural elements by which they are controlled. The first section of this thesis reviews the current literature pertaining to nonapeptide influence on electrocommunication behaviour. AVT has been shown to modulate various electrocommunication behaviours by influencing segregated neural pathways controlling distinct electric displays. In pulse-type weakly electric fish, AVT modulates nocturnal increases in EOD frequency (EODf). Among these same fish, AVT also had an effect on EOD rate changes signalling submission and dominance. In wave-type fish,
AVT modulates the production of two types of “chirps” – transient increases in EOD used as courtship or aggressive signals. AVT decreased the production of aggressive signals and increased the production of courtship signals. AVT also increased the magnitude of the jamming avoidance response (JAR), the communicative function of which is still poorly understood. The second section ofApteronotus this thesis was leptorhynchus aimed at investigating the effects of IT on three electric behaviours in male , a South American wave-type electric fish: baseline EOD, the JAR, and chirping behaviour. Intramuscular injection of IT showed no statistically significant effect on any of the three behaviours. The lack of a response to IT was somewhat surprising since the behavioural functions of AVT and IT systems are related. A number of reasons are discussed that may explain the lack of response, chief among them being the possibility that receptor expression was low in the 4
fish tested. Further exploration of IT and AVT in relation to communication is warranted and weakly electric fish are a particularly advantageous system in this regard.
5
Résumé
Les hormones nonapeptidiques, arginine vasotocine (AVT) et isotocine (IT), jouent un rôle dans la médiation d’un grand nombre de comportements sociaux chez les poissons. Les poissons faiblement électriques gymnotiformes utilisent leur décharge de l’organe
électrique (EOD) pour produire un éventail extensif de signaux électriques sociaux. Ces comportements partagent des éléments neuronaux simples qui les contrôlent. La première section de cette thèse examine la littérature actuelle pertinente à l’influence des nonapeptides sur le comportement électro-communicatif. L’AVT module plusieurs comportements électro-communicatifs, et ceci par l’influence de réseaux neuraux séparés qui contrôlent des signaux électriques distincts. Chez les poissons faiblement électriques de type pulsatif, l’AVT module les augmentations nocturnes de la fréquence des décharges de l’organe électrique (EODf). Chez ces mêmes poissons, l’AVT a aussi un effet sur le taux de changement des signaux de soumission ou de dominance émis par l’EOD. Chez les poissons
à décharge par ondes, l’AVT module la production de deux types de « pépiement » - des augmentations transitoires de l’EOD utilisées tels des signaux d’agression ou de cour. L’AVT a diminué la production de signaux d’agression, et augmenté le taux de signaux de cour
émis. L’AVT a aussi augmenté l’intensité des réponses d’évitements du brouillage (JAR), dont la fonction communicative n’est pas encore assez analysée. La deuxième section de cette thèse vise à étudier les effets de l’isotocine (IT) sur trois comportements électriques chez les Apteronotus Ieptorhunchus mâles, un poisson à décharge électrique ondulée Sud-
Américain; ces comportements étant l’EOD de base, la JAR, et les pépiements. Des injections intramusculaires d’IT ne montrent aucun effet sur ces trois comportements. L’absence de réaction à l’injection d’IT est surprenante puisque les fonctions comportementales des 6
systèmes d’AVT et d’IT sont fonctionnellement liés. Un nombre de raisons sont analysées pour expliquer l’absence de réponse, principalement la possibilité que l’expression du récepteur soit faible dans les poissons testés. Une exploration plus approfondie de l’IT et de l’AVT par rapport à la communication est justifiée, et les poissons faiblement électriques sont un système particulièrement avantageux en cet égard. 7
Acknowledgments and Contribution
I would like to thank, first and foremost, my supervisor Dr. Rüdiger Krahe for guiding me through the ups and downs of my post graduate experience. Thank you, Rüdiger, for your overall support and making me feel comfortable and accomplished in your lab. Thank you for providing me with many opportunities to develop as a researcher and teacher. I could not have done this without your constant and steady support. I would also like to thank the other members of my supervisory committee, Dr. Alanna Watt and Dr. Michael Hendricks, for their constructive input and direction for this thesis. The chirp experiments were designed with help from Dr. Rüdiger Krahe. I am indebted to Dr. Simon Reader and members of his lab, Dr. Adam Reddon and Dr. William Swaney, for sharing their wealth of knowledge, and assistance in developing the ideas for the isotocin experiments. Isotocin was provided by Dr. Simon Reader and the isotocin solutions were prepared by Adam
Reddon. I conducted the experiments and collected the data for this thesis with the help of an undergraduate research student, Zhuo Luan Xu. I would like to thank Dr. Rüdiger Krahe,
Dr. Adam Reddon, and Tyler Moulton for helping me analyze and interpret the data collected. I would also like to extend my gratitude to Dr. Jon Sakata and Dr. Rüdiger Krahe for their insightful conversations that helped structure and guide the review section of this thesis. This thesis was written with editorial help from my supervisor, Rüdiger Krahe. I cannot end without thanking my family and friends for sharing this wonderful experience with me: Hanan, Ibrahim, Nouhad, Khallil, Rayan, Stefan, Lianne, and Layal – Thank you all!
8
General Introduction
Mechanisms underlying the variation and complexity of behaviours expressed within and across vertebrate species has been a central focus in the field of behavioural endocrinology. Studies of neuropeptide influence on social communication behaviour have been especially important in this regard (see Goodson and Bass, 2001; Albers, 2012).
Nonapeptide modulation of communication behaviour has been demonstrated across a broad range of vertebrate taxa such as birds (reviewed in Goodson and Kabelik, 2009), amphibians (reviewed in Leary, 2009), mammals (reviewed in Albers, 2012), and fish
(Goodson and Bass, 2000a,b). However, no consistent pattern for the role of nonapeptides in communication behaviour has yet emerged. As Goodson and Bass (2001) point out, determining a general explanatory model for nonapeptide function is dependent on assessing how these peptides influence social communication. More specifically, it is important to determine the neural mechanisms involved in communication behaviour that are influenced by nonapeptides. To this end, teleost fish may play a very important role, as they represent the most diverse group of extant vertebrates and exhibit an extraordinarily diverse range of social behaviours (Nelson, 2006).
The nonapeptide systems of teleost fish have two distinctive features. First, the two neuropeptide hormones of teleosts, arginine vasotocin (AVT) and isotocin (IT), are almost exclusively produced in the preoptic area and hypothalamus (POA-AH) (reviewed in
Godwin and Thompson, 2012). Second, although teleost fish exhibit a high degree of variation in relative densities of AVT and IT fibers, between and within species (reviewed in Thompson and Walton, 2013), they show similar distribution patterns across species 9
(Goodson et al., 2003). This conservation in AVT and IT systems makes teleost fish a useful system in which to study nonapeptide roles in social behaviour.
Although nonapeptides have been linked to a number of social behaviours within and across species of fish (reviewed in Godwin and Thompson, 2012; Thompson and
Walton, 2013), only a handful of studies have investigated the role of these peptides in communication behaviour. Research pertaining to nonapeptide modulation of communication in fish was introduced by the influential studies done on the plainfin midshipman fish (Goodson and Bass, 2000a,b; Goodson et al., 2003) but recently research has been focused on South American weakly electric fish.
Gymnotiform weakly electric fish generate a weak electric field around their body by which they sense their environment and communicate (Lissmann, 1958). The electric organ discharge and its modulations constitute easily measurable and robust electrocommunication signals that show a wide range of intraspecific and species-specific variation (Turner et al., 2007). Electrocommunication signals are generated by a relatively simple and well-characterized neural circuit (reviewed in Metzner, 1999; Maler et al.,
1991). The ability to evoke and easily measure a wide range of electrocommunication signals make weakly electric fish an excellent system in which to study the role of nonapeptides in communication behaviour.
The first goal of this thesis was to review the current literature pertaining to the role of nonapeptides in the modulation of electrocommunication behaviour and to explore the promise of weakly electric fish in this regard. Key knowledge gaps are discussed and future directions are presented based on related work in other taxa. The few studies that have 10
investigated the effects of nonapeptides in electrocommunication have focused on AVT;
currently, no published study has investigated the role of IT in weakly electric fish social
behaviour. The second goal of this thesis was to address this gap in knowledge. Specifically,
the goal was to investigate the effect of intramuscular injection of IT onApteronotus previously leptorhynchus.reported AVT-dependent electrocommunication behaviours in male
11
Review - The role of nonapeptides in social communication: A case for
gymnotiform weakly electric fish
Introduction
The complexity and diversity of vertebrate social behaviour is generated by a highly conserved, yet highly plastic, neuroendocrine system. Increasing evidence suggests that a core social behaviour network (SBN) of the midbrain and basal forebrain regulates basic social behavior processes that are essential to all vertebrate species (Newman, 1999;
Goodson and Kabelik, 2009; Godwin and Thompson, 2012). Inter- and intraspecific variations of social organization and behaviour are correlated with the functional connectivity, or distributed pattern of activation, of this conserved network (Goodson,
2005). Underlying the modulations of the SBN is a highly plastic neuropeptide system, the arginine vasotocin (AVT) and isotocin (IT) family of neuropeptides – the nonapeptides
(Goodson and Thompson, 2010). Nonapeptide expression, release pattern, and receptor distribution vary across species (Lema and Nevitt, 2004b; Donaldson and Young, 2009), establishing a basis for species-specific behaviour. Temporal patterns of behaviour associated with social context, season, and physiological state have been linked with responses of nonapeptides to extrinsic and intrinsic cues such as communication signals, photoperiod, and steroid hormones (Perrone et al., 2010; De Vries and Panzica, 2006).
While there is mounting evidence for nonapeptidergic regulation of social behaviours within and across species, no consistent pattern has yet emerged to explain how neuropeptides control social behaviour. The role of AVT systems, has however, been linked to large number of communication behaviours (see Goodson and Bass, 2001; Goodson and 12
Kabelik, 2009; for review of hormone modulation of communication across species). As such, Goodson and Bass (2001) have stressed the importance of understanding the conserved or convergent role of nonapeptides in sensorimotor processes underlying social behaviours, particularly those that serve a communicative function.
Teleost fishes provide a number of advantages to studying the link between nonapeptide hormones and social behaviour. Bony fishes represent the most socially diverse group of vertebrates, displaying a remarkable diversity of social and sexual behaviours (Nelson, 2006). Comparative analyses can be made within and between species of fish, in much the same way that has benefited research in amniotes (Goodson, 2005).
Additionally, bony fishes have retained some of the ancestral traits of AVT/IT systems
(Gwee et al., 2008; Urano and Ando, 2011). Nonapeptide-producing cells are restricted to the preoptic area/hypothalamus in bony fishes (Thompson et al., 2008). Also, the general distribution patterns of putative AVT and IT fibers seem to overlap in descending motor pathways and their respective distribution patterns are similar across teleost species (see
Goodson et al., 2003), providing a great opportunity to explore the effects of nonapeptides on neural circuits and descending motor output pathways that influence communication in fish (Thompson and Walton, 2009). Presently, research pertaining to nonapeptide regulation of communication signals in fishes is scarce and mostly focussed on weakly electric fishes (Thompson and Walton, 2013).
The electric organ discharge (EOD) and its modulations exhibited by weakly electric fish constitute easily measurable communication signals that depend on a small number of well-characterized groups of neurons (Metzner, 1999). The range of electric behaviours displayed, the well-developed understanding of the neural basis of such behaviours, and 13
the opportunity to conduct comparative studies among closely related species with
different social systems make weakly electric fish advantageous systems to investigate the
role of nonapeptides in the regulation and production of communication signals (Turner et
al., 2007). The aim of this review is to provide an overview of nonapeptide systems in the
teleost brain and to summarize the current studies of nonapeptide influences on social
behaviours relating to communication in gymnotiform weakly electric fish. Future
directions will be suggested by drawing from related work in other species.
Nonapeptide systems in teleost fish
The nine-amino-acid neurohypophyseal peptides are classified into two groups, th based on the amino acid at the 8 position, as the basic and neutral peptide families. Each
vertebrate, except for jawless fish, expresses at least two nonapeptides, one from each family (Gimpl and Fahrenholtz, 2001). Cyclostomes, representing the earliest vertebrate groups, possess only a single gene for the basic peptide arginine vasotocin (AVT) utilized by all non-mammalian vertebrates so far studied (Gwee et al., 2008). This suggests AVT to
be the ancestral peptide that gave rise to all other neurohypophyseal nonapeptides. After the divergences of agnathans, a tandem duplication occurred in the ancestor of common
jawed vertebrates which gave rise to two hormone paralogs (Gwee et al., 2008). One of
these genes continued to code for the basic hormone, AVT, in most vertebrates (including
amphibians, reptiles, birds, and cartilaginous and bony fishes). The other gene copy
underwent subsequent mutations that eventually gave rise to the neutral OT-family of
nonapeptides, including oxytocin in most mammals, mesotocin (MT) in amphibians,
reptiles, birds, and marsupials, and isotocin (IT) in ray-finned fishes (for further discussion 14
on the molecular characteristics and evolution of AVP/OT systems see Gwee et al., 2008;
Thompson and Walton, 2013).
In teleosts, AVT and IT are almost exclusively produced in the preoptic area-anterior
hypothalamus (POA-AH) of the diencephalon (Batten et al., 1990; Canosa et al., 2011;
Huffman et al., 2012), but project diffusely throughout the central nervous system (Cerda-
Reverter and Canosa, 2009). The preoptic area is structurally and functionally continuous
with the hypothalamus (Meek and Nieuwenhuys, 1998). The POA-AH nonapeptide
producing cells form a paired nucleus adjacent to the third ventricle thatApteronotus is delineated by
leptorhynchuscell size. According to Maler’s nomenclature for brown ghost knifefish (
; Maler et al., 1991; Johnston and Maler, 1992) the anterior ventrolateral portion is composed of small parvocellular neurons, forming the nucleus preopticus paraventricularis pars posterior (PPp). More caudally, larger, magnocellular cells constitute the anterior hypothalamus (AH). The IT and AVT cells are intermingled within the POA-AH but are not co-expressed within neurons. However, nonapeptide producing cells have been shown to co-express a multiplicity of other neurotransmitters and neuroendocrine factors including adrenocorticotropic hormone (ACTH), somatostatin, dopamine, and corticotropin releasing factor (CRF) (Johnston and Maler, 1992).
The behavioural role of each of the nonapeptide producing cell groups (PPp and AH) is poorly understood, however, some evidence exists to suggest these two subsystems of nonapeptide producing cells control different aspects of social behaviour. Across vertebrates, the PPp neurons generally project to and influence regions involved in stress- response (Gilchriest et al., 2000; Herman et al., 2003) as well as subordinance in teleosts
(Greenwood et al., 2008), whereas the magnocellular population of the AH is generally 15
associated with contributing to behaviours related to dominance (Greenwood et al., 2008) and reproductive status (Ota et al. 1996). A functional distinction between the two nonapeptide producing cell groups is, however, difficult to ascertain due to dynamic patterns of neuromodulation - target regions may express different levels of receptor
(Lema et al., 2015) - and the dynamic release and diffusion of nonapeptides from all parts of the neuronal membrane (Landgraff and Neumann, 2004).
Studies tracing the projections of nonapeptide cells in teleosts are very limited
(Thompson and Walton, 2013). We will present the known general fiber distribution here.
More detailed discussion of fiber distribution in relation to weakly electric fish behaviour will be discussed in a later section. Retroactive tract tracing along with immunohistochemical studies report both hypophysiotropic and extrahypothalamic projections of POA-AH neurosecretory cells (Holmqvist and Ekstrom, 1995; Goodson and
Bass, 2001; Saito et al., 2004). Interestingly, single neurons show simultaneous, multiple projections to the pituitary and extrahypothalamic regions, a characteristic exclusive to teleost fishes (Saito et al., 2004; Cerda-Reverter and Canosa, 2009). These projections are suggested to serve as a means for coordinated control of peripheral and central target regions, although it is also possible that nonapeptide release, receptor type, and density differ from region to region (Saito et al., 2004).
Generally, AVT and IT share conserved projection patterns, innervating regions particularly important for social behaviour (Goodson et al., 2001; Saito et al., 2004; Cerda-
Reverter and Canosa, 2009), although IT shows abroader distribution in the brain
(Goodson et al., 2003). Densely neuropeptide-immunoreactive fibers project from the POA-
AH through the preoptico-hypophysial tract and terminate in the neurohypophysis where 16
nonapeptides are released into the vascular system via axon terminals. Rostrally projecting fibers innervate the telencephalon with higher fiber density coursing through the lateral telencephalon than the dorsal regions. Within the diencephalon, fibers project throughout the POA-AH. Fibers also course along the midline to innervate the thalamus. Retrograde labelling techniques have demonstrated POA-AH projections to diencephalic regions involvedEigenmannia in communicative virescens motor output in at least one species of weakly electric fish
( ; Wong, 1997). Common to most fish, a lateral descending projection courses through the midbrain to innervate the torus semicircularis (Thompson and Walton, 2013). Medial projections descend further through the tegmentum to the cerebellum and hindbrain (Goodson et al., 2001). A more detailed account of POA connectivity in relation to electromotor brain regions is discussed below. Although some patterns of projection are conserved, inter- and intraspecific variation in behaviour are reflected in variation in nonapeptide distribution (Godwin and Thompson, 2012;
Thompson and Walton, 2013). However, a number of studies in fishes have demonstrated that variation in behaviour cannot be strictly ascribed to different levels of nonapeptide production and release (Lema, 2010; Lema et al., 2015). One must also consider variation in nonapeptide receptor abundance and distribution.
Currently, four AVT receptors and two IT receptors have been identified in teleost fishes (Konno et al., 2010; Lema et al., 2015, ). The four AVT receptors are made up of two
V1a types, V1a1 and V1a2, and two V2 type receptors, V2a and V2b (Lema et al., 2015). The two IT receptors, ITR1 and ITR2, have only been described in Lamprologine cichlids
(O’Connor et al., 2015); all other studies of IT receptor expression note only one IT receptor type, termed “ITR” (see Lema et al., 2015). Transcripts encoding each of the 17
nonapeptide receptors have been localised, using RT-PCR, to the fish brain (Konno et al.,
2010; Huffman et al., 2012; Yamaguchi et al., 2012; Lema et al., 2015; O’Connor et al.,
2015). Studies of AVT/IT receptor distribution patterns remain scarce in the literature.
Huffman and colleagues (2012) found overlapping expression of V1A-typeAstatotilapia and ITburtoni receptors in the telencephalon, diencephalon, and mesencephalic regions of .
Within the telencephalon, V1A-type and IT receptors are expressed in sensory regions including the olfactory bulb. Expression of these receptors within the diencephalon is more diffuse than in the telencephalon, including sensorimotor regions such as the POA and central posterior nucleus.The presence of multiple nonapeptide receptors in the brain suggests effects on social behaviour in teleosts that can be mediated through any combination of these receptors (Semsar et al. 2001; Lema and Nevitt, 2004b; Santangelo and Bass, 2006).
Most studies dealing with specific receptor effects of AVT on social behaviour focus on V1a-type receptors (Insel et al., 1994; Insel and Fernald, 2004; Donaldson and Li, 2008).
Several studies in ray-finned fishes have administered V1a-type receptor antagonists and observed modulations of aggressive, courtship, and approach behaviours (Semsar et al.,
2001; Thompson and Walton, 2004; Oldfield and Hofmann, 2011). However, other receptor types are known to be differentially expressed in brain regions involved in social behaviour
(Lema et al., 2015), suggesting a high level of complexity in nonapeptide regulation of species-specific social behaviour (see Goodson and Bass, 2001; O’Connor et al.,2015)
18
Communication behaviour
Vocal communication behaviour
Social communication manifests in a number of ways, from vocalizations to electric
signalling. The first neurophysiological evidence of AVT and IT involvement in sexual and
aggressive communicationPorichthys in notatus a teleost fish comes from studies on the vocalizing
midshipman fish, (Goodson and Bass, 2000a,b). Midshipman produce phenotype-specific vocalizations that serve as agonistic or courtship signals. The investigation of nonapeptide effects on intraspecific behaviour is benefited by the presence of three morphologically and behaviourally distinct sexual phenotypes. Type-I, territorial males defend nests under rocks and produce long advertisement ‘hums’ to attract potential mates. All morphs, including type-II sneaker males and females, produce agonistic ‘grunt’ calls. The acoustic behaviour of these fish can be accurately quantified, mimicked through playback, and the neural correlates are well characterized (Godwin and Thompson, 2012).
The physical attributes of vocalizations are directly determined by the temporal properties of a hindbrain pacemaker-sonic motor nucleus (PN-SMN) (Bass and Baker,
1990). Recordings of “fictive vocalizations” from isolated occipital nerves, whose axons arise partially from the sonic motor nucleus, are, therefore, used as a measure of naturalistic vocal-motor activity (Goodson and Bass, 2000b). Variation of vocal output, then, must be mediated through the PN-SMN. A connection between the POA-AH and the
PN-SMN has been described and naturalistic vocal-motor activity can be evoked by stimulation of the POA-AH (Bass et al., 1994). Goodson and Bass (2000a) tested the hypothesis that nonapeptide application into the POA-AH would differentially modulate vocal-motor activity in each of the adult morphs. AVT injection into the POA-AH effectively 19
reduced the duration and total number of fictive vocalizations produced by type-I males.
Administration of a V1-type receptor antagonist facilitated the vocal-motor response by
increasing the burst duration of fictive calls, but not the totaltype number of bursts produced.
This would suggest that AVT in the POA-AH regulates the of calls (long hums or short grunts) produced by territorial males. A further study by this group (Goodson and Bass,
2000b) investigated the effect of AVT injected into a different brain region, the paralemniscal midbrain tegmentum, a candidate region for vocal-acoustic integration. AVT in this region reduced the number of total bursts produced but also increased the latency of burst response to stimulation. These studies, together, demonstrate that the differential effect of AVT on vocal communication is contingent on brain region affected. No significant effects of AVT administration were observed for females or type-II males. The divergence in vocal-motor output and behaviour seems to be due to differences in receptor distribution and or density since type-II males share similar AVT-ir tract profiles with type-
I males but exhibit female-like vocal behaviour (Goodson and Bass, 2000b). AVT appears to modulate the specific type, and overall production of vocal signals in a region-specific manner.
In contrast to AVT, IT had an effect on type-II males and females, but not type-I male vocal- motor behaviour (Goodson and Bass, 2000a). IT injections into the POA-AH decreased the total number of bursts whereas an oxytocin antagonist facilitated the fictive vocalizations.
Behavioural neuroendocrinology studies in communication can benefit from animal systems that have robust and distinct species-specific behaviours that are easily measured and manipulated as well as neural systems that are well-characterized. Weakly electric fish are a prime example of such a system. A vast repertoire of electrocommunication signals 20
exists within and between species of weakly electric fish that share a relatively simple neural system that drives these behaviours (Turner et al., 2007; Silva et al., 2013).
Electrocommunication behaviour
South American weakly electric fish (Order Gymnotiformes) generate an oscillating electric field around their body that they use to actively sense their environment and communicate with conspecifics (see Zupanc and Bullock, 2005). Weakly electric fish can be categorized into two types based on the type of electric organ discharge they produce.
Pulse-type species generate intermittent discharge pulses, the frequency of which can greatly vary. Wave-type species generate continuous, quasi-sinusoidal voltage oscillations that are characterized by a highly regular frequency, amplitude, and waveform (Zakon et al., 2002). Species within both groups are able to modify their EOD during social interactions to communicate sexual status (Black-Cleworth, 1970, Hagedorn and
Heiligenberg, 1985), aggression (Zubizarreta et al., 2012), and social status (Perrone et al.,
2010, Silva et al., 2013). These modulations are often referred to as social electric signals
(SES).
Wave-type species produce two main forms of SES – “chirps” and the well-known jamming avoidance response (JAR) (Zakon et al., 2002). The most common modulations produced are chirps, transient (20-200 ms long) increases in EOD frequency (EODf) that can be accompanied by a decrease in EOD amplitude (Bullock, 1969; Hopkins, 1974; Zakon et al., 2002). Chirps occur spontaneously (Engler et al., 2000), during free-swimming dyadic encounters (e.g., Hupé and Lewis, 2008), and can be evoked with stimulation by a sinusoidal electric signal that mimics a conspecific fish (Zupanc and Maler, 1993; Bastian et 21
al., 2001). Chirp structure such as frequency excursion, duration, and amplitude
modulation can vary and thus six chirp types have been so far described (Reviewed in
Zupanc et al., 2006). The communicative functions of type-I and type-II chirps are
discussed below.
The JAR is characterized by a gradual EOD frequency shift away from the frequency
of a stimulus signal (Figure 1). This EOD shift is usually exhibited in response to the
presence of a conspecific fish with a similar EOD frequency (within ±20Hz of the fish’s own
EODf; Kramer and Kaunzinger, 1991). The JAR is thought to help maintain the
electrolocation ability of a fish that would be “jammed” by a similar EOD. Kramer (1994).
however, advocates the idea of a communication function of the JAR. By increasing the
difference in EOD frequency, a fish can better discriminate EODf and waveform of an
interacting fish (Kramer, 1994). Pulse-type weakly electric fish, like wave-type species,
produce chirps and the JAR, however, they are also able to turn off their EOD as a form of
communication. The cessation in the emission of the EOD signal is termed an “off”.
Both wave- and pulse- type species are principally nocturnal animals (Winemiller
and Adite, 1997) with different patternsApteronotus of circadi leptorhynchusan rhythmicity in EOD production and
modulation. The wave-type fish, , showed a pronounced increase
in spontaneous chirp generation during the dark periods of a 12:12h light:dark cycle
(Zupanc et al., 2001) but no evidence is available to suggest a change in the baseline EOD or
JAR. On the other hand, pulse-type fish increase their baseline EOD rate during the night,
when they are most activeBrachyhypopomus (Kawasaki and pinnicaudatus Heiligenberg, 1989; Perrone et al., 2010). The
enhanced EOD rate in is a necessary antecedent for other
SESs to be produced (Silva et al., 2007). Chirps and interruptions in this species are only 22
observed following the additional increase in EOD rate. Silva et al. (2007) suggest that the
neural mechanisms involved in producing these SESs require a higher level of baseline
firing.
Finally, both wave-type (Zakon et al., 2002) and pulse-type (Perrone et al., 2009;
Batista et al., 2012) are able to increase and decrease their baseline EOD frequency. The
modulation of baseline EOD frequency is species- and context-dependent and can be used
as a sexual and/or aggressiveA. leptorhynchus signal. Since EOD frequency is closely related to size, it is
conceivable that male , in the presence of females, raise their EOD frequency to appear more dominant than they areBrachyhypopomus (Dunlap and Oliveri, gauderio, 2002). In reproduction-related aggressive interactions of dominant fish increase their EOD rate to signal dominanceG. omarorum (Silva et al., 2013). During non-reproduction- related aggressive interactions of , submissive fish decrease their EOD rate to signal submission (Batista et al., 2012).
Segregated neural pathways control electrocommunication behaviour
The neural circuitry involved in producing the EOD and its modulations in
gymnotiform fishes is relatively simple and well characterized (Metzner, 1999). The EOD is
produced by synchronous firing of electrogenic cells, the electrocytes, which make up the
electric organ (Mills et al., 1992; Metzner, 1999). The discharges of the individual
electrocytes are driven by command pulses of a medullary hindbrain pacemaker nucleus
(Pn), the cells of which fire in a one-to-one manner with the electrocytes (Dye and Meyer,
1986; Zupanc and Maler, 1997). The Pn is composed of two main neuron types, pacemaker
and relay cells. Pacemaker cells, which generate the command signal for the EOD, are 23
connected, through mixed chemical and electrical synapses, with each other and with relay cells (Dye and Meyer, 1986; Metzner, 1999; Smith, 2005). The relay cells convey the rhythmic command signal to spinal motoneurons that innervate the electrocytes of the electric organ (in Apteronotids the spinal motoneuron endings comprise the electric organ itself; Bass, 1986).
Two known inputs to the Pn mediate modulations of the EOD frequency to generate distinct behavioural electromotor patterns. One of these inputs originates from the thalamic prepacemaker nucleus which is part of the central posterior nucleus, hence termed the central posterior prepacemaker nucleus (CP/PPn) (Zupanc and Heiligenberg,
1992). The second is a mesencephalic, sublemniscal prepacemaker nucleus (SPPn) (Keller et al., 1991). A lateral group of cells of the CP/PPn sends descending glutamatergic inputs to the Pn (Kawasaki and Heiligenberg, 1988). The glutamatergic inputs depolarize relay cells of the Pn, resulting in a transient increase in the EOD resembling chirps (Kawasaki and Heiligenberg, 1988; Spiro, 1997). Chirp-like modulations of the relay cell activity upon electrical stimulation of this lateral region of the CP/PPn were abrupt and continuous with the stimulation, suggesting a direct connection with the CP/PPn (Kawasaki and
Heiligenberg, 1989). This lateral region is thus termed PPn-C (“C” stands for chirp).
Whereas the PPn-C appears to be involved in generating chirps across all gymnotiforms
(Kawasaki and Heiligenberg, 1990; Zupanc and Heiligenberg, 1992), the role of the SPPn differs between species, controlling different behaviours.
TheEigenmannia SPPn has been shownApteronotus to play a role in the control of the JAR in wavetype fish of the genera and (see Heiligenberg et al., 1996 for a comparison in 24
Eigenmanniabehaviour and neuronal control of the JAR between these genera). Briefly, the SPPn of
provides tonic glutamatergic (non-NMDA) excitation to relay cells of the Pn which maintain the EOD frequency at an elevated level. In the presence of a jamming signal, the SPPn receives an inhibitory GABA-ergic input from a diencephalic structure, the nucleus electrosensorius (nE), which interfaces electrosensory processing and premotor control of electricApteronotus, behaviour (Heiligenberg et al., 1991), to reduce the elevated EOD frequency. In the glutamatergic activity of the SPPn is normally tonically inhibited via nE input. Upon release of inhibition, the SPPn activates, by glutamatergic projections, relay cells to raise the EOD frequency required to perform a JAR (Heiligenberg et al., 1996).
Pulse-type fishes, on the other hand, utilize the SPPn-Pn connectivityHypopomus to generate pinnicaudatusinterruptions in the EOD. Stimulation of the SPPn in the pulse-type species
triggers a sustained depolarization of relay cells as a result of NMDA receptor activation (Spiro, 1997). The consequence of sustained relay cell depolarization is an incoherent firing of relay cells and ultimately an interruption or “off” in the EOD.
If nonapeptides are involved in the regulation of social electric signalling, we would expect them to impinge, directly or indirectly, on at least one of the units of the basic motor control system for SES (Pn, PPn-C, or SPPn).
25
Connections between the electromotor system and POA-AH
Figure 2 provides a schematic representation of the connection between the POA-
AH and electromotor and electrosensory systems discussed in this section. Much attention
has been given to the chirp controlling region of the CP/PPn and its connectivity to regions
regulating sexual communication, including the POA-AH (Johnston and Maler, 1992;
Zupanc and Horschke, 1997a; Wong, 1997; Wong, 2000; Correa et al., 2002; Zupanc,E. virescens 2002;
Kolodziejski et al., 2005). AnterogradeA. leptorhynchus and retrograde tract tracing studies in
(Wong, 1997, Wong, 2000) and (Johnston and Maler, 1992; Zupanc and
Horschke, 1997a; Correa et al., 2002) present similarities and differences between the connections of the POA-AH and the CP/PPn of these species. An early tract-tracing study conducted by Johnston and Maler (1992)A. found leptorhynchus a reciprocal connection between the
CP/PPn and the lateral hypothalamus of . They hypothesized a connection between peptidergic hypophysiotropic nuclei (including the POA-AH) and the CP/PPn which would influence electrocommunication output. This hypothesis set the grounds for further investigation to extend the understanding of POA-AH-CP/PPn connectivity and nonapeptide influence on electrocommunication.
Retrograde and anterograde tract-tracing methods, using neurobiotin as a tracer substance,Eigenmannia were virescens performed to provide a detailed analysis of the connections of the CP/PPn in (Wong, 1997). Following injections into the CP/PPn, retrogradely labelled cells were consistently observed in the AH but rarely in the PPp, providing a link between at least one cluster of nonapeptide producing cells and the electromotor system.
To test for a functional role of the POA-AH in electrocommunication, Wong (2000) used electrical stimulation of the POA-AH in anesthetized animals while recording EOD output 26
near the tail of the fish. Complex EOD modulations, particularly chirps, were elicited by
stimulation of localized regions of the POA-AH and not by global activation of the
telencephalon. The chirp-like modulations elicited by POA-AH stimulation differed from
stimulation of the PPn-C of earlier studies (Kawasaki and Heiligenberg, 1988; Spiro, 1997)
in one key way: response to stimulation of POA was observed as a long-latency after-
response, which followed the termination of the stimulus (Wong, 2000) . This, along with
the finding from the same study that stimulation of the preoptic area evokes bursts of
chirps rather than continuous modulation, suggests that the POA-AH-PPn-C connection is
indirect. A. leptorhynchus
Tract-tracing experiments in also support an indirect connectionA. betweenleptorhynchus these areas,Eigenmannia however, it would seem that the connectivity differs between
and (Zupanc and Horschke, 1997a; Correa andA. leptorhynchus Zupanc., 2002).
Results from anterograde and retrograde tract-tracing investigatons in
present no indication of a projection from the POA-AH to the CP/PPn proper (Zupanc and
Horschke, 1997a). Neurons of the CP/PPn, however, project, bilaterally, to the POA-AH to
innervate both the PPp and the AH. Zupanc and Horschke (1997a) posit that the previously
described projections from the POA-AH to the CP/PPn (Wong, 1997) were a result of
unrestricted application of tracer substance. Application of large amounts of biocytin to the
CP/PPn resulted in the uptake of tracer substance in this complex as well as by neurons in
the vicinity, in turn, retrogradely labelling cell bodies in the POA-AH. By restricting the
tracer substance to regions defined by the cell bodies of the CP/PPn, no retrogradely
labelled cell bodies could be found in either the PPp or the AH (Zupanc and Horschke,
1997a). This was corroborated by a later study that employed a tract-tracing method that 27
allows for precise application of tracer substance under visual control (Correa and Zupanc.,
2002).
Two avenues for POA-AH influence on chirping behaviour have been proposed
(Correa and Zupanc, 2002). The first, a direct route, involves the dendritic arborisation of the PPn-C cells. The dendrites of PPn-C cells extend beyond the boundary of the CP/PPn, defined by the cell bodies within the nucleus (Kawasaki et al., 1988). Dense innervation of varicose fibers immunopositive for several neuropeptides (Sas and Maler, 1991; Weld and
Maler, 1992; Zupanc et al., 1999) overlap with PPn-C dendritic arbors. It is possible, then, that POA-AH projections terminate in the fields occupied by PPn-C dendrites and influence
EOD modulations by direct activation of the PPn-C. The second, an indirect route, involves a diencephalic relay to forebrain structures, the preglomerular nucleus (PG) (Zupanc and
Horschke, 1997b). The pallium of gymnotiform fishes receives almost all of its input from the PG (Giassi et al., 2012). A major component of the PG connectivity is the reciprocal connection between it and the POA-AH (Zupanc and Horschke, 1997b), which presumably couples the endocrine system to motivated behaviour such as electrocommunication signals during courtship and agonistic encounters (Giassi et al., 2012). Finally, recent studies have confirmed the presence of nonapeptidergic projections in the vicinity of the
Pn (Perrone et al., 2014). This would suggest a direct link between the nonapeptidergic system and the electromotor system.
As an extension to the direct involvement of the nonapeptidergic system on electromotor systems, nonapeptides are also involved in modulating the activity of electrosensory structures, which in turn regulate electromotor responses. The dorsal torus semicircularis (TSd), a midbrain structure, receives input from a number of electrosensory 28
regions and integrates into the electromotor system via the nE (Giassi et al., 2012). The TSd
contains neurons that are sensitive to the difference in EODf of neighbouring conspecifics
and is, therefore, considered a major player in processing and regulating the response to
electrocommunication signals, particularly to courtship and aggressive signalsin situ (Giassi et al.,
2012). Huffman and colleagues (2012), using immunohistochemistry and hybridization, have mapped IT- and AVT- receptor protein and mRNA expression to the
TSd of an African cichlid fish. Although it is unclear whether the nonapeptidergic system innervates the TSd or nE in gymnotiform fish, POA innervation of the torus semicircularis is a common feature among teleost fishes (Thompson and Walton, 2013).
Nonapeptide effects on electrocommunication behaviour
Nonapeptides and wave-type fish
A number of previous studies (reviewed in Godwin and Thompson, 2012) provide strong evidence, across and within fish species, for AVT and IT effects on communication.
The tendency of gymnotiform fishes to produce chirping behaviour and the type of chirps produced vary enormously with species and behavioural situation within a species
(Hagedorn and Heiligenberg, 1985). Evidence linking nonapeptide producing cells to chirp- controlling regions, described above, support the notion that AVT and IT have modulatory effects on the propensity to produceA. leptorhynchus chirping behaviour and the types of chirps produced.
Studies of chirps produced by during agonistic and courtship encounters
(Hagedorn and Heiligenberg, 1985) and those evoked by electrosensory stimuli that mimic the discharge of conspecifics (Bastian et al., 2001), identify two main chirp types. Type-I and type-II chirps are characterized by transient increases in EOD with a duration of 29
approximately 20 ms but differ in maximum frequency excursion; type-I chirps are marked by large frequency excursions (between 300-500 Hz), whereas type-II chirp maximum frequency excursions range between 50-150Hz (Zupanc et al., 2006). Type-II chirps, the more commonly produced chirps, are thought to serve as agonistic signals during intrasexual encounters whereas type-I chirps may serve as courtship signals by males during intersex interactions. The baseline EOD frequency of brown ghosts is sexually dimorphic with females discharging at a frequency between 600 and 800 Hz and males discharging at rates between 800 and 1000Hz (Zakon et al., 2002). By subjecting the fish to a “chirp assay”, which consists of stimulating an isolated fish with electrical signals within the range of EOD frequencies produced by that species, one can readily evoke chirps (Dye,
1987; Zupanc and Maler, 1993; Zupanc et al., 2006). Interestingly, male brown ghosts, but rarely females, respond with chirps to electrical stimulation during chirp assay experiments (Dulka and Maler, 1994; Zupanc et al., 2006). Type-I chirps likely function as courtship and mating signals since difference frequencies between 100 and 200 Hz, likely to occur as a result of intersex interactions, evoke this type of chirp (e.g. Figure 3A)
(Bastian et al., 2001). Stimulation of male brown ghosts with small difference frequencies
(a stimulus signal with a frequency within the range of ±12 Hz from the experimental fish), corresponding to same-sex encounters, evoke primarily type-II chirps (e.g., Figure 3B), suggesting a territorial and/or agonistic function of this communicative signal (Bastian et al., 2001).
Bastian et al. (2001) investigated the effect of AVT on the propensity to produce each type of chirp in both sexes. This group also examined the effect of AVT on frequency excursion during JAR behaviour. The AVT experiment included three phases for each fish. 30
The first, control phase, consisted of small difference frequency stimulus presentations
while recording EOD output and chirp production of the fish. Following immediately upon
the control phase were intraperitoneal injections of saline and subsequent stimulus
presentation similar to the control phase. The final phase consisted of another injection, this time of AVT with saline as a vehicle, followed by another period of stimulus presentation. For each fish, the mean number of type-I and type-II chirp counts as well as
JAR excursion evoked during stimulus presentation following AVT injection were
compared to mean chirp counts and JAR excursion following saline injection and
presentation of the same stimuli. The mean number of type-II chirps produced per
stimulation period, by males, was significantly decreased relative to the saline treatment
phase. Interestingly, only a subset of males (5 of 12 males) showed a significant increase in type-I chirp production following AVT injection. The same subset of males showed an
increase in JAR excursion following AVT injection. Chirp production and JAR excursion by
females, however, was not significantly affected by AVT. This experiment clearly
demonstrates a sex-specific, and possibly reproductive status-related effect of AVT on
electrocommunication.
The authors (Bastian et al., 2001) suggest that the decrease in type-II chirp and
increase in type-I chirp production following AVT injection is indicative of a shift from
agonistic to reproductive motivation in male brown ghosts modulated by the nonapeptide
AVT. The switch from type-II to type-I chirps has been correlated to the recruitment of
PPn-C cells (Kawasaki et al., 1988). Type-II chirps are evoked when individual or small groups of PPn-C cells are activated, whereas type-I chirps are evoked when larger populations of these cells are stimulated. It is possible that AVT is involved in recruitment 31
of PPn-C cells, shifting electrocommunication signals from type-II to type-I during courting and sexual interactions (Bastian et al., 2001). The presence of an effect on JAR behaviour, although functionally poorly understood, suggests that AVT is active in modulating multiple neural circuits controlling electrocommunication, the SPPn being a region of interest. Finally, the absence of an effect of this substance on female behaviour could reflect a difference in nonapeptide involved. As mentioned above, AVT is involved in modulating fictive vocalizations produced by territorial male midshipman fish whereas IT modulates vocalizations in females (Goodson and Bass, 2000b). It is of particular interest to understand where in the brain nonapeptides exert their effect and if their distribution is correlated with sex and reproductive status. Studies aimed at determining the distribution of nonapeptide receptors in the brain of brown ghosts are needed. The receptors for AVT and IT have been identifiedA. leptorhynchusin another fish (pupfish; Lema, 2010). The central nervous system transcriptome of is available (Salisbury et al., 2015) and should aid in the effort to identify and determine the distribution of each type of nonapeptide receptor within the brain. A map of receptor distribution can guide targeted activation of specific electromotor regions by nonapeptides to better understand their role in electrocommunication.
Nonapeptides and pulse-type fish
Nonapeptides have been extensively linked to social status in fish, particularly in dominant-subordinate relationships (Fox et al., 1997; Dewan et al., 2008; Greenwood et al.,
2008; Perrone, 2012; Reddon et al., 2015). Evidence from a number of pharmacological and neuroanatomical studies has led some to propose the idea that the relative weights of two 32
subsystems of AVT neurons influence different aspects of behaviour relating to social
statusDanio (Greenwood rerio et al., 2008; Godwin and Thompson, 2012). As an example, in zebra fish
( ), dominant fish express higher levels of AVT in magnocellular neurons whereas subordinates express higher levels of AVT in parvocellular neurons of the POA-AH
(Larson et al., 2006). Weaklycommunication electric fish provide signals a unique opportunity to investigate nonapeptide regulation of involved in establishing dominant- subordinate status. It is surprising that there exists only a single study (Perrone, 2012; reviewed in Silva et al., 2013), that we are aware of, which implicates nonapeptides in the control of communication signals relatedB. gauderio to social status.G. omarorum The study took advantage of two syntopic species of pulse-type fish, and , that form different social structures and establish ranks of EOD rate after contest resolution. G. omarorum
In the post-resolution phase of agonistic encounters of two , a solitary, monomorphic, and highly aggressive species (Richer-De-Forges et al., 2009), submissive individuals will signal their submission by decreasing the baseline EOD frequency that they emit (Silva et al., 2013). On the other hand,B. gauderio dominant males of a gregarious species that displays a polygynous breeding system, (Miranda et al., 2008), sustain an increasedG. omarorum EOD rate duringB. gauderio, the contest through the post-resolution phase (Perrone, 2012).
Both, and produce other, distinct submissive and dominant behaviours (see Batista et al., 2012; Zubizarreta et al., 2012).G. omarorum In regards to establishing social ranks, B.however, gauderio only the submissive signalling of and the dominance signalling of seem to be under the influence of nonapeptides. As reviewed by
Silva et al. (2013), electrical dominance and submiB. gauderiossive signalling are both AVT-dependent, each in a particular way. Predicted dominant were injected with Manning 33
compound (MC), an AVT receptorB. gauderioV1a antagonist. The AVT antagonist prevented the
electrical dominance display in . Because baseline EOD is controlled directly by the Pn, it is possible that direct AVT stimulation of the Pn is responsible for the sustained increase in EOD rate observedG. omarorum in intact dominant individuals. Injections of AVT into predicted subordinate prevented the electrical submission displayed in the post-resolution phase, suggesting that the decrease in EOD rate observedGymnotus in omarorum intact subordinates is attained by inhibition of an AVT input to the Pn. also produce other, unambiguous, submissive social electric signals. Along with the previously mentioned decrease in EOD rate, these fish produce offs and chirps to signal submission
(Batista et al., 2012), which result from the activity of prepacemaker nuclei, as discussed above. The effect of AVT on the production of submissive offs and chirps, however, has not been tested.
The EOD rate of gymnotiform fishes, the basic unit of electrical communication, carries information about an individual’s sex, social status, and physiological state (Caputi et al., 2005) and varies with season, time of day, and social context (Silva et al., 2007).
Recent comparative studies (Perrone et al., 2010; PerroneB. gauderio et al., 2014)G. haveomarorum aimed at exploring the effects of AVT on the basal EOD rate of and and have identified the Pn as a neural substrate through which AVT can modulate diverse electrocommunication behaviour.
Pulse-type electric fish increase their EOD rate during the night when they are most active (Black-Cleworth, 1970) but the magnitude and timing of this night-time behaviourG. is contextomarorum and species-specific (Silva et al., 2013). The nocturnal increase in EOD of
is transient and returns to baseline less than 60 minutes after sunset (Silva et al., 34
2013; Perrone et al., 2014). The function of this transientB. gauderio increase is, however, not well
understood. The nocturnal enhancement of EOD in can last 2 hours during night-time social interaction (Silva et al., 2007; Perrone et al., 2014) and shows significant seasonal variations (Silva et al., 2007). Individuals show an additional increase in EOD rate that is only observed during male-female interactions during the breeding season, resulting in a higher EOD rate increase as compared to dyadic interactions outside of the breeding season (Silva et al., 2007). Arginine vasotocin has been directly linked to the night time increase in EOD rate observed in both species. in vivo in vitro
Perrone et al. (2010) conducted aB. number gauderio. of experiments, both and , linking AVT to nocturnal EOD activity in Intraperitoneal injection of AVT in isolated fish induced an immediate and progressive increase in the EOD rate that lasted
120 minutes, resembling nocturnal increases in baseline EOD rate of intact fish. The magnitude of EOD rate enhancement by AVT was greater in breeding than non-breeding individuals. Interestingly, the additional EOD rate enhancement observed in intact breeding individuals, could be induced in non-breeding males during dyadic interaction, by administering AVT shortly before artificial sunset. Together these results suggest that AVT is involved both in the nocturnal increase of EOD rate as well as the seasonally-dependentin vitro additional EOD rate enhancement observed in breeding context. As for the experiment, perfusion of AVT onto an isolated Pn preparation (without prepacemaker input) reproduced the AVT-induced EOD rate increase, both in magnitude and timing, observed in individuals injected with AVT. Administration of an AVT V1aR receptor antagonist, MC, in the same study (Perrone et al., 2010) had opposing effects to injections of AVT. Application of the antagonist decreased the diurnal EOD rate of isolated individuals, 35
in vitro
non-breeding dyads, and the firing rate of Pn cells. Moreover, MC inhibited the additional EOD rate enhancement in breeding dyads. Altogether these results implicate V1a receptors on neurons of the Pn in the nocturnal enhancement of EOD rate and support the notion that fluctuating endogenous levels of AVT (or AVT receptors) influence the day- night and seasonal variation in behaviour observed in these fish. Diurnal rhythms of nonapeptide release and receptor expression have been explored in another teleost, the
Amargosa pupfish (Lema, 2010). Transcripts encoding AVT, IT, and an AVT V1a2 receptor exhibited distinct diurnal patterns that were paralleled by changes in social behavior in the pupfish.
Upon closer examination, however, a follow-up study (PerroneG. omarorum et al., 2014), was able to show a clear, but distinct effectG. omarorum of AVT on the EOD rate in .
Intraperitoneal injection of AVT to produced a transient increase in vitro of the diurnal EOD rate that lasted less than 30 minutes and was paralleled by an effect of
AVT on Pn firing rate in brainstemG. omarorum slices. AVT V1a receptors are also involved in the AVT- induced EOD rate increase of in vivo as MC injectionsin vitro prior to AVT administration inhibited the EOD enhancement during and trials. The effect of AVT in this species had been missed in the previous study (Perrone et al., 2010) that focused on a much longer time scale. In summary, interspecific differences in AVT modulation of EOD rate (Perrone et al., 2010; Perrone, 2012; Silva et al., 2013; Perrone et al., 2014) support the conclusion that AVT, acting directly on the Pn, modulates electrocommunication behaviour in dramatically different ways across species.
Nonapeptides, and particularly AVT, are involved in generating between-species differences in electrocommunicative behaviours that seem to be reflective of social 36
B. gauderio
structure (Silva et al., 2013; Perrone et al., 2014). It may be important for male ,
in a gregarious system, experiencing social interactions throughout the year, to maintain
long-lasting modulations of its electric signal and toG. signal omarorum dominance to ward off other
males from their territory. By contrast, for solitary , it may be unnecessary to maintain costly EOD modulations, but important to produce unambiguous signals of subordination in order to avoid howphysical conflict.
What remains unclear is AVT is able to exert such dramatically different responses from the Pn of different species. Two possible explanations are apparent: 1)
Differential distribution of nonapeptide receptors give rise to a complex variety of behaviours and phenotypes across species (Goodson, 2008; Thompson and Walton, 2013;
Lema et al., 2015). A compelling next step in this area of study would be to map the distribution and density of nonapeptide receptors in the brain of both species and compare distribution and density patterns, particularly within the Pn. 2) The pattern of AVT fiber distribution and position in the proximity ofG. theomarorum Pn differs between the two fish species
(Perrone et al., 2014). The AVT-ir fibers of were arrangedB. Gauderio in a narrow area along the midline, just dorsal to the Pn, whereas the AVT-ir fibers of showed a wider distribution pattern around the Pn midline (Perrone et al., 2014). As previously mentioned, it has been postulated that the relative development and activity of two subsystems of AVT control different forms of social behaviour: magnocellular cells of the
AH release AVT in circuits that stimulate aggression and courtship behaviour, whereas cells of the PPp release AVT in circuits related to subordinate behaviour (Greenwood et al.,
2008; Godwin and Thompson, 2012). The species-specific distribution of AVT-ir cells near the Pn of each fish could be achieved by differences in the relative weight of PPp and AH 37
cell projections to the Pn (Perrone et al., 2014). Experiments using retrograde tract tracing methods would help to determine whether there is a significant difference in the origin of
AVT-ir fibers found near the Pn of each species.
Conclusion
Nonapeptide regulation of social behaviour is a conserved feature of vertebrates, underlying species-specific, sexual, and context-specific difference in behaviour (Godwin and Thompson, 2012). This high level of variability and wide range of effects have made it difficult to derive general patterns of nonapeptide effects. Comparative studies of nonapeptide actions of related species with different behaviours have been especially valuable in understanding how nonapeptides interact with neural networks to regulate social behaviour (Young et al., 1997; Goodson, 1998a,b; Dewan et al., 2008). Gymnotiform weakly electric fish are a promising system in which to study the role of nonapeptides on social behaviour, particularly relating to communication. The electric organ discharge and its modulations constitute easily measurable communication signals that have been described in a wide range of species and social contexts (Black-Cleworth, 1970; Zakon et al., 2002; Capurro and Pakdaman, 2004; Silva et al., 2007; Turner et al., 2007). The extraordinary diversity of communication signals depends on a small number of well characterized groups of cells: two prepacemaker nuclei (CP/PPn and SPPn) and a pacemaker nucleus (Johnston and Maler, 1992; Kennedy and Heiligenberg, 1994;
Heiligenberg et al., 1996).
The effect of AVT has been linked, directly or indirectly, to each of the three nuclei making up the descending electromotor pathway in gymnotiform fishes (Wong, 2000; 38
Bastian et al., 2001; Perrone et al., 2014). The chirp-controlling region, PPn-C, is under the indirect influence of nonapeptidesA. leptorhynchus (Wong, 2000). AVT has been shown to modulate chirping behaviour in by increasing type-I chirps and decreasing type-II chirps (Bastian et al., 2001). Since chirping behaviour is the most common form of EOD modulation (Zupanc and Bullock, 2005) and nonapeptides are linked to the chirp- controlling region, an exciting avenue for future studies would be investigate the effect of both AVT and IT on chirping behaviour between species and behavioural contexts.G. omarorum, For example, AVT is known to play a role in one type of submissive displayGymnotus in omarorum, namely, the post-resolution decrease in EOD rate (Silva et al., 2013). however, also produce chirps during agonistic encounters to signal subordination (Batista et al., 2012). It is, likely, then, that submissive chirps are under nonapeptide control. No neuroanatomical evidenceA. is leptorhynchus available to support nonapeptide connections to the SPPn, the
JAR controlling region in . However, AVT has been shown to affect the magnitude of JAR excursion in this species, linking, at least indirectly, nonapeptidesG. to the
SPPnomarorum (Bastian et al., 2001). Let’s revisit the submissive electrocommunication of
. Along with a decrease in basal EOD rate and chirping, these fish produce “offs”, a behaviour controlled by the SPPn in this fish, to signal submission (Batista et al., 2012). If nonapeptides are linked to the SPPn, then it is probable that the production of “offs” is under nonapeptide control. It will be interesting to determine if all these submissive behaviours are under nonapeptide control and, if so, through which mechanisms nonapeptides achieve these different outcomes.
The role of nonapeptides on descending motor pathways has been demonstrated across a number of vertebrate species (reviewed in Goodson and Bass, 2001). However, it 39
had remained unclear whether these effects were mediated through sensorimotor or
higher motivational systems (Goodson and Bass, 2001). Evidence for nonapeptide effects
on a distinct locus of the descending motor pathway comes from studies on weakly electric
fish (Perrone et al., 2010; Perrone et al., 2014). Administration of AVT to hindbrain slicesin vivo
containing the Pn evoked Pn-output rate increases that paralleled AVT experiments .
The studies presented in this review tell a story of nonapeptide influence on segregated components of the neural networks that control electrocommunication in weakly electric fish. These studies should serve as a basis for future research aiming to investigate nonapeptide modulation of communication and social behaviour. There are two main future research directions which promise to be particularly fruitful: the mapping of nonapeptide receptors in the brain of weakly electric fish, and the investigation of the role
A.IT leptorhynchusplays in electrocommunication. The central nervous transcriptome is now available for
(Salisbury et al., 2015). This should facilitate the development of the
proper molecular tools to map the distribution of nonapeptide receptors in the brain of this
fish. The effect of IT on communication in fish has only been demonstrated in a single
species, the plainfin midshipman (Goodson and Bass, 2000a,b).The similarities between the
vocal and electrocommunication behaviour of plainfin midshipman and weakly electric fish
(reviewed in Bass and Zakon, 2005), respectively, should encourage investigation into the
role of IT systems in electrocommunication behaviour.
40
Investigating the roles of isotocin in electrocommunication behaviour in a weakly
electric fish, Apteronotus leptorhynchus
Introduction
Nonapeptide systems and social behaviour
Neuropeptides of the arginine vasotocin (AVT) and isotocin (IT) family (and the mammalian homologues arginine vasopressin; AVP and oxytocin; OT, respectively), the so called nonapeptides, influence social behaviour across a broad range of vertebrate groups
(Donaldson and Young, 2009). There is a considerable body of evidence linking the
AVT/AVP system to descending motor pathways involved in social communication, particularly in relation to aggression and courtship (for review see Albers, 2012; Godwin and Thompson, 2012; Thompson and Walton, 2013). In contrast, the role of IT/OT systems has been more generally linked to higher-order sensory processing systems that have downstream effects on social behaviour related to affiliation and social grouping (Ross and
Young, 2009; Goodson and Thompson, 2010; O’Connell and Hofmann, 2011; Reddon et al.,
2012). However, Goodson et al., (2003) have proposed the hypothesis that the behavioural function of IT/OT may be evolutionarily derived from, and similar to, those of AVT/AVP.
Indeed, the effects of both AVT and IT on vocal communication behaviour have been demonstrated in a teleost, the plainfin midshipman fish (Goodson and Bass, 2000a,b).
41
Nonapeptides and descending motor pathways in teleosts
Among teleosts, AVT and IT neurons are localized almost exclusively to the preoptic
area and anterior hypothalamus (POA-AH) (ThompsonPorychthys an notatusd Walton, 2013). Stimulation of
the POA-AH in the plainfin midshipman fish, , evoked naturalistic vocalizations, suggesting a role of AVTP. notatus, and/or IT on the descending vocal motor pathway in this species. Subsequent studies in have provided evidence for the involvement of both AVT and IT in modulating vocal-motor pathways (Bass et al., 2000a,b). Bass and colleagues (2000a) showed that injection of AVT into the POA-AH inhibited fictive vocalizations in territorial males, associated with courtship signalling, but had no effect on sneaker males or females. Conversely, IT injection to the POA-AH inhibited fictive vocalizations in both sneaker males and females, not normally associated with reproductive behaviour, but had no effect on territorial male vocalizations. Furthermore, the distributions of AVT-immunoreactive (AVT-ir)P. notatus and IT-immunoreactive (IT-ir) fibers showed an extensive overlap in the brain of , particularly in vocal-motor regions
(Goodson et al., 2003). The distribution of IT in the brain, however, is wider than that of
AVT. Unlike AVT, IT-ir fibers are found in ascending sensorimotor pathways (Goodson et al., 2003). Although there are inter- and intra-specific differences in the relative densities of
AVT and IT fibers and receptor expression, the general pattern of AVT and IT distribution seems to be conserved across teleost species (Van den Dungen et al., 1982; Batten et al.,
1990; Goodson et al., 2003; Lema et al., 2015). Within teleosts, however, there are only three studies, to our knowledge, that have investigated the role of IT on descending motor pathways involved in social behaviour (Goodson and Bass, 2000a, Goodson et al., 2003;
Thompson and Walton, 2004). 42
Electrocommunication behaviour
Weakly electric fish generate an electric field around their body that they use for
electrolocation as well as for communication (for review see Kramer, 1990; Zupanc and
Bullock, 2005). Weakly electric fish in the order Gymnotiformes are classified into two groups based on the type of electric organ discharge (EOD) they produce. Species that
produce discontinuous voltage oscillations, which can have variable pulse rates, are termed
“pulse-type” fish. Wave-type fish, on the other hand, produce continuous and consistent
voltage oscillations with a quasisinusoidal wave-form. Species within both groups are able
to modulate their EOD during social interactions to communicate sexual or social status as
well as aggression (Hagedorn and Heiligenberg, 1985; Kramer, 1990; Zubizarreta et al.,
2012; Silva et al., 2013). The speciesApteronotus of interest leptorhynchus in this experiment is the wave-type, South
American brown ghost knife fish, . Brown ghost knife fish
produce sexually dimorphic EOD frequencies (EODf): male EODfs ranging from 800-1000
Hz and female EODfs ranging from 600-800 Hz (Bastian et al., 2001). The EOD as well as its
modulations, provide the basis for electrocommunication (Zakon et al., 2002). A. leptorhynchus
The two main forms of EOD modulations exhibited by are chirps
and the jamming avoidance response (JAR) (Zakon et al., 2002). The jamming avoidance
response consists of a gradual shift in EODf away from the frequency of a potentially
jamming signal (Figure 1), i.e., a frequency close to the EODf of the fish (Heiligenberg,
1973). The JAR behaviour is best evoked by stimulating the fish with a frequency close to,
and below, its own EODf (Bastian et al.,A. 2001). leptorhynchus More specifically, the difference frequency
(Df) that most reliably evokes a JAR in is -4Hz (Bastian et al., 2001). 43
Although the communicative function of the JAR is poorly understood, Kramer (1994) suggests that by increasing the difference in EOD frequency, a fish can more easily discriminate EOD frequency and waveform of an interacting fish.
Chirps are characterized as transient (20-200 ms long) increases in EOD frequency that can be accompanied by a decrease in amplitude (Bullock, 1969; Hopkins, 1974). Chirps are predominantly produced by males (Bastian et al., 2001) and can occur spontaneously
(Engler et al., 2000), during dyadic encounters (Hupé et al., 2008), and can be evoked with electric stimulation that mimics the presence of a conspecific fishA. (Dye, leptorhynchus 1987; Zupanc and
Maler, 1993; Bastian et al., 2001). Studies of chirps produced by during agonistic and courtship interaction (Hagedorn and Heiligenberg, 1985) and those elicited under electrical stimulation simulating conspecifics (Zupanc and Maler, 1993; Bastian et al.,
2001) have identified the two most commonly produced chirps as type-I and type-II chirps.
Both type-I and II chirps have a duration of around 20ms but type-I chirps have a much higher frequency excursion (between 300-500Hz) as compared to type-II (between 50-150
Hz) (Zupanc et al., 2006) (e.g. Figure 4). Type-I chirp EOD frequency increase is coupled with a decrease in amplitude (Figure 5A). This decrease in amplitude is not seen in type-II chirps (Figure 5B). The production of type-I and type-II chirps is context-dependent, as demonstrated by subjecting the fish to a “chirp assay” (Dye, 1987; Bastian et al., 2001;
Engler et al., 2001). The chirp assay consists of stimulating a fish with electric oscillations of varying frequencies above or below the fish’s EODf and recording the EOD and modulations produced by the fish. Stimulation with a Df close to a fish’s own EODf (±12
Hz), which simulates a same-sex conspecific, evokes type-II chirps from males (Bastian et al., 2001). Type-II chirps are also more commonly produced during male-male aggressive 44
interactions (Triefenbach and Zakon, 2008). Type-I chirps are more readily evoked by EOD frequencies 50 to 200Hz higher or lower than the fish’s EOD, which mimic female conspecifics (Bastian et al., 2001). Type-I chirps are thus believed to be involved in courtship (Dye, 1987; Triefenbach and Zakon, 2003; Bastian et al., 2001).
Nonapeptide control of electromotor circuits
The neural mechanism involved in producingA. the leptorhynchus EOD and its modulations is relatively simple and extensively characterized for (Kawasaki et al., 1988;
Johnston and Maler, 1992, Metzner, 1999). The EOD is produced by synchronous firing of electrogenic cells which are driven by command pulses of a hindbrain pacemaker nucleus
(Pn) (Dye and Meyer, 1986; Zupanc and Maler, 1997). The Pn fires in a one-to-one fashion with the EOD (Metzner, 1999). Two main prepacemaker nuclei provide input to and modulate the firing rate of the Pn, thereby directly controlling the EOD. Chirps can be evoked by stimulation of a diencephalic region, the prepacemaker nucleus-chirp region
(PPn-C) (Metzner, 1999). The two chirp types can be evoked by differential stimulation of the PPn-C (Engler et al., 2000). Stimulation of single PPn-C neurons resulted in type-II-like
EOD modulations while activation of larger populations of PPn-C cells evoked type-I-like
EOD modulations (Kawasaki et al., 1988). The JAR is controlled by a sublemniscal prepacemaker nucleus (SPPn). The SPPn, which is normally under tonic inhibition, provides glutamatergic input to the Pn (Heiligenberg et al., 1996). Upon release of inhibition, the SPPn stimulates the Pn, consequently increasing the EOD firing rate
(Heiligenberg et al., 1996). 45
Arginine vasotocin has been linked to modulations in baseline EODf, chirping, and
JAR behaviour, involving each of the three nuclei that make up the descending electromotor circuit (Pn, PPn-C, and SPPn) (Wong et al., 1997, Bastian et al., 2001;Perrone et al., 2010;
Perrone, 2012; Silva et al., 2013; Perrone etA. leptorhynchusal., 2014). AVT was shown to modulate the production of type-I and type-II chirps in (Bastian et al., 2001).
Intraperitoneal injections of AVT decreased the number of type-II chirps produced by males and increased the number of type-I chirps produced by a subset of males. No direct connection exists between the POA-AH and the PPn-C, however, Zupanc and Horschke
(1997b) have provided evidence for an indirect link between nonapeptide producing cells and the PPn-C via the preglomerular nucleus. The JAR was also affected by AVT injections
(Bastian et al., 2011).A. leptorhynchus Administration of AVT increased the magnitude of JAR frequency excursion in male , however no neuroanatomical link between the POA-AH and SPPn has been documented. Finally,Brachyhypopomus AVT was show gauderion to have direct effects on the Pn of a pulse-typeB. weakly gauderio electric fish, (Perrone et al. 2014). During night-time, increase their baselinein EODf vivo andwhich in vitroacts as social courtship signal
(Silva et al., 2007). AVT administration, both , increased the diurnal EODf, simulating the nocturnal increase in EODf of intact fish (Perrone et al., 2014). This group also provided evidence for AVT-ir fibers near the Pn, suggesting a direct effect of AVT on the Pn, controlling the nocturnal behaviour.
46
Goal
The focus of exploration of nonapeptide regulation of electrocommunication has
been on the role of AVT. To date, no study has explored the role of isotocin in
electrocommunication of weakly electric fish. Following the hypothesis of Goodson et al.
(2003) that the behavioural functions of IT are similar to those of AVT the goal of this study
was to investigate the role of IT in three AVT-dependentApteronotus behaviours: leptorhynchus. baseline EOD
frequency, the JAR, and type-I/II chirping behaviour in
Materials and Methods
Animal care and housing
Apteronotus leptorhynchus
Male weakly electric fish, , were used for this study.
Animals were obtained from a local tropical fish importer (Belowwater, Montreal, Quebec).
Fish were housed singly or in small groups (2-5 fish), for at least one month prior to experimentation, on a 12:12 hour light:dark cycle. Fish were monitored daily and water conditions were kept within the following ranges: temperature 26-28°C, pH 6.5-8, conductivity 100-400 µS. Each fish was provided with a section of PVC tube, which served as a shelter. Artificial plants were also used as extra cover in each tank. Fish were fed frozen blood worms or Tubifex three times per week. Males were distinguished by their response to a chirp assay. Males are more likely than females to produce chirps in response to mimics of the EOD of a conspecific (Dye, 1987) and only males were found to produce type I chirps (Bastian et al., 2001). Experimental protocols for this study were approved by the McGill University animal care committee. 47
Recording and stimulation
The electric organ discharge and its modulations were measured for each fish using a similar method as that used in previous studies (Bastian et al., 2001; Cuddy, 2010).
Individual fish were placed in a 50 x 32 x 25 cm tank inside a recording chamber which blocked out light to simulate night time. The experimental tank contained water from the fish’s home tank. A heating pad was placed under the tank to maintain a water temperature of 26°C. Fish were confined to a section of PVC tube with the ends and sides cut out and replaced with plastic mesh. This allowed the fish to swim freely but confined their range of motion to within a few centimeters forwards and backwards, and prevented the fish from turning around. Silver chloride recording electrodes were placed at each end of the PVC tube (head and tail orientation). The head-to-tail EOD was amplified with a differential AC amplifier (A-M Systems 1700; Carlsborg, WA) and analog-to-digital converted using an analog/digital interface (NI BNC–2090, National Instruments Inc., Austin, TX) to a desktop computer running Matlab (The Mathworks, Natick, MA) at a sampling frequency of 20 kHz.
The fish were stimulated with EOD mimics of conspecifics using silver chloride stimulating electrodes placed 8 cm apart. The stimulating electrodes were oriented perpendicular to the axis of the fish’s body and placed 10cm away from the fish. Stimuli were generated using a desktop computer running Matlab (sampling rate 20 kHz) and delivered through the same analog/digital interface and an analog stimulus isolator (A-M
Systems Model 2200). The stimulus was calibrated to produce an intensity of 1mV/cm at the skin surface of the fish. 48
Experimental design
Chirp assay
Brown ghost knifefish show a Df-dependent response in chirping behaviour
(Bastian et al., 2001). Type-I chirps are more readily evoked by large Dfs while type-II chirps are more readily evoked by EOD mimics closer to their own EODf. In the current study, the Dfs used were ±5Hz, ±20Hz, ±50Hz, and ±100Hz. To test for dose-dependent effects of IT on electrocommunication behaviour, we used a pretest-posttest design consisting of two phases for each fish: a pre-injection phase and a post injection phase
(Figure 6). Before each experimental session, a fish was placed in the experimental tank for a 15-minute acclimation period. Each experimental session consisted of 8 stimulus presentations that were ordered in a quasi-random fashion: -50Hz, +20Hz, -5Hz, +100Hz, -
20Hz, -100Hz, +50Hz, +5Hz. Each stimulus was presented for 60 s. The fish’s EOD was recorded for 120 s, centered on the stimulus presentation. The 30-s recording period prior to the start of stimulation permitted the fish’s baseline EODf to be obtained. After a 180-s pause, the next recording started, giving a total pause duration between successive stimuli of 240 s. Each experimental session lasted 37 minutes. Body mass and length were recorded following the pre-injection session. After the pre-injection phase, the fish was returned to its home tank for 48 hours before the start of the post-injection phase. This was done to minimize the effects of habituation (Harvey-Girard et al., 2010). Post-injection experimental sessions were preceded by intramuscular injection of saline or saline plus IT.
Subjects were assigned, at random, to one of 4 treatment groups prior to testing, 49
corresponding to different levels of IT concentrations in µg/g of body mass: (1) saline
(control group); (2) low dose (0.5µg/g); (3) medium dose (1µg/g); or (4) high dose
(2µg/g). IT doses were based on earlier IT injection experiments in other species
(Mennigen et al., 2008; Reddon et al., 2012). The experimenters in the current study were blind to the dose received by a given fish. Stock solutions of 0.9% saline, 0.05g/L IT in saline, 0.1g/L IT in saline, and 0.2g/L IT in saline were prepared and each fish received
10uL/g of body mass of the solution that corresponded with their treatment group. For example, a fish in the high dose treatment group (4) with mass = 7g, would receive 70µL of
0.2g/L IT in saline, resulting in a concentration of 2µg/g of body mass. After injection, fish were placed in the experimental tank for 15 minutes of acclimation and then presented with the same 8 stimuli as was done in the pre-injection phase.
Chirp and JAR analysis
A custom-written Matlab script was used to count the number of type I and type II chirps, as well to record the presence and frequency excursion of a JAR. Chirp identification was based on duration and frequency excursion from the baseline EODf. In some cases, particularly with type-I chirps, large amplitude modulation in the recordings prevented the automated counting of chirps and so these chirps were counted manually from a spectrogram of the recording. The large amplitude modulations were likely a result of misaligned recording electrodes picking up beat modulations from the combined stimulus and fish signal. The JAR was calculated using the median EODf during the stimulation period and comparing that to the EODf before and after stimulation. 50
Statistical analysis
Data were organized using Microsoft Excel. Statistical procedures were performed using SPSS (IBM, Somers, NY). Mean percent change in EODf was analyzed using a one-way
ANOVA. Change in mean JAR magnitude was analyzed using a factorial ANOVA. Change in type-I and type-II chirps over stimuli was analyzed using an extension of the Kruskal-Wallis test, the Sheirer-Ray-Hare test. Scheirer-Ray-Hare is a nonparametric equivalent of a factorial ANOVA with replication (Dytham, 2003).
Results
Effect of isotocin on baseline EODf
Apteronotus In total, leptorhynchus pre- and post-injection baseline EODf was measured from 44 male
. Baseline values were recorded prior to the first stimulation during experimental sessions. Percent change of EODf was calculated for each fish as the difference between pre- and post-injection baseline EODf divided by the pre-injection
EODf. The effect of varying levels of IT injection was examined across the 4 treatments. The number of subjects in each treatment group were as follows: saline (control), n = 10; low
(0.5µg/g), n = 11; medium (1µg/g); n = 12; high (2µg/g), n = 11. No significant effect of IT on baseline EODf was observed (Figure 7) (p = 0.669, one-way ANOVA). Across treatment groups, post-injection EODf was lower than pre-injection EODf, but as this pattern was the same in the control group, it was likely an effect of injection and not IT. 51
Effect of isotocin on JAR excursion
The JAR is characterized by a gradual EOD frequency shift away from a potentially jamming signal (Kramer and Kaunzinger, 1991). Figure 1 shows an example of the JAR in response to a -5Hz signal. A previous study showed that the JAR was most readily and maximally evoked by small negative Dfs (-4Hz, Bastian et al., 2001). To investigate the effect of IT on the JAR, we compared pre- and post-injection JAR frequency excursion in a total of 41 fish in response to a -5Hz signal. Substantial amplitude modulations during stimulation made the fine-grained analysis of EODf impossible in 3 of the total 44 fish tested and so JAR data could not be measured from these individuals. No significant effect of IT on JAR behaviour between treatment groups was found (Figure 8) (p = 0.636, between-subjects factorial ANOVA).
Chirp analysis
A total of 27,464 chirps were observed across all stimuli and include pre- and post- injection experiments from 40 fish. Recordings from 3 of the total 44 fish tested showed substantial amplitude modulations during stimulation, so that chirping could not be analyzed. A technical problem occurred midway through the chirp assay during the post- injection phase experiment of 1 fish, and so the data from this fish were not included in chirp analysis. Chirp frequency excursion ranged from 10 Hz to over 1500 Hz. The distribution of frequency excursion showed a trough at 160 Hz that was used to separate type-I and type-II chirps (Figure 4). Type-I chirps are also characterized by decreases in 52
amplitude, shown in Figure 5A, as compared to type-II chirps (Figure 5B). The
experimental males produced mostly type II chirps (25,157/27,464, 92%) across all
stimuli. Type-I and type- II chirps showed different patterns of Df tuning (Figure 3). The
chirp probability is expressed as the proportion of type-I or type-II chirps produced at each
Df out of the total number of chirps produced across all Dfs. The type-II chirp response
showed a clear peak at small Dfs, particularly at -5Hz (Figure 3B). In contrast, the
probability of type-I chirp production was highest at large Dfs (Df = -100 Hz and +100 Hz,
Figure 3A), consistent with published data (Bastian et al., 2001).
Effect of isotocin on type-I and type-II chirp production
To test the effect of IT on chirping behaviour we looked at chirp data in two ways.
First we compared the change in number of chirps produced at each Df across treatment
groups, summarized in Table 1. For each fish, we measured the pre-post injection
difference in number of type-I and type-II chirps across stimuli. Table 2 and 3 show pre-
and post-injection production of type-I and type-II chirps, respectively, for a typical male in
each treatment group. Figures 9 and 10 show the mean change in type-I and type-II chirp
count, respectively, in relation to Df stimulus for each treatment group. Positive values represent a post-injection increase in chirp production; negative values represent a post- injection decrease in chirp production. We could not detect a clear difference between groups in the post-injection change in number of type-I (p = 0.07, Scheirer-Ray-Hare) or type-II (p = 0.59, Scheirer-Ray-Hare) chirps. We did observe a consistent decrease in type-
II chirps across stimuli tested (Figure 10). This, however, can be explained as an effect of 53
injection and not as an effect of IT since the saline injected group showed a similar decrease in type-II chirps.
Injections of another nonapeptide, AVT, were previously found to decrease the number of type-II chirps and increase the number of type-I chirps (Bastian et al., 2001).
This was interpreted as an AVT-induced shift in proportion of chirp type produced.
Following this hypothesis, the second approach to analyzing chirp data was to compare the change in proportion of type-I chirps produced at each Df across treatment groups (Figure
11). The proportion of type-I chirps was calculated as the number of type-I chirps produced at each Df divided by the total number of chirps at that Df. Positive values represent a post-injection increase in proportion of type-I chirps and a decrease in proportion of type-II chirps; negative values represent a post-injection decrease in proportion of type-I chirps produced and an increase in proportion of type-II chirps. No significant difference was observed between treatment groups in the change in proportion of type-I chirps at any Df (p = 0.09, Scheirer-Ray-Hare).
Discussion
This study sought to investigate the role Apteronotusof IT on three leptorhynchus electrocommunication behaviours in males of the weakly electric fish, : the baseline
EODf, the JAR, and type-I and type-II chirping behaviour. Overall, no significant effect of IT was observed on any of the behaviours examined.
The doses of IT used in this study were based on IT injections in goldfish and cichlids (Mennigen et al., 2008; Reddon et al., 2012). To explore whether the chosen doses 54
A. leptorhynchus
were too low for A. leptorhynchus, we ran exploratory experiments with higher doses. We
injected two other male with 4µg/g of body mass and three with 8µg/g of
body mass IT in saline. The between- and within-fish variability in chirp production was
extremely high and no effects of the higher doses could be discerned on either chirping
behaviour, JAR, or baseline EODf.
IT was administered intramuscularly in this study, while previous teleost studies
have generally used intraperitoneal injections. However, Santangelo and Bass (2006) have
shown, in a tropical species of damselfish, that intramuscular injections of neuropeptides
have effects just as rapid as intraperitoneal injections. Moreover, intramuscular injection of
AVT facilitates aggression, in damselfish, by a similar magnitude as intraperitoneal injections of AVT in another teleost species (Lema and Nevitt, 2004a). Nevertheless, it is conceivable that peripheral injections of IT have wide-ranging effects on a number of neural processes that could interfere with electromotor systems. To clarify whether IT modulates electromotor responses when administered in a more targeted fashion, this substance should be applied directly to electromotor regions including the Pn, PPn-C, SPPn.
A number of studies, across vertebrate species, have described sex-specific
distribution patterns of nonapeptidergic fibers, nonapeptide mRNA expression (see De
Vries and Panzica, 2006; Goodson, 2008), and receptor expression (Lema et al., 2015).
Although no general picture of AVT and IT as having opposite effects on social behaviours
in males and females has emerged, Goodson and Bass (2000a) did show such opposite
effects in midshipman fish. In females of that species, injections of IT into the POA-AH
inhibit vocal-motor responses elicited by local electrical stimulation, whereas AVT has no 55
effect. The same modulatory pattern was observed for satellite males, which are female- typical in some dimensions of morphology and behaviour. The modulatory pattern was reversed for territorial males; AVT inhibited fictive vocalizations whereas IT had relatively little effect. It would be interesting to test theApteronotus effect of both leptorhynchus AVT and IT on electrocommunication behaviour of female to see if a similar pattern emerges.
IT receptor expression levels have been shown to vary across seasons in other teleosts; seasonal variation in nonapeptide receptor expression is related to sexual status and development in fishes (Gozdowska et al., 2006; Zhang et al., 2009). Bastian et al. (2001) found that only a subset of males tested showed a response to AVT injection. However, no distinguishing characteristics among responsive males were reported in that study. In the current study, neither the sexual nor the developmental status of the fish was taken into consideration when selecting test subjects. It is possible that males used in this study were at different developmental stages and/or exhibited different sexual status, thereby expressing different levels of the IT receptor (Lema, 2010). It is therefore conceivable that the effects of IT in some individuals were masked in our analysis by the lack of responses in other individuals. Interestingly, in regards to the effect of IT on mean change in number and proportion of type-I chirps, a statistical trend was observed (Figure 9, p= 0.07, and
Figure 10, p = 0.09, Scheirer-Ray-Hare, respectively). The trend suggests a dose-dependent decrease in proportion, as well as count, of type-I chirps produced, although no obvious trend is seen for IT-dependent type-II chirp count. This would support the notion of opposing effects of AVT and IT on electrocommunication behaviour. To test this hypothesis, 56
future work in this area should investigate the effects of IT on the social behaviour of electric fish at different levels of development and differing sexual status. 57
Summary and Final Conclusion
Weakly electric fish (Order Gymnotiformes) represent a group of animals with a wide range of context-, species-, and sex-specific behavioural variation (Black-Cleworth,
1970; Hopkins, 1988; Turner et al., 2007). The electromotor system, controlling electrocommunication behaviour is a relatively simple circuit composed of three main units: the pacemaker nucleus, and two prepacemaker nuclei that modulate the electric organ discharge via the pacemaker. Variation in electrocommunication has been extensively linked to variation in neuroactive substances impinging on the electromotor system (Kolodziejski et al., 2005; Dunlap et al., 2011; Smith, 2013; Silva et al., 2013).
The first aim of this thesis was to summarize the current literature linking nonapeptide function to the electromotor system and electrocommunication in weakly electric fish. The nonapeptide arginine vasotocin has been shown to play a part in modulating electrocommunication behaviour that involves all three basic units of the electromotor system (Bastian et al., 2001; Silva et al., 2013; Perrone et al., 2014). For example, AVT induced changes in chirp-type production, which is under the control of the
PPn-C and increased the magnitude of the JAR, under the control of the SPPn (Bastian et al.,
2001). AVT was also found to modulate dominant and submissive signalling, presumably through direct influence on the Pn (Perrone, 2012; Silva et al., 2013). No study, in the literature, has investigated the functional role of IT in modulating electrocommunication.
Indeed, across teleosts, relatively few studies have explored the effects of IT on social behaviour (reviewed in Thompson and Walton, 2013). Goodson et al. (2003) suggested the hypothesis that behavioural functions of the IT system should be evolutionarily derived, and thus similar to those of the AVT system. This hypothesis was substantiated by 58
similarities found in peptidergic modulation of vocal processes across vertebrate groups.
Additionally, Goodson et al., (2003), provided evidence for extensive overlap of AVT and IT
systems in descending motor pathways controlling communication behaviour. Based on
Goodson and colleagues’ (2003) hypothesis, and the studies linking AVT to the
electromotor system, the second part of this thesis aimed to exploreApteronotus the effect leptorhynchus of
intramuscular injection of IT on electrocommunication in male .
No clear effect of IT was observed on any of the three electrocommunication behaviours studied: baseline EODf, JAR, or chirping behaviour. The lack of an IT-induced response in this study could be due to lack of IT receptor expression in regions that ultimately control the behaviours that have been examined. Nonapeptide receptor levels seem to play a role in intraspecific variation in social behaviour (De Vries and Panzica,
2006; Goodson, 2008; Lema, 2010; HasunumaA. leptorhynchus et al., 2013; Lema et al., 2015). Given that the central nervous system connectivity of has been meticulously characterized (e.g., Maler et al., 1991; Johnston and Maler, 1992; Giassi et al., 2012; Harvey-
Girard et al., 2012), it is high time that nonapeptide receptor distribution be mapped in the brain of this fish. To date, four AVT receptors and one IT receptor have been identified in teleost fishes (Konno et al., 2010; Lema, 2010): two AVT V1a types, V1a1 and V1a2; two V2 type receptors, V2a and V2b; and a single IT receptor. The characterization of nonapeptide receptor types in teleostsA. leptorhynchus and the recent publication of a central nervous systemin situ transcriptome for (Salisbury et al., 2015) should facilitate hybridization and immunocytochemical approaches to mapping receptor distribution and expression levels in the brain of brown ghost knifefish. Knowledge of receptor distribution 59
and expression patterns will provide a promising window into the role of AVT and IT
systems in the regulation of electrocommunication.
Although the focus of this thesis was on the role of nonapeptides in descending
motor pathways, numerous studies have suggested nonapeptide involvement in sensory
processing (Maruska, 2009; Ross and Young, 2009; Reddon et al., 2012; Goodson et al.,
2003). In fact, POA-AH A.fibers leptorhynchus have been shown to project to a number of sensory and motor
regions in the brain of (Johnston and Maler, 1992). These include, but are not limited to: the nucleus electrosensorius, a nucleus upstream of the SPPn, and the preglomerular nucleus, which has been suggested to provide the indirect connection between the POA-AH and PPn-C, controlling chirping behaviour. The detailed knowledge of the neural basis of electrosensory processing (see e.g., Toscano Marquez et al., 2013) and electromotor output (see Zupanc, 2002), coupled with the interesting array of easily quantifiable behaviours described, make weakly electric fish a prime candidate for studying the role of nonapeptides in social behaviour.
60
Figures and tables
61
Figure 1
)
(Hz EODf
Time (seconds) Apteronotus leptorhynchus
Example of JAR produced by (EODf = 786Hz) at 26°C in response to a -5Hz Df stimulus (vertical line represents start of stimulation, lower horizontal dotted line represents the instantaneous frequency of the -5Hz Df stimulus). An instantaneous EOD plot shows a gradual increase in EODf shortly after the start of stimulus presentation (JAR magnitude = 5Hz). 62
Figure 2
4
4
4 * 5 1 2 3 5 2
Schematic diagram of the gymnotiform brain displaying connections between the POA-AH and the electromotor and electrosensory systems involved in electrocommunication behaviour. Thick lines indicate nonapeptidergic projections. Thin lines indicate connections within the electrosensory and electromotor systems. Dashed line around nucleus indicates PPn-C dendritic arborisation around the CP/PPn proper. Asterisks indicate putative connections. Connections are indicated by numbers (1-5). (1) POA-AH nonapeptidergic fibers terminate in the fields occupied by the PPn-C dendritic fibers (Kawasaki et al., 1998). (2) The POA-AH projects to the PG, a diencephalic relay to forebrain structures. The PG sends projections to the CP/PPn, thus serving as an indirect route for nonapeptidergic influence on the electromotor system (Zupanc and Horschke, 1997b). (3) The POA-AH projects directly to the Pn to influence baseline EODf (Perrone et al., 2014). (4) Putative projection of POA-AH fibers to the mesencephalic TSd based on evidence from other teleosts (Huffman et al., 2012; Thompson and Walton, 2013). The TSd, integrates sensory information from a number of regions and then projects to the nE, which interfaces the electrosensory and electromotor systems. (5) The CP/PPn integrates electrosensory and electromotor information and ultimately influences electrocommunication behaviour (Correa and Zupanc, 2002).
63
Figure 3
A 0.018
0.016
0.014
0.012
0.01
0.008
0.006
0.004 Type-I chirp probability chirp Type-I 0.002
0 -100 -50 -20 -5Hz +5 +20 +50 +100 B
0.3
0.25
0.2
0.15
0.1
Type-II probabilitychirp Type-II 0.05
0 -100 -50 -20 -5Hz +5 +20 +50 +100
Stimulus difference frequency (Hz) A. leptorhynchus Behavioural tuning curves for type-I and type-II chirps of in response to stimulus difference frequencies ranging from -100 to +100 Hz. (A) Probability of type-I chirps produced in response to Df (B) Probability of type-II chirps produced in response to Df. Values are total means ± S.E.M for 40 animals. Probabilities across stimuli add to 1 for each (A) and (B). Data collected by A. Mokdad (unpublished). 64
Figure 4
6000
5000
4000
3000
2000
1000
700
Number of chirps of Number 500
300
100 *
30
0
Frequency excursion (Hz) Histogram of frequency excursion measured from chirps. A total of 27 464 chirps were observed across all stimuli and include pre- and post-injection experiments. The histogram includes measurements from 21 619 of the 27 464 chirps. A portion of total chirps (5 845 of 27 464) were manually counted from spectrograms and frequency excursion could not be accurately measured for these chirps. The asterisk indicates the boundary used for classifying type-I (>160 Hz) and type-II (<160 Hz).
65
Figure 5 A
Amplitude Amplitude (mV)
EODf (Hz)
B
Amplitude Amplitude (mV)
EODf (Hz)
Time (seconds) Apteronotus leptorhynchus Examples of type I (A) and type II (B) chirps produced by a male (EODf = 830Hz at 26°C) during a +100Hz Df stimulus presentation. (A) The transient increase in EODf during type-I chirps is accompanied by a decrease in amplitude. The horizontal scale represents time in seconds (stimulus presentation starts at 30 seconds). (B) The transient increase in EODf during type-II chirps is of a smaller magnitude than type-I chirps and is not accompanied by a change in amplitude.
66
Figure 6
A
B
C
Timeline of experiments for each fish. (A) The pre-injection phase consisted of three stages. Fish were placed in the experimental tank for a 15-minute acclimation period (empty bar) prior to the experimental session. The experimental session (grey bar), elaborated in (C), lasted for a total of 37 minutes. Following the pre-injection experimental session, the body mass and total length of the fish was measured and recorded (dark grey bar). (B) The post- injection phase took place 48-hours after the pre-injection phase, which time fish spent in their home tanks. For the post-injection phase, fish were first intramuscularly injected with saline or saline plus IT followed by an acclimation period and experimental session similar to that of the pre-injection phase. (C) The experimental session consisted of 8 stimulus presentations in a quasi-random order: -50Hz, +20Hz, -5Hz, +100Hz, -20Hz, -100Hz, +50Hz, +5Hz (the first two and the last stimulation are shown). Each stimulus presentation (grey bar) lasted 60 s, pre- and followed by a 30 s period of control recording (empty bars). The 30 s delay before the start of stimulation allowed for baseline EODf to be recorded. Stimulus recordings were separated by a 180-s pause period (black bars). 67
Figure 7
Saline Low Medium High .0000 Dose of isotocin injection
-.0020
-.0040
-.0060
-.0080
-.0100
-.0120
-.0140
Mean percent change inchange EODf Meanpercent -.0160
-.0180
-.0200
Effect of isotocin on mean change in baseline EODf. Horizontal axis represents treatment group injected with IT in saline at different concentrations in µg/g of body mass: saline (control), n = 10; low (0.5µg/g), n = 11; medium (1µg/g); n = 12; high (2µg/g), n = 11. Vertical axis represents mean change in EODf post injections normalized to pre-injection EODf values. Mean EODf decreased in all groups and therefore was likely an effect of injection procedure and not IT. No significant effect of IT on change in EODf was found (p = 0.669, one-way ANOVA). Values are group means ±S.E.M.
68
Figure 8
8.0
7.0
6.0
5.0
4.0
3.0
2.0 Mean JAR magnitude (Hz) magnitude MeanJAR
1.0
0.0 Saline Low Medium High
Dose of isotocin injection
Effect of isotocin on mean JAR excursion. Open bars indicate pre-injection trials and filled bars indicate post-injection trials with a -5Hz Df stimulus. Horizontal axis represents treatment group saline (control), n = 9; low, n = 11; medium; n = 11; high, n = 10. Vertical axis represents the JAR excursion magnitude in Hz. No significant effect of IT on JAR behaviour was found (p = 0.636, factorial ANOVA).
69
Figure 9
10
8
6
4
2
-2
-4
Mean change in type I chirp count I in typechirp changeMean -6
-8
-10 -100 -50 -20 -5 +5 +20 +50 +100
Stimulus Df (Hz)
Mean change in type I chirp count in relation to stimulus Dfs ranging from -100Hz to +100Hz. Each line represents a different treatment group [circles = saline (control), n = 9; triangles = low dose, n = 11; diamonds = medium dose, n = 10; squares = high dose, n = 10]. IT injection has no significant effect on the change in mean number of type I chirps produced (p = 0.07, Scheirer-Ray-Hare). Values are group means ± S.E.M.
70
Figure 10
20
10
0
-10
-20
-30 Mean change in type II chirp countchirpII in type changeMean
-40
-50 -100 -50 -20 -5 +5 +20 +50 +100
Stimulus Df (Hz)
Mean change in type II chirp count in relation to stimulus Dfs ranging from -100Hz to +100Hz. Each line represents a different treatment group [circles = saline (control), n = 9; triangles = low dose, n = 11; diamonds = medium dose, n = 10; squares = high dose, n = 10]. Change in the mean number of type II chirps was not affected by IT injection (p = 0.59, Scheirer-Ray-Hare). Values are group means ± S.E.M.
71
Figure 11
.20
.15
.10
.05
.00
-.05
-.10
-.15 Change in proportion of type I chirps type of in proportion Change
-.20
-.25 -100 -50 -20 -5 +5 +20 +50 +100
Stimulus Df (Hz) Change in mean proportion of type-I chirps produced across stimuli. Each line represents a different treatment group [circles = saline (control), n = 9; triangles = low dose, n = 11; diamonds = medium dose, n = 10; squares = high dose, n = 10]. Change in the mean proportion of type-I chirps was not affected by IT injection (p = 0.09, Scheirer-Ray-Hare). Values are group means ± S.E.M.
72
Table 1
Stimulus difference frequency -100 Hz -50 Hz -20 Hz -5 Hz +5 Hz +20 Hz +50 Hz +100 Hz chirp type chirp type chirp type chirp type chirp type chirp type chirp type chirp type Dose I II I II I II I II I II I II I II I II Saline 0.11 -7.00 -1.67 -6.78 1.44 -13.67 --- -8.67 -0.44 -8.78 1.22 -8.22 2.44 -11.44 0.33 -9.00 ±1.07 ±4.05 ±1.01 ±2.20 ±1.00 ±3.84 ±6.23 ±0.73 ±6.02 ±1.42 ±3.19 ±1.43 ±4.81 ±2.32 ±4.83 Low -3.54 -3.27 -1.81 -1.45 -3.23 -0.27 -0.09 5.45 0.18 -4.18 -2.55 0.09 -3.18 -5.55 -3.55 -3.63 ±3.94 ±2.72 ±3.61 ±4.08 ±5.30 ±6.88 ±0.58 ±5.84 ±0.48 ±7.96 ±2,52 ±6.02 ±3.54 ±6.20 ±3.69 ±4.95 Medium 3.90 1.20 -1.30 -4.20 2.30 -3.20 3.30 -10.60 1.40 0.90 1.50 -8.20 2.90 -2.90 3.40 -2.00 ±3.10 ±3.78 ±1.78 ±6.08 ±2.19 ±5.87 ±2.93 ±9.31 ±1.63 ±10.23 ±2.55 ±8.54 ±1.85 ±2.58 ±4.46 ±3.76 High 2.30 -5.5 1.50 -4.1 5.20 -12.20 1.00 -12.70 0.20 -26.40 3.10 -11.60 3.80 -6.70 2.30 -5.10 ±2.21 ±5.62 ±1.19 ±5.31 ±2.86 ±6.97 ±0.79 ±7.88 ±0.20 ±16.32 ±2.12 ±7.37 ±2.85 ±4.48 ±2.14 ±5.51
Group mean change in type-I and type-II chirp count at each Df. Dose represents the treatment groups. No type-I chirps were produced by any fish in the 0 µg/g group at Df = -5Hz, in either pre- or post-injection phase experiments and therefore no values are presented. Values represent group means ±S.E.M 73
Table 2
Stimulus difference frequency Total -100 Hz -50 Hz -20 Hz -5 Hz +5 Hz +20 Hz +50 Hz +100 Hz Dose Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post
Saline 14 30 2 5 0 2 2 5 0 0 1 3 0 2 5 9 4 4
Low 15 17 4 1 0 4 2 6 0 1 0 1 0 1 5 2 4 5
Mediu 17 9 8 1 3 1 1 1 0 0 1 0 1 0 2 0 3 6 m
High 58 42 22 13 1 0 0 4 1 1 0 0 5 5 11 7 18 12
Type-I chirp counts at each stimulus Df produced by a typical male for each IT dose treatment with pre- and post-injection chirp counts. Total is the summed chirp count across Dfs. 74
Table 3
Stimulus difference frequency
Total -100 Hz -50 Hz -20 Hz -5 Hz +5 Hz +20 Hz +50 Hz +100 Hz Dose Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post
Saline 405 309 34 23 35 35 47 33 85 71 76 46 62 55 40 32 26 14
Low 243 305 1 13 33 35 34 43 63 74 48 31 54 62 7 21 3 26 Mediu m 590 586 41 62 66 58 66 85 100 108 97 80 86 89 73 66 61 38
High 388 407 27 29 31 40 58 56 59 81 135 93 19 54 39 34 20 20
Type-II chirp counts at each stimulus Df produced by a typical male for each of the isotocin treatments with pre- and post- injection chirp counts. Total is the summed chirp count across Dfs. 75
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