Fish were anesthetized by immersion in 0.05% tricaine methanesulphonate (TMS) prior to dissection of the brain explant. Briefly, after severing the spinal cord, the skull was carefully opened with surgical scissors to expose the brain.  The explant was attached to a Petri-dish ventral side up at the level of the spinal cord and cerebellum with cyanoacrylate glue, and placed in a bath with ice-cold artificial cerebrospinal fluid (ACSF) of the following composition [mM]: 127 NaCl, 1.9 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgCl2, 26 NaHCO3, 10 Dglucose; gassed with carbogen (95 O2, 5% CO2); pH adjusted to 7.4 with NaOH. The ACSF was modified from rats' ACSF (Spanswick et al., 1998) and it was similar to others (ACSF) in other fish such as *Apteronotus leptorhynchus* (Kotecha et al., 1997) and *C. auratus* (Wilkie et al., 2008). When magnesium-free solution was used, MgCl2 was omitted from the ACSF.

The meninges were removed with fine forceps to expose the ventral telencephalon and access the vPOA; then a transverse cut using a razor blade was made posterior to the hypothalamus to free the brain from the dish. The brain was then transferred carefully to a custom-built recording chamber perfused at room temperature with ACSF at a rate of 2–4 ml/min. The brain explant was mounted with the ventral side up and then held between two custom-made nylon grids where it was allowed to recover for 1 h prior to recordings; all recordings were made in the 7 h following dissection. Neuroanatomical nomenclature in this study follows that of Anglade et al. (1993). Our vPOA corresponded to *nucleus preopticus periventricularis* as depicted in Plate 43 of the goldfish brain atlas (Peter and Gill, 1975).

## **ELECTROPHYSIOLOGICAL RECORDINGS**

Electrophysiological recordings were made based on previous methods for rat spinal cord and hypothalamus (Spanswick et al., 1998). Whole-cell patch clamp recordings using a Multiclamp 700B amplifier (Molecular Devices) in current clamp mode, were obtained from vPOA neurons (*N* = 107) in the *in vitro* forebrain explants at room temperature (∼18–20◦C) from 120 fish. Patch pipettes (5–8 M*-*) were fabricated from borosilicate filament glass (Sutter Instrument Co., Novata, CA, USA) using a horizontal pipette puller (P2000; Sutter Instrument Co., Novata, CA, USA) and filled with intracellular solution of the following composition [mM]: 140 K-gluconate, 10 KCl, 1 sucrose, 2 Na2ATP, 1 EGTA-Na4 plus 10 HEPES and pH adjusted to 7.4 with KOH modified from Spanswick et al. (1998).

Using the anterior commissure and optic chiasm as landmarks, patch electrodes were guided to the vPOA under visual control of a dissecting microscope. Seal formation was monitored on an oscilloscope. Once a gigaohm seal (typically *>* 5 G*-*) was achieved, whole-cell access was made by gentle suction. Series resistance was *<* 25 M*-*.

To measure synaptic connectivity, a bipolar stimulating electrode was inserted into one of the olfactory bulbs (OBs). Postsynaptic potentials (PSPs) in the vPOA were elicited by single pulse electrical stimulation (5–30 V, 0.2 ms pulse duration) of the ipsilateral OB via a stimulus isolation unit (Digitimer Ltd., model DS2). Data acquisition and experimental control was performed using pCLAMP 9.2 software (Molecular Devices). Data were lowpass filtered at 2 kHz and acquired at 10 kHz and later analyzed offline using CLAMPFIT 9.2 software (Molecular Devices).

## **PHARMACOLOGICAL AGENTS**

To characterize the pharmacological properties of the connectivity from the OB to the vPOA neurons, we used 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX; Tocris), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA/kainate) receptor antagonist; and D-2-amino-5-phosphonopentanoic acid (D-APV; Tocris), an N-Methyl-D-aspartic acid or N-Methyl-D-aspartate (NMDA) receptor antagonist. Drugs were made up as stock solutions, CNQX in DMSO (Sigma-Aldrich) and D-APV in distilled water, then, diluted in Mg2+ free ACSF. The final concentration of DMSO was always *<* 0.1%. Typically, Mg2+ free ACSF and the drugs were applied sequentially for 10 min each to the recording chamber before any attempts at recordings to allow sufficient equilibration time.

## **DATA ANALYSIS**

Intrinsic membrane properties of vPOA neurons were characterized by patch clamp electrophysiology to determine whether they constituted distinct populations. After achieving whole cell access, the resting membrane potential (RMP) which is the baseline potential in the absence of any current stimulus was measured in current clamp mode (*I* = 0 nA). In addition, properties related to spontaneous action potential production was measured (**Figure 1A**): Spike amplitude (SA) was measured from the shoulder of the rising phase (∼threshold) to the peak; spike width (Swidth) was measured at the width of half-maximal from the peak to the afterhyperpolarization (AHP); AHP was determined from the threshold to the peak of the hyperpolarization following the action potential (note that only the fast component of the AHP was considered in this study); after-depolarization potential (ADP) was measured from the hyperpolarization peak to the ADP peak. For neurons that showed spontaneous activity, the coefficient of variation of the interspike interval (the interval between successive spikes) was calculated over a 90 s time window (CVspikes = SD/mean). Neurons were then stimulated with 1 s hyperpolarizing and depolarizing current steps (2–30 pA) from a holding potential of −60 mV to measure a number of other

intrinsic membrane properties (**Figures 1B–D**): current threshold (Ithreshold) is the minimum current required to elicit an action potential; spike threshold (Sthreshold) is the number of spikes elicited by the minimum current i.e., at Ithreshold; the presence or absence of a rebound depolarization (RD) after a hyperpolarizing current step; the presence or absence of rebound depolarization spikes (RDS), rebound spike frequency (RS) is the action potential frequency during the RD; the presence or absence of H current (IH) which is associated with non-selective cation channels; rectification (Rec) refers to a non-linearity in the current-voltage (IV) relationship (Siegelbaum and Koester, 2000) (compare insets in **Figures 1C,D**); the input resistance (Rinput), which is the slope of the IV curve; membrane time constant (TC) is the time for the hyperpolarization response to reach two-thirds of its plateau value; soma membrane capacitance (Csoma) is calculated as TC/ Rinput (Abbud and Smith, 1995).

A principle component analysis (PCA; SPSS Inc.; 2006, v.15) was used to reduce the set of intrinsic membrane properties to a number of independent uncorrelated variables. For this analysis, all properties that were characterized by their presence or absence, i.e., RD, RDS, IH and Rec were assigned binary values (0, 1). The PCA variables were then used to cluster (SPSS Inc.; 2006, v.15) the neurons into groups. An unsupervised cluster analysis was performed to classify neurons, similar to previous studies (Ward, 1963; Krimer et al., 2005; Sosulina et al., 2006; Andjelic et al., 2009). This method consisted of grouping individual neurons based on the Euclidean distance between their respective PCA loadings.

Postsynaptic potentials (PSP) data were characterized by the peak amplitude (the height of the evoked PSP measured from baseline to peak), the latency (the time between the OB stimulus and beginning of PSP rise), 10–90% rise time (measured from the shoulder of the rise to the peak) and 90%–10% decay time (determined from 90% of the peak of the PSP to 10% above baseline) (**Figure 2**). To measure variability in the latency and rise time, the mean, standard deviation (SD) and coefficient of variation (CV); (SD/mean) was calculated over 4 stimulus trials in each cell.

All data generated by the PCA were tested for normality and homogeneity of variance, and either an analysis of variance (ANOVA) or a Kruskal-Wallis (KW) analysis was used for between group comparisons where appropriate (SPSS Inc.; 2006, v.15). *Post-hoc* analyses consisted of paired *t*-tests and Tukey's (SPSS Inc.; 2006, v.15). Unless otherwise stated, data are reported as mean ± s.e.m.

## **RESULTS**

## **HETEROGENEITY OF INTRINSIC MEMBRANE PROPERTIES**

To characterize the population of vPOA neurons, a number of intrinsic membrane properties were quantified (see Materials and Methods). We used a Principle Component Analysis (PCA) to determine the set of properties that could best distinguish neuronal subgroups. The PCA revealed seven significant properties (loading factor) [RD (0.92), Rinput (0.87), SA (0.77), Sthreshold (0.80), RS (0.82), IH (0.78), and Rec (0.83)] (**Table 1**). A subsequent cluster analysis of these variables revealed five distinct neuronal subgroups (denoted I, II, III, IV, and V). In the following, we

compare the membrane properties across the different subgroups (**Table 2**).

Since the data from our PCA failed the normality test (*P >* 0*.*05), it was transformed to its square root equivalent; and statistical analyses performed. Neuronal subgroups were found to differ in their Rinput [*F*(4*,* 106) = 325*.*93, *P* = 0.001], TC [*F*(4*,* 106) = 13*.*63, *P* = 0*.*001] and Ithreshold [*F*(4*,* 106) = 3*.*86, *P* = 0*.*006] but not ADP [*F*(4*,* 106) = 1*.*35, *P* = 0*.*25]. *Tukey's post-hoc* analyses showed that Rinput was different in each neuronal cluster with V = III *>* IV *>* II *>* I (**Figure 3A**). Similarly, the TC of neurons in clusters III and V were higher than IV which in turn was higher than those in clusters I and II (**Figure 3B**). In addition, Ithreshold for neurons in cluster I was greater than II which in turn was greater than for IV greater than III and V neurons (**Figure 3C**). Since the rectification (Rec) and RS were categorical variables, *post-hoc* comparisons were performed using KW analyses: rectification was not significant [χ2 (4) = 1*.*55; *P* = 0*.*818] nor was RS [χ2 (4) = 10*.*28; *P* = 0*.*036, Bonferroni correction, *P >* 0*.*005].

Overall, our analyses showed that only Rinput, TC and Ithreshold were significantly different between groups (*P <* 0*.*05), while rectification and RS were not (*P >* 0*.*05). Together, the Rinput, TC and Csoma constitute the passive membrane properties of the cell. Note that the TC is directly proportional to the product of the Rinput and Csoma [TC = Rinput*.* Csoma] (Molleman, 2003). The calculated Csoma (after its transformation to the reciprocal of its square root to normalize the data) was also statistically different between neuronal groups [*F*(4*,* 106) = 25*.*89, *P* = 0*.*001], with *Tukey's post-hoc* indicating that I *>* II *>* IV *>* III = V (**Figure 3D**). Since capacitance is proportional to membrane area (Hille, 2001), the differences we observe between neuronal groups can be at least partially explained by neuronal size.

### **PROPERTIES OF POSTSYNAPTIC POTENTIALS: INPUTS FROM THE OLFACTORY BULB**

Given these putative subgroups of vPOA neurons, we next set out to determine their inputs from the olfactory bulb (OB). Of the 107 vPOA cells tested, 50 received synaptic inputs from the OB. The ratio of connected to unconnected neurons in each cluster was: I: 59% (12/23); II: 60% (20/38); III: 0% (0/4); IV: 53% (17/36); and V: 19% (1/6).

The PSPs were reliably evoked with latencies of approximately 90 ms. The small coefficients of variation (CV) of the latency (1.5%) and rise-time (2.2%) within cells are not consistent with a multi-synaptic pathway (for which latency is expected to be more variable). Given the consistency of the synaptic response, we suggest that these connections are monosynaptic (Spanswick et al., 1998). Since the distance from the OB to the POA is about 9 mm, a latency of 100 ms suggests an estimated conduction velocity similar to the slowest conduction velocity (∼0.1 m/s) reported in olfactory nerves of the tench at similar temperatures (Dubois-Dauphin et al., 1980).

## **BIOCHEMICAL PROPERTIES OF THE POSTSYNAPTIC POTENTIALS OF vPOA**

To characterize the pharmacological properties of the PSP in vPOA (*N* = 13) neurons, goldfish brain explants were perfused sequentially with normal ACSF, Mg++-free ACSF (MFACSF), 20μM D-APV and 10μM CNQX before washing off both drugs with normal saline. The latency, peak amplitude rise and decay times of the evoked PSPs were then measured and compared under the different recording conditions. **Figure 4** shows representative data from a vPOA neuron. The Mg2+free ACSF increased the PSP compared to normal saline. The glutamate antagonist APV partially blocked the evoked response. The residual response was subsequently blocked completely by CNQX, indicating that the PSPs had a dual component and were mediated by glutamate acting on both NMDARs and AMPARs (**Figure 4**). Indeed, when AMPARs alone were blocked with CNQX (data not shown; *N* = 10), no EPSPs were evoked suggesting that activation of NMDARs requires preceding depolarization via AMPARs to overcome voltage-dependent Mg2+ block.

A statistical analysis on data obtained from POA neurons (*N* = 13) did not find significant differences (*P >* 0*.*05) in their PSP latencies and rise times between the different perfusion media, but their decay times differed significantly between MFACSF vs. ACSF [*F*(1*,* 12) = 16*.*11, *P* = 0*.*002] and MFACSF vs. APV [*F*(1*,* 12) = 16*.*59, *P* = 0*.*002]. *Post-hoc* analysis indicated that POA neurons in MFACSF had a longer decay time than in either ACSF [*t*(13) = 3*.*76, *p* = 0*.*002] or APV [*t*(13) = 4*.*07, *p* = 0*.*002] (**Figure 5**). These data show that the evoked PSPs had a biphasic response, with APV partially blocking the slower and longer lasting NMDAR component, revealing a faster and shorter lasting AMPAR component that was completely blocked by CNQX.

## **SECTIONING THE LATERAL OLFACTORY TRACT WHILE RECORDING FROM THE vPOA**

To determine if the OB to vPOA projection is mediated through the MOT, we sectioned the LOT (*N* = 3) while leaving the medial tract intact. We found that PSPs evoked in vPOA neurons by OB stimulation did not differ from those evoked under control conditions for latency, rise time, peak amplitude and decay times (KW analysis; *P >* 0*.*05). Cutting the MOT (*N* = 10) while leaving the LOT intact did not elicit any response consistent with studies which have shown that the MOT innervates targets in area

*Loaded factors in bold show measurements that were subsequently used for our cluster analysis. See definitions in Materials and Methods section and Figure 1. Extraction Method: Principal Component Analysis. Rotation Method: Varimax with Kaiser Normalization. Rotation converged in 8 iterations.*

*RMP (resting membrane potential); TC (membrane time constant); R*input *(input resistance); Cm (membrane capacitance); AHP (after hyperpolarization potential); APA (action potential amplitude); TI (action potential threshold current); ST (number of spikes at threshold current); APD (action potential duration); CV (coefficient of variation); ADP (after depolarization potential). Not shown are variables for rectifying and RS. See definitions in Materials and Methods section and Figure 1.*

dorsalis and area ventralis while the LOT projects only to area dorsalis (Levine and Dethier, 1985). Furthermore, pharmacological manipulations influenced the recorded EPSPs in similar ways for both cut and intact LOTs for latency, rise time, peak amplitude and decay times (KW analysis; *P >* 0*.*05). Sectioning both the medial and LOTs abolished the PSPs completely. This confirms that the pathway we describe from OB to POA is via the MOT.

 Previous anatomical studies have shown direct neuronal pathways from the OB to POA in the teleost brain (Forlano and Bass, 2011). With whole-cell patch clamp recordings, we show for the first time that these connections are functional and glutamatergic. In addition, we show that these synapses involve both N-methyl-D-aspartate (NMDAR) and α-amino-3-hydroxy-5 methyl-4-isoxazolepropionic acid receptors (AMPAR). We also provide evidence, with a thorough characterization of electrophysiological properties, that neurons of the vPOA comprise several subgroups.

The POA is an important hypophysiotropic center that regulates reproduction in vertebrates. Electrical stimulation of this area has been shown to elicit sperm release (Demski, 1983; Dulka and Demski, 1986; Dulka, 1993; Dominguez, 2009); sexual calling (Schmidt, 1968); nest-building and courtship (Demski and Knigge, 1971) in several vertebrate models. Conversely, lesioning the POA impairs reproduction in male goldfish thereby underscoring the importance of this neural system (Hart et al.,

**FIGURE 3 | Intrinsic membrane properties of POA neurons.** Neurons were made up of connected (*N* = 50) and unconnected (*N* = 57) neurons to the OB. **(A)** The input resistance showing group differences. **(B)** The time constant, indicating differences between groups. **(C)** The threshold current

1973; Kyle and Peter, 1982; Kyle et al., 1982; Koyama et al., 1984; Sorensen et al., 1991; Dulka, 1993).

### **HETEROGENEITY OF NEURONAL ELECTRICAL PROPERTIES**

We recorded from neurons in the vPOA while stimulating the OB. Our analysis revealed five subgroups of vPOA neurons, each with distinct intrinsic membrane properties and variable connectivity to the OB.  Thus, this heterogeneity in electrophysiological profiles may reflect functionally diverse classes of vPOA neurons. Further work is required to determine if these electrophysiological "signatures" correspond to chemical phenotypes, exhibiting differential projections and functional roles.

## **PROPERTIES OF POSTSYNAPTIC POTENTIALS: INPUTS FROM OLFACTORY BULB**

Electrical stimulation of the OB evoked PSPs in vPOA neurons. These PSPs gave rise to action potentials at the peak of their responses in some cases. The evoked excitatory PSPs (EPSPs) had consistent and constant latencies and rise times with small coefficient of variation suggesting they arise through monosynaptic inputs from the OB (Spanswick et al., 1998). The conduction velocities of the inputs from the OB to the POA were similar to those found previously in other systems (Gasser, 1956; Potapov and Gusel'nikova, 1976b). The relatively slow conduction is consistent with propagation through unmyelinated olfactory fibers (Westerman and Wilson, 1968; Potapov and Gusel'nikova, 1976a; Farbman, 1992). The conduction velocity of the inputs from the OB to the vPOA was estimated to be 0.1 m/s, which was similar to the pike (Gasser, 1956) and slower, by four times, than that reported by Kandel (1964) in the goldfish for the larger POA magnocellular neurons projecting to the neural lobe of the pituitary gland when stimulated antidromically.

## **BIOCHEMICAL PROPERTIES OF THE SYNAPTIC CONNECTIONS**

Anatomical connections between the OB and vPOA have been established previously through tract-tracings (Levine and Dethier, 1985; Anglade et al., 1993), but their functional nature remains unclear. To investigate the possible role of glutamate in chemical communication, we perfused the goldfish brain explant sequentially with ACSF, APV, and CNQX in Mg2+free ACSF while stimulating the OB to measure the latency, amplitude, rise time, decay time and duration of the evoked potentials in the vPOA (**Figure 4**). Mg2+free ACSF enhanced the evoked EPSPs compared to normal ACSF. The NMDAR antagonist APV partially blocked the EPSPs leaving a fast acting and short lasting component that was subsequently completely blocked by CNQX, suggesting that the evoked PSP was mediated by glutamate acting on NMDARs and AMPARs, respectively. Receptors for AMPA may therefore be required to depolarize the cells (from their resting state) sufficiently to relieve the Mg2+ blockage of NMDARs (Gotz et al., 1997; Spanswick et al., 1998). Bath application of drugs allows for the possibility that the observed effects are indirect and involve peripheral pathways. However, given that the latency and rising phase of the evoked response is very consistent and remains so during drug application, the effects are likely direct.

The complete blockage of the evoked PSPs by the glutamatergic antagonists suggests that glutamate plays an important role in mediating chemical communication between the OB and vPOA. Since the POA is important for the regulation of reproductive behaviors, it may receive pheromonal cues from the OB to integrate milt release and spawning in male goldfish (Kyle and Peter, 1982; Kyle et al., 1982). The use of glutamate signaling through NMDA receptors may therefore be a mechanism to induce the sustained neuronal firing required to trigger an LH surge when sex pheromones are detected. To our knowledge, this is the first pharmacological characterization of second order neurons in the teleost olfactory system linked to reproduction.

A monosynaptic glutamatergic connection from the OB to the vPOA complements and extends our understanding of the neural circuitry involved in the control of goldfish reproduction. Previously, Trudeau et al. (2000b) demonstrated the existence of monosynaptic GABAergic projections from the Vv to the vPOA. Indeed, GABA plays a central role in male goldfish reproduction by suppressing the DAergic inhibition of LH release (Trudeau et al., 1993). This suggests that there are interactions between diverse sets of neurotransmitters and neurohormones that regulate reproduction in male goldfish. The Vv may therefore modulate the glutamatergic inputs from the OB to the vPOA to regulate some aspects of reproductive behavior or hormone release.

## **ROLE OF GLUTAMATE IN GOLDFISH REPRODUCTION**

Previous studies have shown that intraperitoneal injections of male goldfish with either monosodium glutamate (MSG) (Sloley et al., 1992) or NMDA (Trudeau et al., 2000b) or AMPA (Trudeau et al., 2000b; Popesku et al., 2011) rapidly induces LH release. Furthermore, in rainbow trout it has been shown that the LH response to NMDA is blocked by APV or a GnRH receptor antagonist, indicating that glutamate modulates LH release through stimulation of GnRH (Flett et al., 1994), similar to the situation in mammalian models (Kocsis et al., 2003). Moreover, Peter et al. (1980) has shown that MSG injections in goldfish causes cellular degeneration in the POA, demonstrating excitotoxic actions of glutamate on POA neurons. Additionally, in rats it has been shown that glutamate injections in the POA or electrical stimulation of the POA decreases the latency between intromissions thereby increasing ejaculation frequency (Dominguez, 2009). Glutamate in the POA therefore plays an important role in vertebrate reproduction.

### **SECTIONING THE LATERAL OLFACTORY TRACT**

We employed olfactory tract sectioning to determine if the glutamatergic projection to the vPOA was via the MOT or the LOT. The EPSPs recorded in vPOA cells in explants with a transected LOT had the same amplitude and duration as those with the LOT intact. These EPSPs were modulated by APV and CNQX in the same way as in intact preparations. In other experiments, sectioning the MOT while leaving the LOT intact did not elicit PSPs in vPOA neurons indicating that the OB to vPOA projection we have studied is via the MOT and not the LOT. This supports previous studies indicating unequivocally that sex pheromones signals in goldfish are mediated exclusively by the MOT indeed tract tracing studies have shown that while the MOT projects to area dorsalis and area ventralis of the telencephalon the LOT only innervates targets in the area dorsalis (Levine and Dethier, 1985; Sorensen et al., 1991).

## **CONCLUSION**

We describe an electrophysiological basis for classifying neurons of the vPOA. Further, we provide evidence that the synaptic connections from the OB to the vPOA are monosynaptic and glutamatergic. These connections from the OB to vPOA may play a role in facilitating spermiation and steroidogenesis (Peter and Paulencu, 1980; Peter et al., 1980; Kyle and Peter, 1982). While speculative at this point, the olfactory glutamatergic projections we identified may represent pathways that integrate pheromonal signals from females that stimulate reproductive hormone release and male sexual behavior in the spawning period.

## **AUTHOR CONTRIBUTIONS**

Wudu E. Lado, Vance L. Trudeau and David C. Spanswick designed the experiment. Wudu E. Lado and David C. Spanswick performed the experiment. Wudu E. Lado, John E. Lewis and Vance L. Trudeau analyzed the results and wrote the paper.

## **ACKNOWLEDGMENTS**

We thank Dr. Andrew Whyment for invaluable advice and Bill Fletcher for technical assistance during the preparation of this manuscript. We acknowledge with appreciation Dr. Leo Renaud for his early support and encouragement to pursue this study. Funding from the University of Ottawa, NSERC to John E. Lewis and Vance L. Trudeau and OGS to Wudu E. Lado is gratefully acknowledged.

## **REFERENCES**

**Conflict of Interest Statement:** 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.

*Received: 31 March 2014; accepted: 11 June 2014; published online: 30 June 2014.*

*Citation: Lado WE, Spanswick DC, Lewis JE and Trudeau VL (2014) Electrophysiological characterization of male goldfish (Carassius auratus) ventral preoptic area neurons receiving olfactory inputs. Front. Neurosci. 8:185. doi: 10.3389/fnins.2014.00185*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience.*

*Copyright © 2014 Lado, Spanswick, Lewis and Trudeau. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Gender-typical olfactory regulation of sexual behavior in goldfish

## *Yutaro Kawaguchi , Akira Nagaoka , Asana Kitami , Tomomi Mitsuhashi , Youichi Hayakawa and Makito Kobayashi\**

*Department of Life Science, International Christian University, Mitaka, Tokyo, Japan*

#### *Edited by:*

*Ishwar Parhar, Monash University, Malaysia*

#### *Reviewed by:*

*Gustavo M. Somoza, Instituto de Investigaciones Biotecnologicas-Instituto Tecnologico de Chascomus, Argentina Satoshi Ogawa, Monash University, Malaysia*

#### *\*Correspondence:*

*Makito Kobayashi, Department of Life Science, International Christian University, 3-10-2 Osawa, Mitaka, Tokyo 181-8585, Japan e-mail<EMAIL_ADDRESS>
It is known that olfaction is essential for the occurrence of sexual behavior in male goldfish.   It has been considered that olfaction does not affect sexual behavior in female goldfish. In the present study, we re-examined the involvement of olfaction in sexual behavior of female goldfish. Olfaction was blocked in male and female goldfish by two methods: nasal occlusion (NO) which blocks the reception of olfactants, and olfactory tract section (OTX) which blocks transmission of olfactory information from the olfactory bulb to the telencephalon. Sexual behavior of goldfish was induced by administration of PGF to females, an established method for inducing goldfish sexual behavior in both sexes. Sexual behavior in males was suppressed by NO and OTX as previously reported because of lack of pheromone stimulation.  Females treated with both NO and OTX performed sexual behavior normally. These results indicate that olfaction is essential in female goldfish to perform sexual behavior as in males but in a different manner. The lack of olfaction in males causes lack of pheromonal stimulation, resulting in no behavior elicited. Whereas the results of female experiments suggest that lack of olfaction in females causes strong inhibition of sexual behavior mediated by the olfactory pathway. Olfactory tract section is considered to block the pathway and remove this inhibition, resulting in the resumption of the behavior. By subtract sectioning of the olfactory tract, it was found that this inhibition was mediated by the medial olfactory tracts, not the lateral olfactory tracts. Thus, it is concluded that goldfish has gender-typical olfactory regulation for sexual behavior.

 It has been known that many teleost fishes employ sex pheromones to coordinate their reproductive activity with successful fertilization of gametes (Stacey and Sorensen, 2006; Stacey, 2011).  Since most of fish sex pheromones studied are from female to male, the involvement of olfaction in reproductive activity has been intensively studied in male fish, and few studies are conducted on the involvement of olfaction in female reproduction (Sorensen et al., 2005; Lastein et al., 2006; Appelt and Sorensen, 2007; Stacey, 2011; Hayakawa et al., 2012).

Goldfish *Carassius auratus* is a small cyprinid species and intensively used for environmental and physiological studies of behavior. As a result, hormonal, pheromonal, and neural regulation of sexual behavior in goldfish is probably the best understood among fish species (Stacey and Sorensen, 2006; Munakata and Kobayashi, 2010; Stacey, 2011; Kobayashi et al., 2013). Female sexual behavior (egg releasing act) is induced by prostaglandin F2α (PGF) produced in the ovary at the time of ovulation.  For the occurrence of sexual behavior, estrogens are not required by females (Kobayashi and Stacey, 1993), but androgen is considered to be essential for males (Stacey and Kobayashi, 1996). When PGF is injected into non-ovulatory females, these fish are induced to perform female sexual behavior with normal males within several minutes after the PGF injection although no egg release is accompanied in this case (Stacey and Kyle, 1983). Thus, using PGF, sexual behavior can be induced easily in goldfish pairs.

Anatomy of olfactory system of goldfish and crucian carp *Carassius carassius*, closely related species of goldfish, is well studied (Von Bartheld et al., 1984; Hamdani et al., 2000, 2001; Weltzien et al., 2003; Stacey and Sorensen, 2006), which enables surgical block of olfaction in goldfish. In the present study, we examined involvement of olfaction in sexual behavior in male and female goldfish by two methods of olfactory blockage. The first method is occlusion of nasal cavity with glue which blocks reception of olfactants. The second method is olfactory tract section. Since the olfactory bulbs of goldfish are the pedunculated type with elongated olfactory tracts, olfactory information from the olfactory bulbs to the telencephalon can be easily blocked by sectioning olfactory tracts (Stacey and Kyle, 1983; Von Bartheld et al., 1984; Kobayashi et al., 1986, 1994). Using these two methods, we compared the effects of olfactory blockage on sexual behavior between male and female goldfish.

## **MATERIALS AND METHODS**

#### **FISH**

Goldfish *Carassius auratus* were obtained from a local dealer in Saitama Prefecture, Japan. Fish were kept in 800-L stock tanks maintained at 20◦C under 16L/8D photoperiod. Fish were freely fed with commercial goldfish feed once a day. It is known that gonadal maturity of goldfish is generally maintained under these environmental conditions. Most of the stock males were spermiating and had tubercles on their pectoral fins (male secondary sexual characteristic), and females were found to have vitellogenic oocytes in the ovary when randomly sampled and dissected. Fish weighing 14–30 g were used for the experiments. The handling of fish in the present study was endorsed by Animal Experimentation Committee of International Christian University.

#### **SEXUAL BEHAVIOR OF GOLDFISH**

Natural and prostaglandin (PG)-induced spawning (sexual) behaviors are well documented in goldfish (Kobayashi et al., 2002; Munakata and Kobayashi, 2010). In brief, during natural spawning, ovulated females produce PGF in the ovary, and this PGF acts on the brain and triggers the female spawning act (egg releasing act) in the females. PGF and its metabolites are released into the water as sex pheromones which trigger male spawning behavior that are characterized as chasing and culminating in sperm release (i.e., male spawning act). Male chasing is persistent and interspersed with the spawning acts. Spawning acts (complete spawning act) are initiated by the entry of an ovulated female into the floating aquatic vegetation near the surface of the water and where the male follows the female. The female and the male turn on their sides and swim quickly through the vegetation, releasing eggs and sperm. The male always positions itself underneath and in contact with the female during this act. Then, they flip their tails to mix spawned eggs and sperm. Released eggs are characteristically sticky and quickly adhere to the vegetation. Female spawning will continue until most of her ovulated eggs are released, and this may involve hundreds or more spawning acts over several hours. Another type of spawning act of goldfish is called an incomplete spawning act. An incomplete (attempted) spawning act is similar to a complete spawning act except that the fish leave the vegetation without performing gamete release and tail flipping. In the present study, we considered both complete and incomplete spawning acts as normal behavior and were counted equally.

Female goldfish injected with PGF are induced to perform the female spawning act as do ovulated females with sexually mature males, although eggs are not released. Males do not distinguish between ovulated and PG-injected females.

#### **BEHAVIOR EXPERIMENTS**

For the behavior experiments, fish were transferred from stock tanks to 60-L glass acclimating aquaria and kept at 20◦C under 16L/8D photoperiod over the course of 1–10 days. Each behavior test was conducted in 60-L glass observation aquaria provided with artificial floating vegetation made of acrylic yarn, gravel, and an aerated box filter and water temperature maintained at 20◦C and a 16L/8D photoperiod.

 PGF was injected into experimental females at a dose of 0.1μg/0.1μL saline/g body weight for the female behavior experiments, and to partner females at a dose of 10μg/2.0μL saline/fish for the male behavior experiments (Stacey and Kyle, 1983; Saoshiro et al., 2013).

 For female behavior tests, PG-injected experimental females and partner males were paired in each observation aquarium and sexual behavior (total of complete and incomplete spawning acts) was counted for 60 or 90 min (Stacey and Kyle, 1983). Pre-treatment tests were conducted on Day 1 and Post-treatment tests were conducted on Day 3 (**Table 1**). Each experimental fish was paired with the same partner fish in pre- and post-treatment tests. Fish received no treatment or behavior test on Day 2 for the recovery from the treatment and behavior test. Experimental fish and partner fish were kept in 60-L glass acclimating aquaria between the tests.

#### **Table 1 | Experimental design of sexual behavior in goldfish.**

*Fish received no treatment or behavior test on Day 2. OTX, olfactory tract section.*

## **BLOCAKGE OF OLFACTION**

For the blockage of olfaction, two methods were employed in this study: nasal occlusion which blocks reception of olfactants, and olfactory tract section (OTX) which blocks transmission of olfactory information from the olfactory bulb to the telencephalon where the neural center regulating sexual behavior is located (Kyle et al., 1982; Koyama et al., 1985).

Nasal occlusion was carried out according to the method previously reported (Partridge et al., 1976). Fish were anaesthetized by immersion in 0.02% tricaine methanesulfonate (Sigma-Aldrich, St. Louis, MO, USA) solution. The nasal cavities were occluded with denture fix (Shin-Poligrip-SA Mutenka, Earth Chemical, Tokyo, Japan). Then, fish were kept in dechlorinated water for 10–20 min for recovery from anesthesia before behavior tests.

For the complete blockage of olfaction, the nasal cavities of both sides were bilaterally occluded. As one of the controls, one side of nasal cavity was unilaterally occluded. As another control, fish were treated in the same manner without occlusion of the nasal cavities (intact). This method of occlusion was effective in blocking olfaction for 70–80 min since denture fix used in the present study is water-soluble and some bilaterally occluded males started male sexual behavior with PG-injected females around this time, probably due to the leakage of the water to the olfactory epithelium. Therefore, observation period of experiment using this method was set to 60 min.

It is well established that PG pheromone acts on the olfactory epithelium of sexually mature male goldfish and elicits chasing (courtship) of the males (Stacey and Sorensen, 2006). To assess the completeness of nasal occlusion, we examined the suppression of chasing of male goldfish in the presence of the PG-injected females. We tried other methods of olfactory blockage, such as cauterization of olfactory epithelium and application of silver nitrate solution on the epithelium, but these techniques were not successful (Kobayashi, unpublished data) and nasal occlusion was effective for 70– 80 min. The methods that are effective for the olfactory blockage in males, nasal occlusion and olfactory tract section, were applied to the experiments of females in the present study.

Olfactory tract section was carried out according to the method previously reported (Stacey and Kyle, 1983; Kobayashi et al., 1986, 1994). Fish were anaesthetized with a 0.02% tricaine methanesulfonate solution. A four-sided flap was cut in the frontal bone using a disc saw in order to expose a pair of the olfactory tracts. After removal of a square flap of the frontal bone, the tracts were bilaterally or unilaterally sectioned at two places with Wecker's scissors, and the resultant sections were removed (OTX). Olfactory tracts were subdivided surgically into a lateral olfactory tract (LOT) and a medial olfactory tract (MOT) that can be further separated into lateral medial-olfactory tract (lMOT) and medial medial-olfactory tract (mMOT). Specific subtract section was also carried out. The cavity resulting from operation was filled with gelatin sponge (Spongel, Yamanouchi Pharamaceutical Co. Tokyo, Japan). Sham operations were performed in the same manner without cutting the olfactory tracts.

 On Day 1, experimental males were paired with PG-injected partner females in observation aquaria, and male sexual behavior (sperm releasing act) was counted over a period of 60 min (pre-treatment test). On Day 3, experimental males were paired with PG-injected partner females after one of the following three treatments, bilateral nasal occlusion, unilateral nasal occlusion, and intact, and male sexual behavior was observed for 60 min (post-treatment test).

## **EFFECTS OF OLFACTORY TRACT SECTION ON MALE SEXUAL BEHAVIOR (EXPERIMENT 2)**

In this experiment, involvement of olfaction in sexual behavior in male goldfish was examined by OTX which blocks the transmission of olfactory information from the olfactory bulb to the telencephalon. On Day 1, experimental males were paired with PG-injected partner females in observation aquaria, and male sexual behavior was counted over a period of 90 min (pretreatment test). After the behavior tests, experimental males received either OTX or sham operation. On Day 3, experimental males were paired with PG-injected partner females, and male sexual behavior was observed for 90 min (post-treatment test).

## **EFFECTS OF NASAL OCCLUSION ON FEMALE SEXUAL BEHAVIOR (EXPERIMENT 3)**

In this experiment, involvement of olfaction in sexual behavior in female goldfish was examined by nasal occlusion which blocks the reception of olfactants from the environment. On Day 1, experimental females were injected with PGF, paired with partner males in observation aquaria, and female sexual behavior (egg releasing act) was counted over a period of 60 min (pre-treatment test). On Day 3, experimental females were injected with PGF, paired with partner males after one of the following three treatments, bilateral nasal occlusion, unilateral nasal occlusion, and intact, and female sexual behavior was observed for 60 min (post-treatment test).

## **EFFECTS OF OLFACTORY TRACT SECTION ON FEMALE SEXUAL BEHAVIOR (EXPERIMENT 4)**

In this experiment, involvement of olfaction in sexual behavior in female goldfish was examined by OTX. On Day 1, experimental females were injected with PGF, paired with partner males in observation aquaria, and female sexual behavior was counted over a period of 90 min (pre-treatment test). After the behavior tests, experimental females received either OTX or sham operation. On Day 3, experimental females were injected with PGF, paired with partner males, and female sexual behavior was observed for 90 min (post-treatment test).

## **EFFECTS OF NASAL OCCLUSION AND OLFACTORY TRACT SECTION ON FEMALE SEXUAL BEHAVIOR (EXPERIMENT 5)**

In this experiment, effects of simultaneous treatments of nasal occlusion and OTX were examined in female goldfish. On Day 1, experimental females were injected with PGF, paired with partner males in observation aquaria after bilateral nasal occlusion, and female sexual behavior was counted over a period of 60 min (pre-treatment test). After the behavior tests, experimental females received either OTX or sham operation. On Day 3, experimental females were injected with PGF, paired with partner males after bilateral treatment of nasal occlusion, and female sexual behavior was observed for 60 min (post-treatment test).

## **EFFECTS OF LOT AND MOT SECTION ON FEMALE SEXUAL BEHAVIOR (EXPERIMENT 6)**

In this experiment, effects of LOT and MOT section on sexual behavior were examined in female goldfish. On Day 1, experimental females were injected with PGF, paired with partner males in observation aquaria, and female sexual behavior was counted over a period of 60 min (pre-treatment test). After the behavior tests, experimental females received either LOT section or MOT section. On Day 3, experimental females were injected with PGF, paired with partner males after bilateral nasal occlusion, and female sexual behavior was observed for 60 min (post-treatment test).

### **EFFECTS OF LATERAL MOT AND MEDIAL MOT SECTION ON FEMALE SEXUAL BEHAVIOR (EXPERIMENT 7)**

In this experiment, effects of lMOT and mMOT section on sexual behavior were examined in female goldfish. On Day 1, experimental females were injected with PGF, paired with partner males in observation aquaria, and female sexual behavior was counted over a period of 60 min (pre-treatment test). After the behavior tests, experimental females received either lMOT section or mMOT section. On Day 3, experimental females were injected with PGF, paired with partner males after bilateral nasal occlusion, and female sexual behavior was observed for 60 min (post-treatment test).

### **STATISTICS**

Data among groups were statistically compared by ANOVA and Newman-Keuls test. Data between groups were statistically compared by Mann-Whitney *U*-test. Data within a group were statistically compared by paired *t*-test. Level of significance was 0.05 for all statistical tests. Statistical test could not be applied to some data of groups because mean values were zero or close to zero with very low deviation.

## **RESULTS**

## **EFFECTS OF NASAL OCCLUSION ON MALE SEXUAL BEHAVIOR (EXPERIMENT 1)**

Intact fish and unilaterally occluded fish actively performed male sexual behavior in pre- and post-treatment tests on Day 1 and Day 3, respectively (**Figure 1**). Levels of sexual behavior in these fish showed no change between Day 1 and Day 3 (*p >* 0*.*05). Level of sexual behavior in male goldfish significantly decreased after bilateral nasal occlusion (*p <* 0*.*05, compared to pre-treatment level and to levels of intact and unilaterally occluded fish of Day 3).

  Pre, Pre-treatment (open column). Post, post-treatment (solid column). Each column represents the mean and s.e.m. Difference compared to pre-treatment (asterisk), compared to intact fish (solid circle), and compared to unilaterally occluded fish (open circle). Level of significance, *p <* 0*.*05.

 Pre, Pre-treatment (open column). Post, post-treatment (solid column). Each column represents the mean and s.e.m. Difference compared to pre-treatment (asterisk), compared to sham-operated fish (solid circle). Level of significance, *p <* 0*.*05.

3 (*p >* 0*.*05). Level of sexual behavior in male goldfish significantly decreased after OTX (*p <* 0*.*05, compared to pretreatment level and to level of sham operated fish of Day 3).

## **EFFECTS OF NASAL OCCLUSION ON FEMALE SEXUAL BEHAVIOR (EXPERIMENT 3)**

Intact fish actively performed female sexual behavior in pre- and post-treatment tests on Day 1 and Day 3, respectively (**Figure 3**). Levels of sexual behavior in intact fish showed no change between Day 1 and Day 3 (*p >* 0*.*05). Level of sexual behavior in female goldfish significantly decreased after unilateral nasal occlusion (*p <* 0*.*05 compared to pre-treatment level). Female goldfish of bilateral occlusion showed no sexual behavior on Day 3.

## **EFFECTS OF OLFACTORY TRACT SECTION ON FEMALE SEXUAL BEHAVIOR (EXPERIMENT 4)**

Both sham-operated fish and OTX fish actively performed female sexual behavior in pre- and post-treatment tests on Day 1 and Day 3, respectively (**Figure 4**). Levels of sexual behavior in these fish showed no change between Day 1 and Day 3 (*p >* 0*.*05).

## **EFFECTS OF NASAL OCCLUSION AND OLFACTORY TRACT SECTION ON FEMALE SEXUAL BEHAVIOR (EXPERIMENT 5)**

Females with bilateral nasal occlusion showed almost no sexual behavior on Day 1 (**Figure 5**). Sham-operated fish with bilateral occlusion showed almost no sexual behavior on Day 3. OTX fish with bilateral occlusion actively performed sexual behavior on Day 3.

**female goldfish.** Pre, Pre-treatment (open column). Post, post-treatment (solid column). Each column represents the mean and s.e.m. Difference compared to pre-treatment (asterisk), *p <* 0*.*05.

## **EFFECTS OF LOT AND MOT SECTION ON FEMALE SEXUAL BEHAVIOR (EXPERIMENT 6)**

Females before treatment actively performed sexual behavior on Day 1 (**Figure 6**). LOT-sectioned fish with bilateral nasal occlusion showed no sexual behavior on Day 3. MOT-sectioned fish with bilateral nasal occlusion actively performed sexual behavior on Day 3.

## **EFFECTS OF LATERAL MOT AND MEDIAL MOT SECTION ON FEMALE SEXUAL BEHAVIOR (EXPERIMENT 7)**

Females before treatment actively performed sexual behavior on Day 1 (**Figure 7**). Levels of sexual behavior in lMOT-sectioned fish and mMOT-sectioned fish with bilateral nasal occlusion

**FIGURE 5 | Effects of nasal occlusion and olfactory tract section on female sexual behavior in female goldfish.** Pre, Pre-treatment (bilaterally occluded). Post, post-treatment (bilaterally occluded and either sham-operated or olfactory-tract sectioned). Each column represents the mean and s.e.m.

**FIGURE 6 | Effects of olfactory subtract section on female sexual behavior in female goldfish.** LOT section, Lateral olfactory tracts were sectioned. MOT, Medial olfactory tracts were sectioned. Pre-treatment (open column). Post, post-treatment (solid column). Each column represents the mean and s.e.m.

significantly decreased on Day 3 (*p <* 0*.*05, compared to pretreatment level).

## **DISCUSSION**

The present study indicates that olfaction is the key prerequisite for the occurrence of sexual behavior both in male and female goldfish, but its regulation for sexual behavior is different between male and female. In male goldfish, blockage of olfaction by nasal occlusion and OTX severely reduced activity of sexual behavior as previously reported (Partridge et al., 1976; Stacey and Kyle, 1983) because of lack of pheromonal stimulation. Since males with unilateral nasal occlusion actively performed sexual behavior as did intact males, chemical or physical damage of nasal occlusion on fish is considered to be negligible.  These males did not show vigorous chasing that intact males normally do to PG-injected partner females, and sexual behavior of these males occurred mostly when the partner females rose into the aquatic vegetation near these anosmic males. It is considered that anosmic males sometimes show sexual behavior by visual stimulus from females (Norm Stacey and Peter Sorensen, personal communication), and not due to incompleteness of nasal occlusion and OTX. Thus, we examined the involvement of olfaction in female sexual behavior using the same methods that were used for the experiments for males.

Female sexual behavior was strongly suppressed by bilateral nasal occlusion which blocks the reception of olfactants even under the stimulation of PGF. Level of sexual behavior was also reduced by unilateral nasal occlusion. The results of these experiments indicate that the olfaction is essential for the female sexual behavior to occur. However, OTX fish performed female sexual behavior after PG-injection as reported previously (Stacey and Kyle, 1983) although these females were unable to receive olfactory information from the olfactory bulbs. Since OTX did not affect the occurrence of female sexual behavior in PG-injected female goldfish in the previous study (Stacey and Kyle, 1983), it has been considered that olfaction is not essential for the female sexual behavior in goldfish. Nevertheless, nasal occlusion suppressed the female sexual behavior. Results of experiments of nasal occlusion and OTX look contradictory, but one of the possible interpretations we propose is as follows. Blockage of olfaction in females exerts strong inhibition of sexual behavior mediated by the olfactory pathway from the olfactory epithelium to the telencephalon via the olfactory bulb and the olfactory tract even in the presence of PGF stimulation. When females were treated with nasal occlusion and OTX simultaneously, the fish actively performed sexual behavior. Therefore, it is considered that OTX blocked the pathway and removed the inhibitory signaling for the behavior. Reduction of female sexual behavior in unilaterally occluded female may be caused by some inhibition from the occluded side of olfactory epithelium unlike the case of unilaterally occluded male goldfish. Sexual behavior of unilaterally occluded male can receive enough amount of pheromonal stimulation by one side of olfactory epithelium and perform full range of sexual behavior. We further examined the pathway of the inhibitory signaling in female goldfish by olfactory subtract section. Section of LOT did not remove the inhibition of sexual behavior in bilaterally occluded females, but by MOT section, bilaterally occluded females performed sexual behavior, indicating that the inhibition in the olfactory pathway is transmitted via MOT. When lMOT and mMOT was individually sectioned, bilateral nasal occlusion caused partial inhibition of female sexual behavior, suggesting that both lMOT and mMOT are involved in mediating the inhibitory signaling.

It is known that MOT mediates pheromonal information and that LOT mediates information of food odors in male and female goldfish and crucian carp (Stacey and Kyle, 1983; Hamdani et al., 2001; Weltzien et al., 2003). It is interesting that MOT is involved in regulation of sexual behavior both in male and female goldfish although its function is different, stimulatory in males and inhibitory in females. A clear functional difference of the olfactory bulb between male and female is reported in crucian carp.   It is also interesting to study how olfaction is involved in sexual behavior of females in other teleost species.

The present study demonstrated the involvement of olfaction in female sexual behavior in goldfish. However, biological significance of olfactory regulation in female goldfish is not clarified. There are some possible explanations of female olfactory system for the regulation of sexual behavior. One of the possible functions could be detection of special odorants, such as sex pheromone from males.  In the case of female goldfish, PGF triggers female sexual behavior and a male sex pheromone may not be a trigger but could facilitate the behavior. It is known that a large amount of androstenedione is released from sexually mature male goldfish into the water (Sorensen et al., 2005) and that PG-injected female goldfish show rise into vegetation (initial movement of female spawning act) more frequently in the presence of sexually mature males than in the absence of males (Appelt and Sorensen, 2007). It is possible that androstendione from males function as a sex pheromone which stimulates the initiation of female sexual behavior.

Another possible explanation is that female goldfish searches appropriate site and substrate for oviposition using sense of olfaction as well as vision and tactility. Normally females, not males, decide the site of oviposition in goldfish, and female goldfish need to select appropriate oviposition substrate which secures development of embryo and also appropriate site of water quality. Female goldfish may use olfaction for examining water quality of the spawning site. When olfaction is blocked in female goldfish which fails to examine the water quality of the spawning area, sexual behavior is inhibited even if the fish is stimulated by PGF. Such hypothesis can be applied in male tilapia *Oreochromis niloticus* which makes nest for spawning (Uchida et al., 2005).  Female salmonid fishes are known to stop nest-digging behavior and following spawning behavior when pH of the water is slightly lowered even when other environmental conditions are suitable for spawning (Ikuta et al., 2003). It is possible that these females sense the acidity by olfaction. It is of interest to examine whether water quality change may affect sexual behavior in intact and OTX female goldfish.

In conclusion, the present study demonstrated the involvement of olfaction in female sexual behavior in goldfish although it has been considered that olfaction does not affect the behavior. When reception of olfactants is blocked, female sexual behavior is severely suppressed. It is considered that lack of olfaction exerts inhibition of the behavior mediated by the olfactory pathway. Olfactory tract section removes this inhibition resulting in resumption of sexual behavior. Biological significance of this inhibitory system of female individual is unknown. Further behavioral and ecological studies are necessary for better understanding of the regulation of sexual behavior by olfaction in fishes. Also, it is interesting to study the gender typical-olfactory function could be induced heterotypically in goldfish since heterotypical sexual behavior and hormone secretion could be induced in goldfish (Stacey and Kobayashi, 1996; Kobayashi et al., 1997) and crucian carp *C. auratus langsdorfii* (Kobayashi and Nakanishi, 1999) which are considered to have sexually bipotential brain (Kobayashi et al., 2013).

## **ACKNOWLEDGMENTS**

We are grateful to Prof. Naoyuki Yamamoto of Nagoya University for valuable comments and advices in conducting this study and for critical reading of the manuscript.

## **REFERENCES**

Zielinski, B. S., and Hara, T. J. (2007). "Olfaction," in *Sensory Systems Neuroscience, Fish Physiology, Vol. 25,* eds T. Hara and B. Zielinski (New York, NY: Academic Press, Elsevier), 1–43. doi: 10.1016/S1546-5098(06) 25001-5

**Conflict of Interest Statement:** 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.

*Received: 25 January 2014; accepted: 09 April 2014; published online: 30 April 2014. Citation: Kawaguchi Y, Nagaoka A, Kitami A, Mitsuhashi T, Hayakawa Y and Kobayashi M (2014) Gender-typical olfactory regulation of sexual behavior in goldfish. Front. Neurosci. 8:91. doi: 10.3389/fnins.2014.00091*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience.*

*Copyright © 2014 Kawaguchi, Nagaoka, Kitami, Mitsuhashi, Hayakawa and Kobayashi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Pairmate-dependent pup retrieval as parental behavior in male mice

#### *Mingkun Liang1,2, Jing Zhong1,2, Hong-Xiang Liu1, Olga Lopatina1, Ryusuke Nakada2, Agnes-Mikiko Yamauchi 2 and Haruhiro Higashida1 \**

*1 Department of Basic Research on Social Recognition and Memory, Research Center for Child Mental Development, Kanazawa University, Kanazawa, Japan 2 Department of Biophysical Genetics, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan*

#### *Edited by:*

*Sonoko Ogawa, University of Tsukuba, Japan*

#### *Reviewed by:*

*Aldo Lucion, Universidade Federal do Rio Grande do Sul, Brazil Tomoko Soga, Monash University, Malaysia*

#### *\*Correspondence:*

*Haruhiro Higashida, Department of Basic Research on Social Recognition and Memory, Research Center for Child Mental Development, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japan e-mail: haruhiro@ med.kanazawa-u.ac.jp*

Appropriate parental care by fathers can greatly facilitate healthy human family life. However, much less is known about paternal behavior in animals compared to those regarding maternal behavior. Previously, we reported that male ICR strain laboratory mice, although not spontaneously parental, can be induced to display maternal-like parental care (pup retrieval) when separated from their pups by signals from the pairmate dam (Liu et al., 2013). This parental behavior by the ICR sires, which are not genetically biparental, is novel and has been designated as pairmate-dependent paternal behavior. However, the factors critical for this paternal behavior are unclear. Here, we report that the pairmate-dependent paternal retrieval behavior is observed especially in the ICR strain and not in C57BL/6 or BALB/c mice. An ICR sire displays retrieval behavior only toward his biological pups. A sire co-housed with an unrelated non-pairing dam in a new environment, under which 38-kHz ultrasonic vocalizations are not detected, does not show parenting behavior. It is important for sires to establish their own home territory (cage) by continuous housing and testing to display retrieval behavior. These results indicated that the ICR sires display distinct paternity, including father-child social interaction, and shed light on parental behavior, although further analyses of paternal care at the neuroendocrinological and neurocircuitry levels are required.

**Keywords: parental behavior, paternal care, pup retrieval behavior, paternity, mouse**

## **INTRODUCTION**

According to Schor and others, "a stable, well-functioning family that consists of two parents and children is potentially the most secure, supportive, and nurturing environment in which children may be raised" (Schor and American Academy of Pediatrics Task Force on the Family, 2003; Fortunato and Archetti, 2010; Benbassat and Priel, 2012). Thus, the role of a father in the home is highly significant, and currently, the physical absence of the father in the home is seen as a major problem facing families worldwide (Feinberg, 2002; Fleming et al., 2002; Amato, 2005; Benbassat and Priel, 2012; Morrongiello et al., 2013; Bornovalova et al., 2014). This raises questions regarding which factors determine paternal care and how they are maintained.

Although a number of animal models have been used in experimental studies of parental care (Reburn and Wynne-Edwards, 1999; Carter et al., 2009; de Jong et al., 2009; McGraw and Young, 2010; Ozawa et al., 2010; Kuroda et al., 2011; Mogi et al., 2011; Saltzman and Maestripieri, 2011; Lambert et al., 2013; Tachikawa et al., 2013; Yoshida et al., 2013), given its value for genetic studies, a mouse model of paternal behavior may be especially useful (Hager and Johnstone, 2003; Jin et al., 2007; Liu et al., 2013).

Previously, we reported that the outbred ICR strain is uniparental and is a good model for studies of parental behavior (Jin et al., 2007; Liu et al., 2008, 2013; Higashida et al., 2012a), because these mice actively reproduce offspring and exhibit easily monitored pup retrieval after separation (Fujimoto et al., 2013; Liu et al., 2013), which is a reliable indicator of parental behavior (Gammie, 2005; Wynne-Edwards and Timonin, 2007; Yoshida et al., 2013). We demonstrated that male ICR mice display robust parental care, which is induced by signaling from the pairmate dam, after separation from the pups (Liu et al., 2013). We demonstrated that this signaling is mediated through as yet unidentified olfactory pheromonal cues and auditory 38-kHz ultrasonic vocalization (USV) cues (Liu et al., 2013), that the male response can be modified hormonally via oxytocin (Akther et al., 2013), that CD38 in the nucleus accumbens is critical (Akther et al., 2013), and that the central cholinergic system is involved (Fujimoto et al., 2013). However, the factors influencing singly isolated sires in which there is no direct communicative interaction between dams and sires remain unclear.

In the present study, to simplify fatherhood evaluation, we used an all-or-nothing type of pup retrieval behavior by calculating the percentage of sires that displayed retrieval behavior (Liu et al., 2013). We investigated paternal behavior in terms of the types of conditions that can induce or maintain paternal retrieval behavior by sires when the males are isolated before the offspring are delivered by pregnant mates, and the males are held separately to prevent them being sires by physically separating them from other family members for 3 days. Then, family ties are formed with or without mate information. In other experiments, we examined isolation from pups under different housing conditions in which either pairmate dam and pup olfactory information is present or excluded.

## **MATERIALS AND METHODS**

### **ANIMALS**

Male and female Slc:ICR, C57BL/6, and BALB/c mice were obtained from Japan SLC, Inc. (Hamamatsu, Japan) via a local distributor (Sankyo Laboratory Service Corporation, Toyama, Japan). The ICR mice were originally obtained from Charles River Laboratories in 1965 and since then bred in Japan with the alternative name Swiss CD1. The offspring of these mice were born in our laboratory colony, weaned at 21–28 days of age, and housed in same-sex groups of 3–5 animals until pairing (Liu et al., 2013). The animals were paired and kept in our laboratory under standard conditions (24◦C; 12-h light/dark cycle, lights on at 08:00) with food and water *ad libitum*. The mice were housed together continuously in standard mouse maternity cages. The experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Kanazawa University.

### **BEHAVIORAL TESTING**

Virgin males and females were paired at 45–55 d. A single male and a single female were continuously housed together in a standard mouse maternity cage from the mating period until the delivery of pups. In some experiments, the males were separated in new cages 1 day before parturition to prevent formation of family relationships and kept in the new cages for 3 days. Then, the males were allowed to meet their pups with or without pairmates from day 3 to day 5. All family units composed of a new sire (first-time father), dam, and their first litter were experimentally naïve.

One male parent was placed for 10 min in the original cage or new cage alone or with his pairmate (separation environment). Five pups were randomly selected from the litter and placed individually at a site remote from the nest in the original cage. The sires were returned to the original home cage or a new cage in the presence of their five biological or foster pups to assess parental behavior. Parental retrieval behavior (percentage of sires exhibiting retrieval) was examined for 10 min following reunion. The behavioral tests were performed in a randomly mixed sequence of experimental groups. Experiments were usually performed at 10:00–15:00. We defined retrieval as positive if the sires carried all 5 pups to the original nesting place or within two thirds of the distance between the nest and the place at which the pups had been placed (Liu et al., 2013). We also observed other parental behaviors (grooming, crouching, and huddling) as defined by Gubernick and Alberts (1987, 1989). The animals in this and subsequent experiments were tested only once.

## **MEASUREMENT OF USVs**

Experiments were carried out in a soundproof chamber measuring 600 × 500 × 500 mm (model MC-050/VA; Muromachi Kikai, Tokyo, Japan). USVs were detected with a condenser microphone (Type 7016; Aco, Tokyo, Japan) and a preamplifier (type 4116; Aco) designed for sound pressure level (SPL) measurements between 20 Hz and 90 kHz. A 4-kHz band-pass filter was used to minimize background noise during recordings; however, most WAV files still contained a considerable amount of "non-USV" signal. Extraneous noise was identified and removed from the sonograms as far as possible. When a rater found an ultrasound signal that was difficult to interpret, the call was evaluated by a minimum of one additional trained observer and identification required a consensus by all raters. Each sonogram was then evaluated with a series of automated parameters. The microphone was placed 50 cm above the cage in a soundproof chamber and connected to an amplifier (model UMA-2; Muromachi Kikai). Acoustic signals were transmitted to a vocalization analyzer system (model MK-1500; Muromachi Kikai) with functions such as an analog-to-digital converter (192 kHz), frequency filters, a digital fast-Fourier-transform analyzer, and signal input—output terminals. Input signals were visualized on SpectraLAB (Sound Technology Inc., State College, PA) in the analyzer system on a personal computer. USVs were recorded as WAVE files and analyzed; the number of calls, frequency, and wave width (*>*40 ms) were measured using a USV monitor (Muromachi Kikai).

#### **STATISTICAL ANALYSIS**

The data were calculated as the means or the means ± s.e.m. Two-tailed Fisher's exact probability test was used for single comparisons of retrieval behaviors. The remaining data were analyzed by two-tailed Student *t*-test.

## **RESULTS**

It has been reported that parental behavior in mice is dependent on the strain (Wright and Brown, 2000). Therefore, we first examined and compared parent–pup family units in three strains, i.e., ICR, C57BL/6, and BALB/c mice, under various experimental settings. The data are summarized in **Table 1**. Maternal nurturing behavior was observed in dams of all three strains, in a strainnonspecific fashion, except for the low rate of retrieval by the BALB/c dams. In contrast, paternal behavior was variable between the strains. No retrieval behavior was observed by BALB/c sires (*n* = 15). C57BL/6 sires displayed retrieval during reunion after single-separation in new cages (approximately 40%, *n* = 15). However, isolation together with the partner in new cages did not potentiate but rather decreased this rate to 13.3% (*n* = 15). This parental behavior suggests that C57BL/6 males display mateindependent paternal behavior. Interestingly, 38-kHz USVs were

#### **Table 1 | Parental behaviors in three strains of mice.**

not recorded from any dam–sire pairs of C57BL/6 and BALB/c strains separated in new cages for 10 min. These results indicated that pairmate-dependent care is specific to the ICR strain. Therefore, in the following experiments, we examined various critical conditions under which ICR strain males did or did not show paternal behavior.

#### **RETRIEVAL BEHAVIOR BY SIRES SEPARATED ALONE IN HOME CAGES**

The experimental paradigms for each experiment are shown schematically in each figure. In **Figure 1**, we first reproduced our previous results (Liu et al., 2013). Male and female ICR strain mice were paired and housed together continuously in a standard mouse maternity cage (**Figure 1A**). The mice were left undisturbed during the first 3 days after the birth of their pups (**Figure 1B**), during which they displayed distinct paternal and maternal behaviors as described previously (Liu et al., 2013). The sire and dam nursed the pups. This involved nest-building, pup retrieval, licking, and huddling over the pups and lactating. However, as described in the Methods section, we mainly analyzed the male's retrieval behavior, as a parental role, in the following experiments.

The sire in the first family was left alone in the vacated cage during the period of separation (**Figure 1C**), whereas the pups and dam were removed and placed in a new cage (**Figure 1D**) separated from the family cage. After 10 min, the five selected pups of the sire (biological offspring) were returned to the nursing cage in a remote area away from the nest, where the sire was present (**Figure 1E**). The sire retrieved the offspring over 10 min (86% of the sires, *n* = 15; **Figures 1F,S**).

If the non-biological (foster) pups (**Figure 1N**) of the third family (**Figures 1M,N**) were introduced into the vacated home cage with the second sire (**Figures 1I,K**) in the second family (**Figures 1G,H**), instead of the biological pups (**Figures 1H,J**), 33% of the 15 sires displayed pup retrieval (**Figures 1L,S**; twotailed Fisher's exact probability test between sires toward biological (F) and non-biological (L) pups, *P <* 0*.*01).

When a sire from the third family (**Figure 1N**) was placed and isolated for 10 min in the home cage of the second family (**Figure 1O**), the third sire did not retrieve any of the foster (second family's) pups (**Figures 1R,S**; *n* = 15, two-tailed Fisher's exact probability test between unrelated sires (R) and sires with non-biological (L) or biological (F) pups, *P <* 0*.*05 and *P <* 0*.*0001, respectively). These results suggested that paternal pup retrieval behavior in the home cage is maintained by biological family cues of their mate dams and remaining pups.

## **RETRIEVAL BEHAVIOR BY SIRES AFTER SEPARATION IN NEW CAGES**

Male parental care in **Figure 1** may have been induced by the fact that the males were left in the nursing environment during parent–pup separation. To select out pup information during isolation, we used the co-housing paradigm presented in **Figure 2**. We examined whether sires developed paternal behavior following time spent with the family. Pup retrieval increased on a daily basis after parturition, while dams displayed a higher retrieval ratio from the first day of parturition than the sires (**Table 2**).

The sires alone (**Figure 2D**) or together with the mate dams (**Figure 2H**) were placed in a new cage for 10 min, whereas the pups alone (**Figure 2G**) or together with dams (**Figure 2C**)

were left in the home cage. Then, the sires were returned to the home cages in which five pups remained (**Figures 2E,I**). The male's retrieval behavior was undiminished when housed with the pairmate (66%, *n* = 30; **Figures 2J,U**) but was strongly reduced when housed alone (24%, *n* = 41; **Figures 2F,U**). As expected, a high level of sire care was displayed after isolation in the new environment together with mate dams and pups (as the whole family (**Figures 2K,L**) (66%, *n* = 15; **Figure 2U**): twotailed Fisher's exact probability test between sires separated alone (F) and together (J) or as a whole family (N), *P <* 0*.*001, equally.

The latter was specifically associated with co-habitation with the pairmate dam during the separation period (**Figure 2H**), because negligible retrieval behavior was apparent if the sire was housed with the dam of another brood (**Figures 2O–T**; 20%, *n* = 15); two-tailed Fisher's exact probability test shows no significant difference between sires separated together with unrelated dams (T) and alone (F); and separated together (J), *P <* 0*.*01; and separated as a whole family (N), *P <* 0*.*05, **Figure 2U**). Thus, it appears that the mate dam provides some signal(s) during the separation period to induce parental behavior in the sire, in agreement with the results reported previously (Liu et al., 2013). Whereas parental care by the dam is independent of the presence of the male or the housing environment, that by the male is strongly dependent on cues from the pairmate dam and/or home cage.

We recorded USVs (with *>*40 ms in wave width) to determine their role as one form of critical interactive information in this paradigm. We detected 38-kHz USVs identical to those reported previously (Liu et al., 2013) under isolation conditions in new cages for 10 min between sires and mate dams at a frequency of 25.9 ± 4.8 calls/10 min (*n* = 8, **Table 3**; *P <* 0*.*01 from other values, two-tailed Student *t*-test). No identical 38-kHz USVs were recorded between sires and unrelated dams. Instead, 30–80-kHz USVs were recorded infrequently at 40.7 ± 26.7 calls/10 min (*n* = 11) between unfamiliar couples. These 30–80-kHz USVs were emitted when a sire was co-housed with a virgin female at 313.6 ± 64.9 calls/10 min (*n* = 11, *P <* 0*.*001 from two other values, twotailed Student *t*-test). These data clearly support the suggestion that paternal retrieval is essentially triggered by the pairmate's 38-kHz USVs.

#### **RETRIEVAL BY ISOLATED BEFORE PAIRMATE PARTURITION**

The retrieval behavior displayed by males may have been induced by family formation in the nursing cage environment. To assess this possibility, data were obtained from parting males (**Figures 3**, **4**) that remained with the paired pregnant females 1 day before parturition of their first litter and were then separated into a new cage (**Figures 3A–C**). The males were then isolated alone for 3 days (**Figure 3E**). When sire paternal retrieval was examined immediately on day 3 in the sire home cage (**Figure 3K**), 21.4% of sires with no prior contact with their biological pups and pairmate dam, i.e., the paternity unformed state (**Figure 3E**), displayed retrieval behavior (*n* = 42; **Figure 3N**). Next, when the isolated males were relocated in the home cage

**FIGURE 2 | Paternal retrieval test in ICR mice isolated in new cages.** Paired couples were kept in rearing cages from mating to postnatal day 3 (P3) **(A,B,O,P)**. In **(C,D)**, the pups and the mating dam were left in their home cages **(C)**, and the sire alone was placed in a new holding cage **(D)**. In **(G,H)**, the pups were kept in the original cage, and the parents were placed in a new cage **(H)**. In **(K,L)**, the whole family was moved to a new cage **(L)**. In **(Q,R)**, the sire was kept during the separation period **(Q)** with a non-mating dam of another family **(O,P)**. After isolation for 10 min in each cage, pup retrieval behavior over a

#### 10-min period was observed in each case **(E,I,M,S)**. The number of sires displaying retrieval behavior was scored **(F,J,N,T)**. The numbers of positive mice/number of mice tested are shown in parentheses and expressed as percentages **(U)**. Two-tailed Fisher's exact probability test: between sires separated alone **(F)** and together **(J)** or as a whole family **(N)**, ∗∗∗*P <* 0*.*001 equally; between sires separated together **(J)** and as a whole family **(N)**, ∗∗*P <* 0*.*01; between sires separated alone **(F)** and co-housed with unrelated dams **(T)**, not significant (n.s.); sires separated as a whole family **(N)** and co-housed with unrelated dams **(T)**, ∗*P <* 0*.*05.

#### **Table 2 | Percentages of sire's or dam's exhibiting retrieval behavior during the postpartum period.**

*Number of mice tested are shown in parentheses. \*, \*\*Significantly different from day 1, \*P < 0.05 and \*\*P < 0.01, respectively, two-tailed Fisher's exact* **Table 3 | Number of USVs recorded from cages of sires co-housed with different types of females for 10 min.**

*USVs (with >40 ms in duration) were recorded in n pairs.*

*\*Judging from the previous results (Liu et al., 2013), 38-kHz USVs appear to be emitted from pairmate dams and 30–80-kHz USVs from sires.*

*\*\*P < 0.01 or \*\*\*P* = *0.001, from pairmate dams, unrelated dams or virgin females, respectively, two-tailed Student's t-test.*

and stayed with the family (pups and pairmate dam) for 3 days (**Figure 3F**), the rate of retrieval in their home cage was only 4% (*n* = 25; **Figures 3G–J**). Although the sire lived together with the family for 3 days, such treatment made no contribution to the formation of paternity (two values in **Figure 3O** were equally very low; no significance, two-tailed Fisher's exact probability test).

To further analyze the relevance of family interaction during the stay as a whole family on postnatal days 3–5 (P3–P5) (**Figure 3**), we used the short-term pup exposure method (twice for 3 h for a total 6 h a day; **Figure 4**) to acquire or learn the process of paternity for the family. Males were isolated in new cages prior to parturition (**Figures 4B,C**) and kept in the cages for 2 days (**Figures 4D,E**). Then, pairmate dams and pups were relocated to the male's cage, and the whole family was kept there for 3 days (**Figure 4F**). Retrieval behavior was displayed by 8 (62%) of 13 sires (**Figures 4G–J**). The high level of retrieval appears to

*probability test.*

have been caused by continuously living in new cages that had been established as the male's territory.

remained until postnatal day 2 (P2). The male was kept continuously in the new cage until P2 **(E)**. From P3 to P5, the sire was introduced to the

In this suitable condition, we examined whether the presence of the dam was necessary for parental behavior by the isolated males. From P3 to P5, the pups and dam were kept together in their original home cages (**Figure 4K**), but the pups were temporarily transferred to the sire's cage twice for 3 h (a total of 6 h) per day (**Figure 4L**), and the males were otherwise alone for the rest of the day (18 h; **Figure 4M**). These sires showed retrieval behavior at a very high rate (17 (85%) of 20 sires tested; **Figures 4O–Q**). In both cases, the sires displayed a very high frequency of retrieval after living as the whole family or only with pups shortly in new cages that had, nevertheless, been established as the territory and established nest of the male, although no significant differences were observed between two types of sire (J and Q in **Figure 4R**; not significant, two-tailed Fisher's exact probability test). Furthermore, these results indicated that direct interaction with the mate dam is not necessary if the home territory is established by the sires.

Finally, we further examined the impact of territorial information on male retrieval behavior. Family cues were learned by individual sires in a manner identical to that shown in **Figure 4** (**Figures 5A–F**) during P3–P5, but in this case, via short exposure by transferring of their biological pups with their dams in new cages to the nursing cage with the sires. Then, retrieval behavior was examined under two housing conditions: in the sire's home cage in which the sire had stayed continuously (**Figures 5F,G,I–L**), or in a new cage (to the sires) in which the mate dams and pups had been staying (**Figures 5G,H,M–P**). In the home cages, 10 (50%) of 20 sires showed retrieval (**Figure 5L**), whereas only 3 (15%) of 20 sires in new cages displayed retrieval behavior (*P <* 0*.*05 between testing in old (L) and new (P) cages shown in **Figure 5Q**, two-tailed Fisher's exact probability test). In both cages, nests were established by the sire and dam. However, the new cages established by the sires' mate dams were quite new to the sires, even if the cages were fully filled with the mate dam's olfactory information.

values in O are equally very low: no significance (n.s.) between **(J)** and

**(N)**, two-tailed Fisher's exact probability test.

## **DISCUSSION**

The studies described here were performed to test several hypotheses that had not been explored previously (Akther et al., 2013; Fujimoto et al., 2013; Liu et al., 2013), pertaining to the various conditions responsible for parental behaviors other than the communicative interaction between sires and dams. Four findings are of particular interest: (1) among the mouse strains tested, the mate-dependent paternal retrieval behavior was observed only in

her pups **(D)** and remained until postnatal day 2 (P2) **(D)**. The male was kept in a new cage before meeting the pups **(C)** and kept until P2 **(E)**. The dam and pups were introduced in the sire's own (new) cage and stayed as a whole family until P5 **(F)**. Instead of the whole family, in another experiment, only pups in home cages with their dams **(K)** were transferred retrieval behavior was scored **(J,Q)**. The numbers of positive mice/number of mice tested are shown in parentheses, and the numbers of sires displaying retrieval were expressed as percentages **(R)**. Note that the retrieval rate in two cases **(J,Q)** was high enough to have no significance (n.s.), two-tailed Fisher's exact probability test.

the ICR strain (**Table 1**), and acquisition of such paternal behavior increased slowly following parturition of the dam (**Table 2**); (2) the ICR sires displayed parental retrieval behavior only for their own biological pups (**Figure 1**); (3) interaction between the sires and unrelated non-mating dams is not effective (**Figure 2**) and does not involve 38-kHz USVs (**Table 3**); (4) it is important for the sire to establish its home cage (territory) by continuous housing to display parental retrieval behavior (**Figures 3**–**5**).

After separation from pups in the home or new cages with the sires alone or together with the pairmate dam, the sires displayed retrieval behavior, as shown in **Figures 2J,N**, in agreement with previous reports (Liu et al., 2013). We designated this behavior of the sire as mate-dependent parental behavior. In the present study, this particular behavior was specific to the ICR strain and was not observed in two other laboratory strains, i.e., C57BL/6 and BALB/c mice. Therefore, the ICR strain's mate-dependent retrieval is not a general behavior observed equally in all mice but is strain-specific. However, this does not reduce the value of our findings because the observed paternal behavior is unique. Furthermore, when considering human society, human males are not completely and genetically predisposed to display parental behavior. In this context, the behavior of the ICR strain may be a more suitable and novel model for investigating paternal behavior, comparing the genetically determined paternity, observed in animals such as voles or California mice (de Jong et al., 2009; Ahern et al., 2011).

The ICR sires displayed parental retrieval behavior only for their biological pups, indicating that they can discriminate between their biological and non-biological offspring. This discrimination likely depends on odor or USV (Kuroda et al., 2007, 2011). The characteristic 38-kHz USVs were not recorded during co-housing of ICR sires with non-mate dams, suggesting that the sires can distinguish the mate from non-mate dams or that the dams can distinguish the mate from non-mate sires. These results strongly support our suggestion that 38-kHz USVs are critical and have context for sires to induce retrieval behavior.

In these experiments, we examined the olfactory information of pups and cages (homes) for the sires prior to separation from the mate dam and their offspring. In habituation as a family, the presence of the mate was not completely essential. Interestingly, we estimated that the territory information is much more important to sires than the pheromones in the cages once they had established their home cage. Surprisingly, when the sires were continuously housed in their newly established home

**FIGURE 5 | Paternal retrieval test in ICR mice isolated prior parturition from the mating pair and then united with pups only.** A paired couple was kept in a rearing cage from mating to 1 day before parturition **(A)**. The male was kept in the old cage **(B)** before meeting the pups and kept until P2 **(D)**. The female was kept in a new cage **(C)**, delivered her pups, and remained until postnatal day 2 **(E)**. The pups were transferred twice for 3 h (total 6 h) a day **(G)** from the dam's (new) cage **(H)** during P3 to P5. During

cages, they displayed paternal retrieval. In sharp contrast, if the cage was new to the sire, even though the dam's and sire's olfactory information was there, the sire failed to display retrieval behavior. These observations suggested that territory establishment is critical to maintaining paternity (Wright and Brown, 2000).

Pup retrieval as a parental behavior is rare among laboratory mice that are not genetically monogamous (Wright and Brown, 2000; Kalueff et al., 2007). We found conditions in which the ICR sires retrieved their pups related to their family structure. This unique ability of the ICR sires will contribute to the increased survival rate after reproduction and to the high level of social attachment and interaction. We have recently reported that central cholinergic cellular signaling (Fujimoto et al., 2013) and CD38 and oxytocin signaling in the nucleus accumbens (NAcc) (Akther et al., 2013) are critical for the expression of paternal care of the ICR mice. We also demonstrated the modulatory roles of the mPOA and VP on parental behavior in rodents (Akther et al., 2014). These published findings suggest that the neural circuitry mediating paternal behavior includes the mPOA, VTA, NAcc, and VP, and may be similar to those that mediate maternal behavior as proposed by Numan and others (Numan et al., 2005; Lee and Brown, 2007; Wynne-Edwards and Timonin, 2007; Numan and Stolzenberg, 2009). In addition, it is particularly interesting to test if mPOA galanin neurons regulate mate-dependent parental behavior in the ICR strain (Wu et al., 2014). Further neuroendocrinological and neurocircuitry analyses in ICR mice will be useful for understanding disorders with social impairment, such as autism spectrum disorders and schizophrenia (Insel, 2010; Munesue et al., 2010; Riebold et al., 2011; Feldman et al., 2012; Salmina et al., 2013).

## **AUTHOR CONTRIBUTIONS**

two-tailed Fisher's exact probability test).

Haruhiro Higashida designed experiments. Mingkun Liang, Jing Zhong, Hong-Xiang Liu, Olga Lopatina, Ryusuke Nakada, Agnes-Mikiko Yamauchi, and Haruhiro Higashida performed animal experiments. Haruhiro Higashida and Mingkun Liang wrote the manuscript.

the rest of the time, the sires stayed alone in the home cages **(F)** and the dams were with the pups **(H)**. Pup retrieval behavior over a period of 10 min was examined at P5 (**I–L** and **M–P**, respectively). The number of sires displaying retrieval behavior was scored **(L,P)**, and the numbers of positive mice/number of mice tested are shown in parentheses. Pup retrieval was expressed as percentages (**Q**; ∗*P <* 0*.*05 between old **(L)** and new **(P)** cages,

## **ACKNOWLEDGMENTS**

This work was supported in part by the Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Agency and by the Strategic Research Program for Brain Sciences from Ministry of Education, Culture, Sports, Science, and Technology, Japan.

## **REFERENCES**

**Conflict of Interest Statement:** 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.

*Received: 24 February 2014; accepted: 11 June 2014; published online: 11 July 2014. Citation: Liang M, Zhong J, Liu H-X, Lopatina O, Nakada R, Yamauchi A-M and Higashida H (2014) Pairmate-dependent pup retrieval as parental behavior in male mice. Front. Neurosci. 8:186. doi: 10.3389/fnins.2014.00186*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience.*

*Copyright © 2014 Liang, Zhong, Liu, Lopatina, Nakada, Yamauchi and Higashida. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Effects of early life adverse experiences on the brain: implications from maternal separation models in rodents

## *Mayumi Nishi\*, Noriko Horii-Hayashi and Takayo Sasagawa*

*Department of Anatomy and Cell Biology, Nara Medical University, Kashihara, Japan*

#### *Edited by:*

*Tomoko Soga, Monash University Sunway Campus, Malaysia, Malaysia*

#### *Reviewed by:*

*David Walker, Ritchie Centre, Australia*

*Kumi Kuroda, RIKEN, Japan*

#### *\*Correspondence: Mayumi Nishi, Department of Anatomy and Cell Biology, Faculty of Medicine, Nara Medical University, Kashihara, Nara 634-8521, Japan e-mail<EMAIL_ADDRESS>
During postnatal development, adverse early life experiences affect the formation of neuronal networks and exert long-lasting effects on neural function. Many studies have shown that daily repeated maternal separation (MS), an animal model of early life stress, can regulate the hypothalamic-pituitary-adrenal axis (HPA axis) and affect subsequent brain function and behavior during adulthood. However, the molecular basis of the long-lasting effects of early life stress on brain function has not been fully elucidated. In this mini review, we present various cases of MS in rodents and illustrate the alterations in HPA axis activity by focusing on corticosterone (CORT). We then show a characterization of the brain regions affected by various patterns of MS, including repeated MS and single time MS at various stages before weaning, by investigating c-Fos expression. These CORT and c-Fos studies suggest that repeated early life stress may affect neuronal function in region- and temporal-specific manners, indicating a critical period for habituation to early life stress. Next, we introduce how early life stress can impact behavior, namely by inducing depression, anxiety or eating disorders, and alterations in gene expression in adult mice subjected to MS.

**Keywords: maternal separation, HPA axis, depression, corticosteroid, gene expression, behavior, epigenetics**

## **INTRODUCTION**

As our contemporary society changes rapidly, changes in family structure can have a large influence on the mother–child relationship, as well as on other social environmental factors. In adult patients with various neuropsychiatric disorders, childhood abuse including sexual and/or physical abuse and neglect, is one of the most serious causes (Bremne and Vermetten, 2001; Heim and Nemeroff, 2001; Teicher et al., 2006). Adverse experiences occurring during critical periods of development, such as perinatal life, harmfully influence behavior, and physiological functions, including growth, metabolism, reproduction, and immune responses. Stressful environments in early life may induce permanent rather than transient consequences in animals. Previous studies have indicated that early unfavorable events augment the risk of behavioral disorders in adulthood, including neuropsychiatric disorders, such as depression (Kendler et al., 2002) and psychosis (Morgan et al., 2007). In rodent and primate models, adverse environments during the neonatal periods seem to play a critical role in developing the brain systems important to regulate behavior and stress responsiveness. In particular, the responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis can be deteriorated by interrupting usual mother-pup interactions, which may induce persistent changes in the neurobiology, physiology, and emotional behavior in adult animals (Ellenbroek et al., 1998; Lyons et al., 1998; Pryce et al., 2005; Enthoven et al., 2008; Nishi et al., 2013).

In this mini review, we will focus on the response of corticosterone (CORT), an end product of the HPA axis in rodents, and c-Fos expression for examining the activated brain regions induced by maternal separation (MS), a model of rodent early life stress. Furthermore, we will also present alterations of behavioral aspects and alterations in gene expression.

## **EARLY MS**

The inventive studies of Levine and colleagues, and consequently of Meaney, Plotsky, and their collaborators have demonstrated that changes in rodents' early postnatal experiences can induce profound long-lasting effects on emotionality and stress response (Levine, 1967; Meaney, 2001; Plotsky et al., 2005), which have spurred the employment of the rodent MS for investigating early life stress. This early life stress model is based on the evidence that unfavorable events in early life cause the vulnerability for developing various kinds of diseases in later life. In this type of study, MS should be carefully discussed in comparison to the appropriate control group, which may or may not be undisturbed from mother.

The procedure of MS showed a variety of the duration (e.g., 60 min–24 h) and the number of days (e.g., 1–14 days, 15–21 days) for the separation experiences among laboratories (Biagini et al., 1998; Caldji et al., 2000; Barreau et al., 2004; Arborelius and Eklund, 2007; Carrera et al., 2009; Tjong et al., 2010). In MS paradigm, many experiments, but certainly not all, have demonstrated that separation of pups from their mothers during the early postnatal period permanently increased anxiety-like behaviors in adulthood (Francis et al., 1999; Huot et al., 2001, 2004; Menard et al., 2004). As to the HPA axis activity, the response to stress is relatively low during early postnatal life (Walker et al., 1991; Levine, 2005), while MS could lead to life-long hyperactivity of the HPA axis (Holmes et al., 2005; Lippmann et al., 2007; Aisa et al., 2008; Marais et al., 2008). In contrast, short-term disturbance (e.g., 15 min), which has been called "handling," appeared to reduce anxiety-like behaviors, decrease HPA axis tone and reduce the response to stress in adulthood (Levine, 2005; Plotsky et al., 2005). The process of handling may imitate natural mice rearing, whereby the mother leaves her pups for short periods of time to collect foods. Thus, the short-term MS, handling, might be considered a more natural event.

The effect of MS also varies depending upon whether pups are separated in a group of littermates during MS or isolated singly. Miyazaki and colleagues recently reported that rat pups isolated singly from the mother during PND7 to PND11 presented disturbance of cortical function, whereas pups separated but gathered from PND7 to PND11 showed no cortical disruption (Miyazaki et al., 2012).

## **CHARACTERIZATION OF MATERNALLY SEPARATED ANIMALS**

#### **SERUM LEVEL OF CORT**

In rodents, there is an unique period during which the HPA axis shows a rapid regression known as the stress hyporesponsive period (SHRP) (Levine, 2001). This period extends from PND4 to PND14 in rats and from PND2 to PND12 in mice. During the course of SHRP, ACTH in increased and baseline plasma glucocorticoid levels are lower than normal (Rosenfeld et al., 1991). Because, during ontogeny, the maintenance of low and stable levels of CORT is necessary for normal growth and development of the central nervous system (CNS), the SHRP is hypothesized to be neuroprotective against stress-induced excessive stimulation of glucocorticoid receptors (GRs) (Sapolsky and Meaney, 1986; Sapolsky, 1996). In rodents, the presence of the mother appears to suppress HPA axis activity, which primarily preserves the SHRP. Indeed, even during the SHRP, MS is a compelling inducer of a stress response. Meaney and his colleagues suggest that the quality of the mother-pup interactions, such as increased maternal licking, grooming, and arched-back nursing, is an important aspect for the preservation of this dampened HPA axis activity (Francis et al., 1999). The disturbance of SHRP induced by MS could cause an excessive exposure of the brain to high concentrations of glucocorticoids and activation of GRs, which may subsequently regulate brain and behavior in later life. Enhanced secretion of stress-induced CORT was observed in pups separated from their mothers for 1 h on PND2 to PND9 (McCormick et al., 1998). Nevertheless, a recent study indicated that repeated MS for 8 h daily from PND3 to PND5 rapidly desensitized the HPA axis activity of neonatal mice (Enthoven et al., 2008). We also reported that repeated MS for 3 h daily from PND1 to PND14 did not elevate a baseline level of CORT on PND14, whereas a single-time MS for 3 h at PND14 raised a baseline CORT level (**Figure 1**) (Horii-Hayashi et al., 2013). In contrast to the effects of MS on neonatal animals, repeated MS for 3 h daily from PND1 to PND14 significantly raises a CORT level in adulthood, as reported by many studies (Ryu et al., 2008; Jahng et al., 2010; Horii-Hayashi et al., 2013).

### **ACTIVATED BRAIN REGIONS ANALYZED BY c-FOS EXPRESSION**

The expression of the immediate early gene product c-Fos is a reliable molecular marker to investigate neuronal activation. The examination of c-Fos expression has revealed that many brain regions are activated by MS, which differs depending on age and the type of stress. We recently analyzed the c-Fos expression induced by repeated MS and single-time MS during different developmental stages and time periods. Mice were exposed to 3 h repeated MS daily from PND1 to PND14 or from PND14 to PND21, or to single-time MS at PND14 or PND21 (Horii-Hayashi et al., 2013). We clarified that MS activated many brain regions and that c-Fos expression patterns

changed developmentally (**Figure 2**). Single-time MS at both ages activated many regions of the hypothalamus and limbic forebrain, while the pattern of c-Fos expression in the repeated MS groups were significantly different on PND14 and PND21. In repeated MS of PND14 mice, the c-Fos expression levels in many regions were markedly increased compared with age-matched controls, excepting the VMH, Arc, BST, DG, Ce, MePV, and MePD. By contrast, in repeated MS on PND21 mice, c-Fos expression was reduced to control levels in all observed brain regions except for the LS and CA3. These findings suggest that repetition of a homotypic stimulus suppresses c-Fos expression by PND21, but that such suppression is barely observed on PND14. Moreover, in animals exposed to repeated homotypic stress during the postnatal period, increase in adrenal CORT secretion does not always associate with increased c-Fos expression in the PVN. Such developmental differences in c-Fos expression detected in the repeated MS groups may be associated with a developmental critical period for stress responses involving the HPA axis, during which animals are more susceptible to MS and other environments. In rodents, the critical period is the first two postnatal weeks. Thus, in early life, a repeated stress will be unlikely to suppress c-Fos expression. In turn, inappropriately activated c-Fos target genes may drastically alter how neurons function in critical neural circuits. Indeed, the suppression of increased c-Fos expression in repeated MS of PND14 mice was observed in specific regions (BST, Ce, MePD, and MePV) that form anatomical neural connections. These regions are referred to as an extended amygdala, which are closely associated with anxiety, fear, and psychiatric disorders (Davis et al., 2010). Therefore, even at PND14, repeated homotypic stress may reduce neural activity in the circuit of the extended amygdala. Moreover, in the SFO, where neurons are influenced by osmolality, calcium, and sodium concentrations in the systemic circulation (Smith and Ferguson, 2010), c-Fos expression was increased in both repeated and single-time MS mice, as compared to controls, on PND14. However, there were no changes in any of the groups

group). In both RMS and SMS, the sampling point is just after MS procedure. ∗*P <* 0*.*05 vs. control; #*P <* 0*.*05 vs. RMS. MPO, medial preoptic area; PVN, paraventricular nucleus; SFO, subfornical organ; DM, dorsomedial hypothalamic nucleus; VMH, ventromedial hypothalamic

anterior part of the basolateral amygdaloid nucleus; Ce, central amygdaloid nucleus; MePD, posterodorsal part of the medial amygdaloid nucleus; MePV, posteroventral part of the medial amygdaloid nucleus; Pir, piriform cortex.

on PND21. This difference may reflect the increased resistance of physical growth to the hyperosmolality induced by deprivation of lactation.

### **BEHAVIORAL CHANGES INDUCED BY MS IN RODENTS**

Early life adverse experiences including MS is one of the greatest contributing factors for mental health problems across life stages (Levine, 2005), relating not only to risk for mental health disorders but also to transdiagnostic features common in many psychological disorders (Glaser et al., 2006). I will introduce some of the behavioral aspects observed in animal model of MS.

## *Depression- and anxiety-like behaviors*

Numerous studies have demonstrated a strong relationship between traumatic events during early life and development of behavioral abnormalities later in life. Early life adversity, such as that induced by MS, child physical, sexual, and emotional abuse, and general neglect has been linked to serious psychiatric impairment in adulthood (MacMillan et al., 2001). Particularly, a stressful life event such as early parental loss is associated with unipolar and bipolar depression, as well as anxiety disorders, beyond familial or genetic factors (Kendler et al., 1992; Agid et al., 1999; Furukawa et al., 1999; Heim and Nemeroff, 2001). Many human studies have reported that major depression and anxiety disorders are frequent in adults with a history of childhood abuse (Stein et al., 1996; Felitti et al., 1998). There have been numerous reports of the behavioral changes induced by MS in animal studies. Neonatal MS induces permanent alterations in the characteristics of the HPA response to stress in the offspring later in life (Ladd et al., 1996; Vazquez et al., 2000). Many studies of repeated MS during the first 2 weeks of neonatal life showed depression- and anxiety-like behaviors in adulthood (Newport et al., 2002; Daniels et al., 2004; Lee et al., 2007; Ryu et al., 2009). In these studies, ambulation and rearing decreased, immobility during a forced swim test increased, and time spent in the closed arms of an elevated plus maze increased.

## *Fear response*

Until recently, no one had investigated how early experiences affected fear retention and extinction development, although these forms of emotional learning could be critically involved in the pathogenesis and treatment of mental health problems. Recent several studies showed that the timing of the maturation of fear learning is not set in static, but can be dynamically regulated by early experiences. Although the exact mechanisms are still unknown, when rats are reared under stressful conditions then they exhibit adult-like fear retention and extinction behaviors at an earlier stage of development (Callaghan et al., 2013). Chocyk et al. reported that MS decreased freezing time in both contextual and auditory fear conditioning in adolescent and adult rats (Chocyk et al., 2014). These results suggest that early life stress may permanently affect fear learning and memory.

#### *Food intake and response to food deprivation*

Previous studies showed that repeated MS during the first 2 weeks after birth may not permanently affect food intake and body weight gain of the offspring as long as the pups are reared in a group (Iwasaki et al., 2000; Kalinichev et al., 2002; Ryu et al., 2008). In contrast, post-weaning social isolation promotes food intake and weight gain of adolescent MS pups, with impacts on anxiety-like behaviors (Ryu et al., 2008). Anhedonia to palatable food, one of the major symptoms of depression, was reported in adolescent MS pups with disruption of the mesolimbic dopaminergic activity in response to stress (Noh et al., 2008). Another study showed that sustained hyperphagia observed in the MS pups subjected to a fasting/re-feeding cycle repeated during adolescent period of MS pups induced a binge-like eating disorder, in which increased activity of the HPA axis responding to such metabolic challenges appeared to play a role, at least partly, in mediation with the hypothalamic neuro peptide Y (NPY) (Jahng, 2011).

## **GENE EXPRESSION**

Many animal studies, including MS, have improved our knowledge of gene-environment interactions and elucidated the pathways that program an animal in response to its early life experiences (Meaney and Szyf, 2005). Epigenetic mechanisms involving DNA methylation, post-translational modification of histone proteins and non-coding RNAs (most notably micro-RNA) are major candidates for regulating gene expression and integrating intrinsic and environmental signals in the genome (Jaenisch and Bird, 2003). Murgatroyd and colleagues showed that in the parvocellular subdivision of the paraventricular nucleus of the hypothalamus, MS in mice persistently upregulates *Avp* gene expression associated with reduced DNA methylation of a region in the *Avp* enhancer. This early life stress-responsive region serves as a binding site for the methyl-CpG binding protein 2, which in turn is regulated through neuronal activity. They also found that the ability of methyl-CpG binding protein 2 to control transcription of the *Avp* gene and induce DNA methylation occurred by recruiting components of the epigenetic machinery (Murgatroyd et al., 2009; Murgatroyd and Nephew, 2013). Other groups investigated DNA methylation levels at a specific sequence motif upstream of the GR gene (Nr3c1) in the hippocampus of offspring, and found that subjecting pups to a single 24 h MS increases methylation levels (Kember et al., 2012). The epigenetic alterations of these genes suggest that the HPA axis could be dysregulated by MS. Importantly, however, the DNA methylation differences were also often strain specific (Kember et al., 2012). Taken together, these findings demonstrate the importance of investigating environmental effects on a range of genetic backgrounds, emphasizing the need for the further examination of environmental, genetic, and epigenetic interactions.

## **CONCLUSIONS**

Adverse environments and experiences during the neonatal period can dramatically affect the development of the HPA axis that underlies adaptive behavioral responses. MS experiments, as a model of early life stress, demonstrate that CORT levels and c-Fos expression change depending upon the different experimental conditions of MS, e.g., age at testing and frequency of repetition. Furthermore, separation conditions (isolation with or without a littermate) could also influence the results of the MS experiments. MS can induce various behavioral changes manifested in later life, which could be caused, at least in part, by alterations in gene expression, particularly through epigenetic mechanisms.

## **ACKNOWLEDGMENTS**

This work was supported by Grants-in-Aid for Scientific Research (23390040 to Mayumi Nishi and AstraZeneca Research Grant 2009). We thank Dr. Julian G. Mercer, a chief editor of J Neuroendocrinology, for permitting the reuse of our own figures published in J Neuroendocrinology.

## **REFERENCES**

**Conflict of Interest Statement:** 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.

*Received: 24 February 2014; accepted: 30 May 2014; published online: 17 June 2014. Citation: Nishi M, Horii-Hayashi N and Sasagawa T (2014) Effects of early life adverse experiences on the brain: implications from maternal separation models in rodents. Front. Neurosci. 8:166. doi: 10.3389/fnins.2014.00166*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience.*

*Copyright © 2014 Nishi, Horii-Hayashi and Sasagawa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Developmental changes in the neural responses to own and unfamiliar mother's smiling face throughout puberty

Tsunehiko Takamura, Shota Nishitani, Takashi Suegami, Hirokazu Doi, Masaki Kakeyama and Kazuyuki Shinohara\*

*Unit of Basic Medical Sciences, Course of Medical and Dental Sciences, Department of Neurobiology and Behavior, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan*

#### Edited by:

*Sonoko Ogawa, University of Tsukuba, Japan*

#### Reviewed by:

*Craig H. Kinsley, University of Richmond, USA Jakob Frie, Karolinska institute, Sweden*

#### \*Correspondence:

*Kazuyuki Shinohara, Unit of Basic Medical Sciences, Course of Medical and Dental Sciences, Department of Neurobiology and Behavior, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, 852-8523 Nagasaki, Japan<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience*

Received: *09 October 2014* Accepted: *21 May 2015* Published: *03 June 2015*

#### Citation:

*Takamura T, Nishitani S, Suegami T, Doi H, Kakeyama M and Shinohara K (2015) Developmental changes in the neural responses to own and unfamiliar mother's smiling face throughout puberty. Front. Neurosci. 9:200. doi: 10.3389/fnins.2015.00200* An attachment relationship between boys and their mother is important for subsequent development of the ability to sustain peer relationships. Affective responses to attachment figure, especially mother, is supposed to change drastically during puberty. To elucidate the neural correlates underlying this behavioral change, we compared the neural response of boys at three different developmental stages throughout puberty to visual image of their own mothers.  We measured their neural response in the anterior part of the prefrontal cortex (APFC) to their own mother's smile compared with an unfamiliar-mother's. We found that in response to their own mother's smiling, the right inferior and medial part of the APFC (Ch6) was activated in the pre-puberty group. By contrast, the left inferior and medial (Ch4) and superior (Ch2 and Ch5) APFC were activated in the middle-puberty group, which is presumably linked to empathic feelings fostered by memories of mutual experience with own mother. These findings suggest that different patterns of APFC activation are associated with qualitative changes in affective response to own mother around puberty.

Keywords: attachment relationship, development, near-infrared spectroscopy (NIRS), prefrontal cortex (PFC), puberty

## Introduction

The attachment relationship between mother and child is crucial for the ability to maintain an intimate relationship with others (Scaramella and Leve, 2004; Kriss et al., 2012). As an attachment figure, the presence of a care-giver, and in particular a mother, endows children with psychological security, and they seek physical and mental proximity to their mother for protection and emotional support when under threat (Bowlby, 1969).

During puberty, many maturational changes in physical appearance occur, which are derived from secondary sexual characteristics, changing relationships with family members, reorganization of sexual and social identity, and self-perception (Mendle et al., 2007).  Indeed, around puberty, children tend to spend more time with peers than family (Csikszentmihalyi et al., 1977; Steinberger, 1989) and are more concerned about maintaining peer relationships (Koepke and Denissen, 2012), and consequently become more independent from their parents (Fujisawa et al., 2012). Nevertheless, the mother continues to be an important figure. For example, deterioration of the maternal relationship during this period can increase the risk for emotional and behavioral problems in later life (Brumariu and Kerns, 2010).

Recently, several studies have identified neural correlates of positive affect that are induced in early childhood (de Haan and Nelson, 1997; Carver et al., 2003; Carlsson et al., 2008; Minagawa-Kawai et al., 2008; Nakato et al., 2011; Dai et al., 2014) by images of a child's own mother. Of particular relevance, Minagawa-Kawai et al. (2008) reported that infants show increased activation in the anterior prefrontal cortex (APFC), including the orbitofrontal cortex, in response to the smile of his/her mother, which presumably suggests that a mother's smile has rewarding value to her infant.

Importantly, recent studies have suggested that the mesolimbic reward circuit, which is strongly linked to the APFC, is reorganized throughout puberty (Todd et al., 2011). As already stated, developmental psychology literature has noted a qualitative change in the relationship between mother and child around puberty. On the basis of these findings, it is plausible to suggest that APFC activation to a mother's smile may change dynamically throughout puberty. However, most previous studies on maternal neural responses (de Haan and Nelson, 1997; Carver et al., 2003; Carlsson et al., 2008; Minagawa-Kawai et al., 2008; Nakato et al., 2011; Dai et al., 2014) have focused on early developmental stages (from early infancy to preschool years). Thus, despite the dramatic physiological and psychological changes that occur during puberty, to the best of our knowledge, no study has investigated the developmental course of maternal neural representation around puberty.

Hence, the overall aim of our study was to investigate the developmental course of APFC activation to a mother's smile. We examined different developmental stages throughout puberty, a time when the mother and child relationship changes markedly, as it is a period of rebelliousness during which attachment styles are altered. In addition, brain structures develop and are reorganized due to dynamic changes in sex steroid hormone levels during this period. Using near-infrared spectroscopy (NIRS), we compared PFC activation in response to a mother's smile in boys at three developmental stages. To exclude the confounding factor of facial familiarity, we adapted the experimental and analytic procedures reported in Minagawa-Kawai et al. (2008). Specifically, in each trial, neutral faces of the child's own mother and an unfamiliar woman were presented as baseline stimuli, and brain activation induced by the target smiling face was calculated by subtracting baseline oxyhemoglobin (oxyHb), which should reduce familiarityrelated bias (Minagawa-Kawai et al., 2008).

We focused on the neural substrate of boys, because girls begin to show menstrual changes in sex steroid hormones after puberty. These developmental and menstrual hormonal changes in girls makes it difficult to control a number of parameters, and does not allow accurate assessment of developmental changes in APFC activation during puberty.

## Materials and Methods

## Participants

A total of 102 healthy and naive pubertal boys participated (**Table 1**). The participants were classified into three groups: pre-, middle-, and post-puberty according to chronological age. The groups largely corresponded to Tanner stages (TS) (Marshall and Tanner, 1970; Fujieda, 1993), although sexual maturity was not assessed by the Tanner scale, which is based on physical examination. Pre-puberty boys were recruited from the 3rd grade of elementary school (approximately 9 years of age), and classified into the early stage of sexual maturity (approximately TS1). Boys at the 2nd and 3rd grade of junior high school (approximately 14 years of age) were recruited as middlepuberty boys, and classified into the middle stage of sexual maturity (approximately TS3). Boys at the 1st and 2nd grade of university (approximately 20 years of age) were recruited as postpuberty boys, and classified into the late stage of sexual maturity (approximately TS5). At all developmental stages, participants were recruited from several schools in different geographical areas of Nagasaki prefecture in Japan, representing students from a range of socioeconomic backgrounds. All subjects were righthanded on the basis of the Edinburgh Handedness Inventory (Oldfield, 1971).  All participants and their parents gave written informed consent after being informed of the purpose of the experiment. The experimental protocol was in accord with the tenets of the Helsinki Declaration, and was approved by the Ethics Committee of the Nagasaki University Graduate School of Biomedical Sciences.

## Stimuli

To prepare the visual stimuli, neutral and smiling facial expressions from the participants' mothers were recorded for approximately 3 min in a quiet experimental room prior to NIRS recordings, using digital video camera (GZ-MG40; Victor). To record a smiling face, the mother was asked to smile as if talking to her child: raise the corners of her mouth so that her smiling expression was most expressive, and look straight at the camera to provide eye contact stimuli (Ekman et al., 1990). To record a neutral face, the mother was asked not to

*Mean* ± *standard deviation.*

show any facial expression. The video camera was positioned approximately 135 cm in front of the mother. Each video image was edited using Canopus Edius J (Thomson Canopus Co., Ltd., Japan) to obtain 30 s video stimuli of the mother with neutral and smiling expressions. To control physical characteristics among the mothers, each video image was edited using the following criteria: (1) the gaze was fixed approximately on the center; (2) the upper part of the body was visible; and (3) the image was recorded against a white background. Video stimuli were presented with no sound because of high variance in auditory information. To standardize facial expression across participants, several movie recordings were made, and the stimulus video created using recordings in which typical and defining smile features described by Ekman et al. (1990) were clearly visible.

## Procedure

#### Visual Presentation Task

During NIRS recordings, participants passively viewed videos of own (i.e., own condition) and unfamiliar (i.e., unfamiliar condition) mother's smiling. The video of smiling face was always preceded by the video of neutral expression of the same person. Their own mother's face was also used as an unfamiliar mother's face for another participant in the same developmental group. Thus, each mother's face was used once in the own and unfamiliar conditions. Video stimuli were presented on a monitor (17 in) located at a distance of approximately 50 cm from the participants.

After NIRS probe placement, each participant performed the visual presentation task with NIRS recordings taken. The task began with a blank screen showing a black background. A white hairline cross was presented for 30 s, and then the video stimulus of their own mother's neutral face was presented for 30 s as baseline period. Then, the video stimulus of the same mother's smiling face was presented for 30 s. After 30 s of a blank screen, a white hairline cross was again presented. Another video stimulus of an unfamiliar mother's neutral face was then presented for 30 s, followed by the same mother's smiling face. Finally, after 30 s, the unfamiliar mother's smiling face disappeared and 30 s of blank screen was presented.

For half of all participants, the unfamiliar mother's face was presented prior to the own mother's face to control residual presentation order effects. Participants had no task to perform and were asked to simply watch the video stimuli on the screen. None of the participants could identify the person presented as the unfamiliar mother.

#### NIRS Recordings

During the visual presentation task, hemoglobin concentrations were measured at a sampling rate of 0.5 Hz using the 10- Ch NIRS system (NIRO-200; Hamamatsu Photonics, Japan; wavelengths 775, 810, and 850 nm, pathlength 18 cm). The modified Lambert–Beer law was used for calculating the oxyHb and deoxyhemoglobin (deoxyHb) concentration changes. NIRS probes were attached to the forehead according to the International 10–20 electrode system used in electroencephalography (EEG), such that a horizontal line through Fp1-Fpz-Fp2 matches the lowest two detectors in our NIRS system. Two emitters and eight detectors were aligned, as previously reported (Kida and Shinohara, 2013; Kida et al., 2014), resulting in 10 recording sites (channels). This position enabled assessment of the APFC. Our previous study using the same position (Kida and Shinohara, 2013) demonstrated spatial registration of the NIRS probe and channel locations using the NFRI toolbox (Okamoto and Dan, 2005; Singh et al., 2005) implemented in NIRS\_SPM software (Ye et al., 2009), and identified the corresponding Brodmann's area.

#### Stimulus Evaluation

After NIRS recordings, participants were asked to rate favorable impressions of own and unfamiliar faces on a visual analog scale (VAS) of 0–10 (0 = none and 10 = most), specifically, the happiness and comfort they felt while viewing the faces in each condition.

### Salivary Testosterone Collection and Analysis

Saliva samples were collected from each participant into small polypropylene tubes, during 11:00 and 14:00 on the day of the experiment to control for diurnal variation in testosterone concentrations. Saliva samples were frozen and stored at −80◦C. Testosterone was assayed in duplicate by ELISA (Salimetrics, State College, USA). The intra-assay variation coefficient was 8.9%, while the inter-assay variation coefficients for high and low controls were 7.0 and 14.0%, respectively.

#### NIRS Data Analysis

Before the statistical analysis, we identified the trials and channels that included artifactual fluctuation with sharp change of oxyHb and deoxyHb concentration. Following the precedence of the previous fNIRS studies (Peña et al., 2003; Takizawa et al., 2008), we identified waveform fluctuation as artifact whose point-topoint concentration change was larger than 5µM/L·m. Note that this is more stringent criterion than those adopted in the previous studies (Peña et al., 2003; Takizawa et al., 2008). We further checked the results by visual inspection of the waveforms.

The data from participants whose data included artifacts in more than three channels were discarded from the final analysis. Other exclusion criteria included the failure to obtain mother's facial stimuli (3 participants), equipment failure (2 participants), and excessive head and bodily movement (12 participants). 17 participants were excluded from the analysis, resulting in 27 prepuberty, 31 middle-puberty, and 27 post-puberty boys (**Table 1**).

Here, we mainly focus on the results based on oxyHb concentration changes, as we consider this the most sensitive parameter of hemodynamic responses (Malonek et al., 1997; Hoshi et al., 2001; Strangman et al., 2002; Shimada et al., 2005; Doi et al., 2013). At the same time, we also present deoxyHb results for completeness.

In quantifying the concentration change, mean concentration of oxyHb/deoxyHb during the last 20 s of the baseline was first calculated for each individual. Then, the concentration change was computed by subtracting the mean concentration during the baseline from that during the last 20 s of the smile presentation (Schroeter et al., 2004; Kida et al., 2014). All the statistical analyses were conducted with PASW Statistics 18.0 (SPSS Inc., IL USA).

## Results

## Concentration Change of oxyHb Overall Developmental Trend

Changes in oxyHb concentration in response to mother's smiling face were analyzed by Three-Way repeated measures analysis of variance (ANOVA) with channel (10 channels) and condition (own or unfamiliar) as within-participant factors, and developmental stage (pre-, middle-, or post-puberty group) as the between-participant factor. A significant interaction among the three factors [F(18, 738) = 1.70, p = 0.03] was found, while the other main effects or interactions were not statistically significant (ps > 0.20). This indicates that there are differential patterns of neural activation to own and unfamiliar mother's smiles across the three age groups. To examine this Three-Way interaction, we performed a Two-Way repeated measures ANOVA separately for each developmental stage, with withinparticipant factors of channel (10 channels) and condition (own or unfamiliar).

## Pre-puberty Group

In the pre-puberty group (**Figure 1A**), the main effects of channel [F(9, 234) = 1.50, p = 0.15] and condition [F(1, 26) = 0.07, p = 0.79] were not significant, but there was a significant interaction between them [F(9, 234) = 1.97, p = 0.04] (**Figure 1B**). Subsequent analysis showed that for Ch3 [F(1, 260) = 5.87, p = 0.02], the unfamiliar condition exhibited a significantly greater oxyHb increase than the own condition, while for Ch6 [F(1, 260) = 3.93, p = 0.05] (**Figures 2A,B**), the own condition yielded a significantly greater oxyHb increase than the unfamiliar condition. Furthermore, for Ch8 [F(1, 260) = 2.90, p = 0.09], the oxyHb increase was marginally significant. No significant condition effect was found for the other channels (ps > 0.43) (**Figure 1B**). In summary, in the pre-puberty group, oxyHb increased in the right medial and inferior APFC in response to their own mother's smile compared with an unfamiliar mother. In contrast, in the left APFC, oxyHb increased more in response to an unfamiliar mother's smile than their own.

## Middle-puberty Group