**1. Introduction**

In order to survive, all animals must detect, interpret and respond to an array of sensory information in their immediate environment [1]. Animals need to locate and assess the quality of food, detect and avoid predators, and identify mates and competitors [1]. Animals can gain information about these resources or threats, as well as convey information to other individuals, via numerous modalities, including vision and olfaction. For many mammals, particularly rodents, olfaction is most likely the dominant sensory modality [2,3]. Social behaviors, including parent/offspring interactions, are also strongly regulated by olfactory cues (e.g., prairie voles (*Microtus ochrogaster*) [4] and Syrian hamsters (*Mesocricetus auratus*) [5]).

While mammalian maternal care is essential for offspring survival, mammalian paternal care is rare (5%–10% of species [6]). This is most likely because the costs associated with paternal care (e.g., predation risk [7], increased energetic expenditure [8], loss of mating opportunities [9], and reduced survival [10]), and the inability of males to physically associate with offspring during prenatal development [11], are major limiting factors in the evolution of paternal care [12]. However, paternal males can significantly influence offspring growth, survival, and cognitive and behavioral

development [6]. Consequently, it is necessary to understand what factors play a role in the expression of paternal care when it does occur.

The ability to detect, recognize, and discriminate olfactory cues of social significance between fathers and o ffspring is dependent on complex neural mechanisms that may be regulated by specific olfactory genes. Here, I consider the role of olfaction in the expression of paternal care behavior in rodents. I first describe the behavioral machinery [13] of paternal care in general, and how this is connected with brain regions associated with the detection and interpretation of olfactory cues. I then discuss the genetic regulation of olfaction in the di fferent olfactory systems in general. Finally, I discuss how olfactory cues might regulate the expression of paternal care behavior, highlighting the roles that 10 di fferent olfactory genes may play in the expression of paternal care behavior. This list is not exhaustive, and there are likely multiple other genes that could be equally important. While indirect paternal care (e.g., alarm calling, [14]) is an important component of the paternal repertoire, I only focus on direct paternal care behavior (retrieval, huddling, nest building and grooming) because indirect paternal care does not require direct pup contact, whereas direct paternal care behavior does. I rely extensively on studies from laboratory mice, which are not naturally paternal, because the predominant literature on olfactory regulation of paternal care behavior comes from these studies, and the literature is decidedly depauperate on how the genetic mechanisms of olfaction moderate paternal care in biparental species. Nevertheless, these studies provide a starting point for those interested in mechanisms underlying the expression of paternal care in biparental species.

#### **2. Neural Regulation of Paternal Care Behavior**

#### *2.1. Brain Regions Implicated in the Regulation and Expression of Paternal Care*

The most important brain region associated with paternal care behavior is the hypothalamic medial preoptic area (MPOA [15,16]). The MPOA is anatomically connected to the bed nucleus of the stria terminalis (BNST) and the amygdala [17,18], as well as the lateral preoptic area (LPOA [19]), and the adjoining substantia innominata (SI [20]). Lateral e fferent neurons project from the MPOA to the LPOA and SI, and pass through the lateral hypothalamus (LH) to the ventral tegmental area (VTA [19]). Neuronal disruption to the central MPOA and the lateral e fferent neurons [21,22] can disrupt paternal care behavior, specifically retrieval behavior. Di fferent subregions or neuron populations in the MPOA may a ffect an individual's responsiveness to pup-specific odor cues [23].

The amygdala may also play an important role in the regulation of paternal care because of its distinctive neuronal heterogeneity, specifically, the caudal olfactory cortex [24]. The olfactory tubercle receives direct information from the main olfactory bulb (OB), whereas the medial amygdalar nucleus (meA), which lies adjacent to the olfactory tubercle, receives direct information from the accessory olfactory bulb (AOB [25]).

#### *2.2. Olfactory Systems and Associations with Brain Regions Implicated in Paternal Care*

Mammalian olfactory systems are complex, remarkably precise (one odorant receptor gene expressed per cell; [26]) and allow mammals to recognize and discriminate a large diversity of odorant molecules [27]. There are two main, anatomically and functionally distinct chemoreceptor systems [28], namely the main olfactory system (MOS) and the vomeronasal system (VNS). It has been suggested that the MOS primarily detects volatile odorants from the environment [29,30], whereas the VNS primarily detects non-volatile odorants from conspecifics [28,30,31], although both systems can, to a degree, detect both types of odor cues [30]. The Grueneberg ganglion, a chemosensory organ that appears to mediate behavioral responses to alarm pheromones in rodents [32], and the septal organ of Masera, a patch of sensory epithelium separate to the MOE that may have a dual role in surveying food or conspecific sexual odors [33], are not considered here.

#### 2.2.1. The Main Olfactory System (MOS)

The nose houses the MOS, which consists of the main olfactory epithelium (MOE; Figure 1). This is the primary site for the detection of volatile odorants [1]. The main olfactory sensory neurons (OSNs) in the MOE, of which there are approximately 10 million in vertebrates, are located directly in the nasal airstream; thus, stimulus access simply requires passive respiration or a sniffing action [34]. The 500–1000 olfactory receptors (ORs [1,27,35]) that belong to the rhodopsin-like G-protein-coupled receptor superfamily (GPCRs [26,35,36]), are located in the cell membranes of the OSNs, and bind specific odorant ligands [35]. While the ORs are responsible for detecting chemosensory cues, they are also involved in axonal guidance to the brain [36,37].

**Figure 1.** Schematic representation of transmission of olfactory information via the main olfactory system (solid black arrows) or the vomeronasal system (broken arrows) to corresponding brain regions. Black circles in the MOE indicate the broad localization of olfactory sensory neurons. Note: locations of brain regions not exact and for pictorial purposes only. AOB: accessory olfactory bulb; BNST: bed nucleus of the stria terminalis; HPC: hippocampus; LS: lateral septrum; MOB: main olfactory bulb; MOE: main olfactory epithelium; MeA: medial amydala; LPOA: lateral preoptic area; MPOA: medial preoptic area; NAcc: nucleus accumbens; SI: substantia innominata; VNO: vomeronasal organ; VTA: ventral tegmental area.

Neurons that express the same OR type converge at similar sites (glomeruli) within the OB [38], forming synapses with mitral [36,38] or tufted cells [39] and conveying olfactory information from the MOE to the OB [40]. The signals from each OR are transferred to the anterior olfactory nucleus, the cortical amygdala [30] and a small number of pyramidal cells that form clusters in the piriform cortex (PC) of the olfactory cortex [41]. The PC cells project to the orbitofrontal cortex, amygdaloid cortex, prefrontal cortex, perirhinal cortex and entorhinal cortex, through which they access the hippocampus (Figure 1; [42]). Androgen and estrogen cellular receptors are expressed in the PC [43], suggesting a responsiveness to hormones that may regulate sexual and paternal behaviors [3]. Importantly, olfactory bulbectomy negatively affects paternal care in male prairie voles [44].

#### 2.2.2. The Vomeronasal System (VNS)

Closely associated with the MPOA is the accessory olfactory or VNS (Figure 1; [45]). The VNS is primarily involved with the reception and decoding of olfactory cues, providing a relatively direct pathway to the amygdala [46], BNST [39] and hypothalamic areas [47].

The vomeronasal organ (VNO; Figure 1) of the VNS is an extraordinarily sensitive structure [48]. In rodents, the morphological complexity of the VNO is greater than any other mammal [49]. The VNO detects both volatile and non-volatile olfactory signals [2], and neurons that express the same receptor form multiple glomeruli within the AOB [34]. Neural projections transfer the olfactory signals to

several brain regions via the AOB [50], including the corticomedial amygdala [24] and the BNST (Figure 1; [51]). Projections from the AOB also extend to the medial (Figure 1) and posteromedial cortical (C3) amygdaloid nuclei [51] and the ventral hypothalamus [52]. Interestingly, there is sexual dimorphism in the AOB [53], meA [54] and BNST [54,55].

The MPOA then receives these impulses (Figure 1), activating Galanin-expressing neurons (MPOAGal [22]), and this cascade of impulses then activates neurons in the LPOA and SI (Figure 1; [20]). From here, LPOA descending efferent neurons, which are localized in the dorsal LH [19], trigger neurons in the VTA (Figure 1). The VTA is part of the dopaminergic reward system, and is associated with reinforcement learning, with unweaned offspring being a strong reinforcing stimulus to males [56]. This pathway likely influences the processing of pup-related olfactory cues, and mediates and regulates pup-directed aggression [57]. Disruptions to this pathway inhibit infanticide and promote paternal care [22,58].

#### **3. Genetic Regulation of Olfaction**

#### *3.1. Genetic Regulation in the Main Olfactory System*

Mammals can recognize and discriminate thousands of odor molecules due to a large multigene family (±1400 functional genes [29]) in the MOE that encodes the ORs. Each OSN expresses only one or a few odorant receptor genes [2,36,59,60], and the genes are randomly monoallelically expressed (i.e., half express the maternal allele while the other half express the paternal allele [36,59]). The ORs are bound to the G-protein *<sup>G</sup>*α*olf* [30] and are typically not very selective. Thus, an OSN typically responds to a range of related odor cues (i.e., combinatorial in nature [61]). In mammals, ORs fall into two major groups (phylogenetic clades; [62]): "Class I" ORs (known as fish-like receptors as they were first identified in fish) comprise approximately 10% of functional ORs, while "Class II" ORs (mammalian-like receptors) comprise approximately 90% [40]. It is thought that a subset of some of these ORs from both classes, respond to volatile compounds in food, thereby influencing foraging behavior and food preferences [63].

In addition to the ORs, trace amine receptors (TAARs) are also expressed by neurons localized in the MOE [63–65], and are activated by distinct combinations of volatile amines, many of which occur in urine [66]. There are between 15 and 17 TAARs found in rodents [64,66]. TAARs are expressed in a small number of OSNs [66] and, like ORs, they are expressed in a mutually exclusive manner [64]. Interestingly, TAARs in the MOE are localized to Gαolf–expressing sensory neurons that can stimulate cyclic adenosine monophosphate (cAMP) pathways, indicating that they couple to canonical olfactory pathways [66]. In rodents, Gαolf is highly expressed in the medium spiny neurons of the striatum, which houses the dopamine 1 receptor [67] and is critical for transduction of the ORs and complete olfactory function [68].

Lastly, a set of molecularly atypical neurons residing in the MOE expresses other non-GPCR receptors (guanylate cyclase GC-D) that are encoded by membrane-spanning 4-pass A (Ms4a) genes [63]. While every Ms4a protein detects specific odors [69], each is likely to play a role in regulating the social acquisition of food preference via olfactory cues [70].

#### *3.2. Genetic Regulation in the Vomeronasal System*

In the VNS, the VNS sensory neurons (VSNs) are located away from the nasal airstream, and activation of the neurons thus requires a vascular pumping mechanism [71]. This mechanism enables the VNO to take up non-volatile stimuli that are investigated by direct nasal contact [34]. The VSNs are among the most sensitive of mammalian chemoreceptors [72]. In contrast to the main olfactory genes, VNO receptors detect only a limited group of ligands (differential tuning hypothesis [73]). There are 250–300 functional vomeronasal receptor genes [74], in at least three families, including vomeronasal type 1 receptor genes (V1Rs), vomeronasal type 2 receptor genes (V2Rs), and formyl peptide receptor genes (FPRs). As for the ORs in the MOE, the V1Rs and V2Rs encode G- protein-coupled transmembrane proteins [28,39,74].

The main receptor proteins of the VNO consist of the V1R (±150) and V2R (±160) families of vomeronasal receptors [75], each derived from individual genes [29]. The two families of receptors are expressed in anatomically distinct neuronal populations of the VN epithelium [2] that coincide with di fferent zones of G-protein expression (V1R = apical zone of the epithelium, express Gαi2, dark purple in Figure 1; [28]; V2R = basal zone of the epithelium, express G0<sup>α</sup>, (light purple in Figure 1; [39]), and each VNO neuron expresses only a single receptor protein [75]. In addition, the two classes project to anatomically and functionally separate sub-regions of the AOB, suggesting di fferential processing of vomeronasal stimuli [76]. V1R-expressing neurons project to the anterior sub-region of the AOB, while V2R-expressing neurons project to the posterior sub-region of the AOB [2]. However, neural projections coming from each of these two regions then project to and overlap at the level of the amygdala, the accessory olfactory tract and the BNST [77].

Unlike OSNs, the VSNs are highly selective for individual molecules [2], although due to their highly diverse nature, V1Rs typically respond to a wide variety of di fferent odor molecules [75,78], and are known to respond to the urinary volatiles 2,3-dehydro-exo-brevicomin (DB) and 2-sec-butyl-4,5-dihydrothiazole (BT, [72]). Both V1Rs and V2Rs are thought to detect olfactory cues that are related to conspecifics [28,31]. For example, 129/SvEv male mice with a cluster of V1R genes genetically deleted show reduced sexual behavior [79]. Interestingly, H2-Mv (a class of major histocompatibility complex (MHC) proteins) is coexpressed in V2Rs in rodents [80], with M10 and M1 family proteins being expressed exclusively in the V2Rs [81]. Indeed, it has been suggested that correct V2R expression relies on the M10s [81]. H2-Mv genes are not randomly expressed, and certain combinations of genes are located with particular V2Rs [82], which could explain an individual's responsiveness to particular MHC-associated chemosignals [2].

The FPRs are another family of olfactory neurons expressed by localized VNO neurons [63,83]. Interestingly, FPR olfactory expression is restricted to rodents [84], and expression of these receptors occurs in a punctate and monogenic pattern in the VSNs [83], which is characteristic of the transcription of olfactory chemoreceptor genes [85]. Within the vomeronasal (VN) epithelium, Fpr-rs3, -rs4, -rs6 and –rs7 are transcribed by neurons in the apical zone, coincident with V1Rs, while Fpr-rs1 is transcribed by neurons in the basal zone, coincident with V2Rs [83]. It has been suggested that FPRs play a role in the detection of pathogens or pathogenic states [83].

#### **4. Olfaction and Paternal Care Behavior: Suggested Genetic Regulation**

Numerous candidate genes, many coding for hormone expression, influence paternal care behaviors (e.g., estrogen receptor alpha (ER α) [86]). However, the regulation of paternal care is likely under multisensory control, and olfactory stimuli from pups should be neurally integrated to allow males to recognize o ffspring [23] and provide paternal care accordingly. The use of odor for distinguishing kin relationships [87,88] and for paternal kin discrimination (e.g., golden hamsters (*Mesocricetus auratus*) [89]) is well documented in rodents. For example, disruption or damage to the OB diminishes paternal care behaviors in biparental male prairie voles [45]. However, several olfactory genes, or genes that regulate olfactory processes, could be involved in the regulation of paternal care behaviors (Table 1).

The MOS likely has less of an influence on the expression of paternal care behaviors since MOE receptor gene sequences are conserved across both paternal and non-paternal vertebrate species [30]. However, VNO receptors detect a limited group of ligands [73], and there is species-specific variation in VNO receptor diversity [30], suggesting that the di fferential detection and signaling in the VNO could be important for the expression of paternal care behavior in biparental species compared to non-paternal species. However, it is equally plausible that the MOS and VNS work synergistically in identifying, recognizing, and discriminating pup odor cues, and that paternal care is mediated by both systems. Below, I discuss 10 genes that likely work mutually to regulate the expression of paternal care in male rodents.

**Table 1.** Genes involved in the regulation and expression of paternal care behaviors in rodents, their theorized olfactory location, functioning associated brain regions and interactions with other genes, proteins or hormones (all references provided in text).


V1R: vomeronasal type 1 receptor; V2R: vomeronasal type 2 receptor; OB: main olfactory bulb; AOB: accessory olfactory bulb; MPOA: medial preoptic area; BNST: bed nucleus of the stria terminalis; LPOA: lateral preoptic area; SI: substantia innominate; LH: lateral hypothalamus; KO: knockout; VNO: vomeronasal organ; MOS: main olfactory system; OR: olfactory receptors; OT: oxytocin; MOE: main olfactory epithelium; VTA: ventral tegmental area; SON: supraoptic nucleus; SVZ: subventricular zone; NAcc: nucleus accumbens; FPR: formyl peptide receptor genes; CNG: cyclic nucleotide-gated; BDNF: brain-derived neurotrophic factor; AVP: arginine vasopressin.
