In more basal vertebrates (paraphyletic taxon Anamnia), composed by agnathans, fish and amphibians, magnocellular neurosecretory neurons express homologs of OT (mesotocin, isotocin, glumitocin, valitocin, aspargtocin) and VP (vasotocin) (Acher, 1978; Donaldson and Young, 2008). These neurons reside in the ancestral preoptic nucleus (PON; Diepen, 1962; Figure 1), which became recently a subject of genetic studies, using transgenic fish models (Gutnick et al., 2011; Herget et al., 2013). Magnocellular neurons of adult Anamnia are quite randomly distributed within the PON, existing intermingled with other types of cells. However, there is a ventro-dorsal gradient in size and morphology of neurons—while ventrally located neurons are rather small, more dorsally residing ones are bigger, and neurons reaching the upper pole of the PON are gigantic (Polenov, 1974; Garlov, 2005). This gradient reflects a “physiological regeneration” of the nucleus, which is caused by short periods of increased secretory activity (migration in fish and seasonal changes in frogs) and subsequent death of the gigantic neurosecretory neurons (Polenov, 1974; Garlov, 2005). This cell loss is, hence, compensated by newly born neurons (Chetverukhin and Polenov, 1993; Polenov and Chetverukhin, 1993). Although in non-mammalian species of vertebrates pronounced adult neurogenenesis is reported for various brain regions (see Kaslin et al., 2008 and Refs therein), in mammals this process is rather unique. Here it occurs only in specific areas, such as the subventricular zone and the dentate gyrus of the hippocampus (Ming and Song, 2011) as well as in the peptidergic hypothalamic arcuate nucleus, where cell turnover occurs at a low rate (Kokoeva et al., 2005).

In advanced vertebrates (monophyletic taxon Amniota: reptiles, birds, and mammals), there is a clear partition of magnocellular neurons in two separate nuclei—the paraventricular (PVN) and supraoptic (SON) nuclei (Meyer, 1935; Diepen, 1962; Figure 1). Some authors further subdivide the SON into main- and retrochiasmatic or postoptic part. However, the latter is absent in most evolutionarily conserved reptiles such as turtles (Fernández-Llebrez et al., 1988), and the retrochiasmatic part exists only in ancient mammals, such as platypus, that lack the typical SON. The PVN—in contrast to Anamnia's PON—is in rats composed by up to eight parts, and three of them comprise predominantly the magnocellular neurons (Swanson and Sawchenko, 1983; Armstrong, 2004; Simmons and Swanson, 2008). Although such strict territorial segregation is typical for rodents (especially for rats), but there are no reports on such segregations in other mammalian species, including humans (Swaab et al., 1993). Besides the main nuclei, PVN and SON, Amniota also possess groups of magnocellular neurons, termed accessory nuclei (AN)², located in the territory between SON and PVN. There is some inconsistency in the naming of groups and their recognition as independent groups or parts of the PVN or SON. Some authors, for example, consider the “anterior commissural nucleus” (ACN) as an independent AN (Rhodes et al., 1981; Grinevich and Akmayev, 1997) while others classify it as division of the PVN (Swanson and Kuypers, 1980). Importantly, an AN of similar localization and composition (such as circular, fornical, and dorsolateral) exists in reptiles and various mammals (see Grinevich and Polenov, 1994). However, in birds—a highly specialized group of Amniota—the main and AN are not clearly bordered, and the subdivisions of PVN and SON as well as the AN are not homologous to those in other representatives of Amniota (Oksche and Farner, 1974; Grinevich and Polenov, 1994). Importantly, studies in rats (Rhodes et al., 1981) revealed that about 1/3 of all magnocellular neurons locate in AN, thereby pointing to their functional significance. In that line, we showed recently that the dorsolateral AN is the main source of OT projections to the central amygdala and is certainly involved in the attenuation of fear responses via OT release within this target structure (Knobloch et al., 2012).

The cause of the formation of a polycentric OT system in evolution is unclear. It could be speculated that the presence of the AN intermediate to the PVN and SON reflects the process of separation of the ancestral PON into the PVN and SON, leaving remnant cell groups in between. During this separation the dorsal part of the PON—the magnocellular preoptic nucleus—likely remained as PVN in amniotes as was recently shown in larval and adult zebra fish by comparing gene expression profiles with mammals (Herget et al., 2013). As for the SON, it was speculated that neurons located in the ventral PON migrate in ventro-lateral direction to the place of the later SON (Herget et al., 2013), leaving remaining cells of further AN. It is interesting that in one of the most primitive modern mammals—monotreme platypus Ornithorhynchus anatinus, most of the magnocellular OT neurons reside in the stream between the PVN and the retrochiasmastic part of the SON and never form the main nuclei found in other mammals (Ashwell et al., 2006).

The process of PON divergence in reptiles (paralleled by the first appearance of AN) coincides with the process of forebrain development (encephalization) and the respective formation of large fiber tracts connecting brainstem and spinal cord to the forebrain. The migrating magnocellular neurons and growing axonal bundles, such as the medial forebrain bundle could have been interfering with each other, as proposed in the following. During the embryogenesis of Amniota, magnocellular neurons possibly migrate along radial glia from the 3rd ventricle into ventro-lateral direction; the association of radial glia and magnocellular neurons was reported in the wallaby, the representative of marsupial mammals (Cheng et al., 2002). Similar migrations are known for the radial development of spinal cord, cerebellum and cortex (Hatten, 1999; Nadarajah and Parnavelas, 2002; McDermott et al., 2005) and are also observable in cell culture studies where neuroblasts migrate back and forth until finding their destination (Hatten, 1990). The bidirectional movement of magnocellular neurons might have been physically blocked by the growing fibers of the solid medial forebrain bundle (phylogenetically evolving in amphibians and reptiles; Herrick, 1910; Nieuwenhuys et al., 1982), thereby hindering neuronal migration from the supraoptic region back to the 3rd ventricle and entrapping cells (i.e., SON) latero-dorsally to the optic tract. This process of magnocellular nuclei formation in the embryogenesis (resembling phylogenetic development in accordance to Ernst Haeckel's law), in any case, requires further scientific investigations employing genetic and viral approaches combined with time-lapse imaging.

As emphasized above, the evolutionarily oldest preserved OT processes contact the ventricle system (Figures 2, 4C). But given their rather low rate in mammals, the high OT concentrations in the CSF—exceeding those in blood (Kagerbauer et al., 2013)—likely arises from another source. Due to the fact that the CFS is composed of 1/3 extracellular fluid and 2/3 of blood plasma, the extracellular fluid, enriched by OT released from somas and dendrites of OT neurons (Ludwig and Leng, 2006) is most probably the main source of OT in the CSF (Landgraf and Neumann, 2004).

From an evolutionary point of view it is remarkable that OT homologs are present in primitive invertebrates species (such as annelids, nematods, mollusks, insects; van Kesteren et al., 1995; Satake et al., 1999; Stafflinger et al., 2008; Garrison et al., 2012), although no pituitary or other typical neuropeptide pathway through the body is available. Hence, the functional significance of evolving diverse distribution modes is not clear. However, it has been postulated that neuropeptides may initially have served as primitive neurotransmitters or modulators of neurotransmission (Jackson, 1980)—a functional implication that is still an aspect in mammalian species. Importantly, about 80% of the brain regions surrounding the ventricle system and the subarachnoid space express OT receptors in mammals. Therefore, diffusion of OT within the fluid of extracellular space (at least to a certain spatial extent) could be underlying behavioral effects of this neuropeptide in mammals, as found in countless studies with intracerebroventricular administration of OT during the last 30 years (Veening et al., 2010). It is assumed that intranasally applied in pharmacological doses (which are ~1000 times higher than the OT blood concentration; Huang et al., 2013; Neumann et al., 2013) OT may reach the CSF and exert substantially delayed and long lasting effects (starting from 30 to 45 min after application and lasting ~60–90 min) as was shown for various neuropeptides by Born et al. (2002). However, due to the short half-life of about 20 min of brain OT (Mens et al., 1983) it is unlikely that somatodendritically released OT reaches distant extrahypothalamic regions within a narrow time frame to achieve defined and rapid behavioral responses.

Simple uni- and bipolar cells forming ventricular contacts have been replaced during evolution by cells with extended dendritic trees (see Figure 2). This shift might have facilitated and intensified somatodendritic release of OT (Pow and Morris, 1989; Ludwig and Leng, 2006), which allows auto- and paracrine action of OT within OT-ergic nuclei under specific demand such as lactation (Landgraf and Neumann, 2004). Dendritically released OT is stimulating coordinated OT neuron activity during lactation, resulting in a pulsatile bolus release of OT into the blood (Lincoln et al., 1973). In parallel, OT release might be induced from axons in extrahypothalamic regions. This assumption was confirmed experimentally with 30 Hz optical stimulation, resembling the bursting activity of OT neurons during suckling (Wakerley and Lincoln, 1973; Poulain and Wakerley, 1982) and inducing axonal OT release in various brain regions (Knobloch et al., 2012, 2014)⁵.

There is a general agreement that parvocellular OT neurons project extensively toward the brainstem and spinal cord to form synaptic contacts with local neurons (Swanson and Sawchenko, 1983). However, these neurons are distinct from magnocellular ones in that they are not releasing OT into the systemic blood circulation. Although the presence of parvocellular OT-like neurons within the PON of Anamnia, e.g., teleost fish, was sporadically reported (Goodson et al., 2003; Thompson and Walton, 2013) the evolutionary transformation of this cell lineage has not been studied. Therefore, we leave this subject for further analysis, which will require the identification of genetic markers to specifically target parvocellular OT neurons.

During the pioneer times of neuroendocrine pathway research, ascending OT-ergic fibers were found in a limited number of extrahypohalamic forebrain regions such as the amygdala, bed nucleus of stria terminalis (BNST) and septal nuclei of rats (Buijs, 1978; Sofroniew, 1980), non-human primates (Atunes and Zimmerman, 1978; Kawata and Sano, 1982; Caffé et al., 1989; Wang et al., 1997) and human (Fliers et al., 1986) in addition to prominent descending brain stem- and spinal cord-innervating fiber tract. However, these studies suffered from technical limitations (such as deficient immunohistochemical feasibility) so that ascending fibers could only be revealed to a minor extent (Buijs, 1978; Sofroniew, 1980; Fliers et al., 1986). However, recent reports from Larry J. Young's (Ross et al., 2009) and our group (Knobloch et al., 2012), employing fluorogold- and viral-vector based techniques, respectively, clearly demonstrated that magnocellular OT neurons extensively innervate major forebrain regions in voles and rats. Interestingly, the number of OT axons in most forebrain regions is rather limited (Knobloch et al., 2012), hence explaining that they had been overlooked. The enormous number of OT molecules per large dense core vesicle (~85,000; Leng and Ludwig, 2008) and the extremely high (nM range) OT receptor affinity (Akerlund et al., 1999) still allows OT to sufficiently exert its effects in various forebrain regions. In line with this assumption, we demonstrated the functionality of sparse OT fibers in vitro and in vivo, as we showed that OT is released focally within the structure of demand, e.g., the lateral division of the central amygdala, and, hence, is capable to modify both microcircuit activity and amygdala-dependent behavior, namely conditioned fear response (Knobloch et al., 2012).

Interestingly, the focal, axonal OT release is, in spite of its spatial precision, not defined to a direct (synaptic) cell communication—a finding which is consonant with the initial idea of the Scharrers, who believed that the neurosecretory colloid can be released along the axon into the peri-axonal space (Scharrer, 1936; cited from Watts, 2011). Our hypothesis that OT acts non-synaptically is based on the fact that the onset of both electrophysiological and behavioral responses occur delayed, thereby exceeding the time typically needed for synaptic transmission (1–10 ms) and ranging within seconds in the central amygdala (Knobloch et al., 2012, 2014) and other brain regions, for example, in the anterior olfactory nucleus (personal communication from Dr. Wolfgang Kelsch, Central Institute of Mental Health and Heidelberg University). Importantly, a similar second-range delay of cellular responses was recently demonstrated after evoked somatodendritic release of VP from magnocellular PVN neurons, pointing on a similar non-synaptic, diffusion-like neuropeptide pathway that allows for interpopulational crosstalk within about 100 μm distance (Son et al., 2013). Besides the kinetics, the spatial distribution of large dense core vesicles, containing OT, also point on a non-synaptic transmission. The vesicles are not located in the active zones of pre-synapses in the few OT synapses found in the SON (Theodosis, 1985; Knobloch et al., in preparation) and ventromedial hypothalamic nucleus (Griffin et al., 2010) and OT receptors could not be attributed to the postsynaptic membrane. Taking all these arguments in account we propose that OT release from axons of magnocellular neurons in forebrain regions occurs by non-synaptic fashion. However, this should be further confirmed by the time-lapse imaging, implementing recently developed techniques for monitoring, docking and release of large dense core vesicles (de Wit et al., 2009; van de Bospoort et al., 2012). These techniques will also allow to dissect the role of glutamate-containing synaptic vesicles in OT neurons (Hrabovszky et al., 2006; Kawasaki et al., 2006), which remain enigmatic as no one was able to show fast synaptic transmission from axons of magnocellular OT neurons either in the hypothalamus or extrahypothalamic places (Knobloch et al., 2012, 2014).

Axonal projections to diverse brain areas are likely provided by distinct subgroups of OT neurons, implying an anatomical heterogeneity of OT neurons (Knobloch and Grinevich, personal observation). It is remarkable that there have been few if any studies on collaterals of OT neurons to different areas. Despite this, our ongoing research (manuscript in preparation) allows us to assume that in certain situations of life, such as love or fear, distinct populations of OT neurons may be activated, which—via specialized axonal projections—modulate specific brain areas and ultimately distinct behaviors in a pro-social or in-group supporting way. Indeed, recently we could show that associative fear learning induces the activation of a small subset of OT neurons, which specifically project to the central nucleus of the amygdala and, furthermore, evoked OT release from their axons within the central nucleus of amygdala readily attenuates fear response (Hasan et al., 2013; Kernert et al., 2013).

With respect to the evolution, there is a unique observation in a representative of the highly specialized and diverse group of teleost fish: in trout several mesotocin (and vasotocin) neurons project toward the forebrain (Saito et al., 2004). In analogy to rats (Knobloch et al., 2012), the authors furthermore demonstrated, using in vitro electrophysiology combined with biocytin-filling of cells, that magnocellular neurons of trout project to the posterior pituitary and—at the same time—to telencephalon and thalamus (Saito et al., 2004). This unique feature can be seen as an evolutionarily early advancement that later re-appeared in amniots. Indeed, ascending mesotocin or OT projections have been clearly demonstrated only in reptiles (Thepen et al., 1987; Silveira et al., 2002) and different mammals (Sofroniew, 1980; Fliers et al., 1986; Ross et al., 2009; Knobloch et al., 2012).

Since the turn of the last century the extract of the posterior pituitary has been known to stimulate contractions of the uterus and mammary glands (Oliver and Schäfer, 1895; Dale, 1909; Ott and Scott, 1910; Schäfer and Mackenzie, 1911). Subsequent comparative studies between numerous species conducted in the first half of the 20th century revealed that in both mammalian and non-mammalian species OT/mesotocin stimulates the activity of smooth muscle in reproductive tracts (Figure 5), furthermore the egg laying, sperm movement, ejaculation, as well as uterus contraction and milk let down in placental and non-placental mammals (Moore, 1992; Sebastian et al., 1998). Importantly, in non-placental marsupials OT and its homolog mesotocin co-exist in the hypothalamus. Together, they stimulate long lasting milk ejection (Nicholas, 1988), thereby prevailing different phases of the milk secretion to regulate lactation from neighboring breasts asynchronously, which is necessary for the contemporaneous development of offspring of different age (Nicholas, 1988; Sebastian et al., 1998).

Beside these neuroendocrine effects, countless publications convincingly demonstrate that in mammals OT is a key peptide for orchestrating reproductive, pro-social and in-group supporting behavior (Bosch and Neumann, 2012; Lukas and Neumann, 2013 and references therein). Based on that, OT is considered as a positive factor for species propagation (Lee et al., 2009) in all vertebrates. We here give a brief overview on aspects of OT involvement without providing a comprehensive analysis but rather a correlative view on the central OT pathways and the corresponding non-apeptide-mediated behaviors in vertebrates.

In a specialized marine teleost fish, the plainfin midshipman fish, Goodson and colleagues showed that central isotocin and vasotocin modulate social vocalization, in a sex- and type-specific manner (Goodson and Bass, 2000). Isotocin applied to the preoptic area of the anterior hypothalamus (the primary regions for endocrine and behavioral integration, e.g., in vocal production) modulates reproduction-unrelated social vocalization in females and type I males, both of which typically do not display parental care. In contrast, vasotocin applied to type II males, which are parental, modulates social vocalization according to the reproductive context—a courtship situation or the defense of the nest, eggs and hatchlings. Furthermore, isotocinergic axons were found in the ventral telencephalon and numerous hypothalamic and brainstem regions, which are components of ascending auditory pathways (Goodson et al., 2003). Unfortunately, there are no studies on the contribution of OT homologs to reproductive behaviors in agnathans, such as hagfish and lampreys, in cartilaginous fish (e.g., sharks and rays) or in primitive actinopterygians (e.g., sturgeon, beluga etc.). This gap makes it impossible to draw any definite conclusion about behavioral role of isotocin and its homologs in the first steps of vertebrate evolution.

In amphibians, especially in the evolutionarily advanced Anura, receptors for mesotocin are spread over brain regions implicated in reproductive behavior (Do-Rego et al., 2006). In addition, mesotocin is thought to stimulate the synthesis of neurosteroids, which target brain circuits controlling male calling and, again, reproductive behaviors (Do-Rego et al., 2006). Since in fish (except teleost) and amphibians isotocin/mesotocin projections reaching extrahypothalamic or reproduction-related brain regions could not be demonstrated, it is likely that these nonapeptides act trans-ventricularly, especially since courtship and reproductive behaviors do not require immediate effects and may last several days or weeks, depending on the species.

In reptiles, reports on OT effects are limited to nesting behavior (Carr et al., 2008). As in other nesting animals, typical nesting behavior in turtles consists of a sequence of actions such as nest-site selection, nest-site preparation, egg-cavity construction, oviposition and nest covering (Carr et al., 2008 and refs therein). Surprisingly, systemic application of OT (intramuscular injection) led to an atypical behavior with decoupled oviposition and nesting behavior, a phenomenon termed “false nesting” (Tucker et al., 1995). In turtles OT application evokes nest-covering behavior that precedes oviposition for up to 417 h (Carr et al., 2008). This study demonstrates that OT is powerful enough to induce nesting behavior even without egg laying. Involved central OT targets have yet not been dissected yet, and our literature search revealed only limited report on OT effects in reptilian reproductive behavior. However, further inside to this uniquely located group of animals—situated between basal vertebrates and mammals—would indisputably be beneficial for our understanding of the evolutionary role of OT homologs on the formation of behaviors as reptiles being the first group that carry a polycentric OT system with advanced multipolar neurons projecting extrahypothalamically. Presumably due to these achievements, reptiles display an extreme divergency of sexual behaviors, ranging from monogamous to “harem” behaviors (Bull, 2000; Godwin and Crews, 2002).

In birds, as shown in zebra finches, mesotocin seems to be a key peptide for the prolongation of time spent in large groups and—most importantly—with familiar conspecifics (Goodson et al., 2009). Furthermore, pro-social behavior elicited by central mesotocin infusion was dependent on the mesotocin receptor density in the lateral septum of female birds (Goodson et al., 2009). In fact, the reported effects of mesotocin resemble effects of OT on pair bonding observed in voles (Carter et al., 1995; Insel and Young, 2000). As in mammals with their specific OT fiber pattern, it is likely that also mesotocin-expressing species possess long-range axons to respective brain regions, such as to the lateral septum in birds, and regulate behavior with spatial precision.

In non-mammalian vertebrates vasotocin and its homologs modulate reproductive behavior and, in fact, seem to hold an even more important role than OT-like neuropeptides. Vasotocin is involved in the induction of vocalization, courtship behavior (like male amplectic clasping behavior), female sexual receptivity, alternative mating and many more social behaviors (Moore, 1983; Wilczynski et al., 2005; Balment et al., 2006; Soares et al., 2012). Such diverse effects in non-mammalians are not surprising since many extrahypothalamic vasotocin-expressing regions and the arising wide-spread projections are comparable to the extrahypothalamic VP system of mammals (de Vries and Miller, 1998). Summing up the impact of both peptides—OT/OT homologs and VP/VP homologs—in different species, it seems that the latter holds a dominant role in regulating reproductive behavior in fish and amphibians, while OT-like peptides are more important in birds (Goodson et al., 2012) and mammals (Lee et al., 2009), which display more complicated reproductive rituals. Nevertheless, the picture seems to be very complex as in many behavioral and cognitive aspects both peptides modulatory interact (Neumann, 2009; Bosch and Neumann, 2012; Stoop, 2012) and furthermore, as constituting a sexual dimorphic systems, vary in their relative priority in males or females (Veenema et al., 2013).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.