l -Amino acid decarboxylase- and tyrosine hydroxylase-immunoreactive cells in the extended olfactory amygdala and elsewhere in the adult prairie vole brain

Journal of Chemical Neuroanatomy 43 (2012) 76–85

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L-Amino

acid decarboxylase- and tyrosine hydroxylase-immunoreactive cells in the extended olfactory amygdala and elsewhere in the adult prairie vole brain Eman I. Ahmed, Katharine V. Northcutt 1, Joseph S. Lonstein * Neuroscience Program, 108 Giltner Hall, Michigan State University, East Lansing, MI 48824, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 August 2011 Received in revised form 25 October 2011 Accepted 26 October 2011 Available online 2 November 2011

Neurons synthesizing dopamine (DA) are widely distributed in the brain and implicated in a tremendous number of physiological and behavioral functions, including socioreproductive behaviors in rodents. We have recently been investigating the possible involvement of sex- and species-specific THimmunoreactive (TH-ir) cells in the male prairie vole (Microtus ochrogaster) principal bed nucleus of the stria terminalis (pBST) and posterodorsal medial amygdala (MeApd) in the chemosensory control of their monogamous pairbonding and parenting behaviors. These TH-ir cells are not immunoreactive for dopamine-beta-hydroxylase (DBH), suggesting they are not noradrenergic but possibly DAergic. A DAergic phenotype would require them to contain aromatic L-amino acid decarboxylase (AADC) and here we examined the existence of cells immunoreactive for both TH and AADC in the pBST and MeApd of adult virgin male and female prairie voles. We also investigated the presence of TH/AADC cells in the anteroventral periventricular nucleus (AVPV), medial preoptic area (MPO), arcuate nucleus (ARH), zona incerta (ZI), substantia nigra (SN) and ventral tegmental area (VTA). Among our findings were: (1) the pBST and MeApd each contained completely non-overlapping distributions of TH-ir and AADC-ir cells, (2) the AVPV contained surprisingly few AADC-ir cells and almost no TH-ir cells contained AADC-ir, (3) approximately 60% of the TH-ir cells in the MPO, ARH, and ZI also contained AADC-ir, (4) unexpectedly, only about half of TH-ir cells in the SN and VTA contained AADC-ir, and (5) notable populations of AADCir cells were found outside traditional monoamine-synthesizing regions, including some sites that do not contain AADC-ir cells in adult laboratory rats or cats (medial septum and cerebral cortex). In the absence of the chemical requirements to produce DA, monoenzymatic TH-ir cells in the virgin adult prairie vole pBST, MeApd, and elsewhere in their brain may instead produce L-DOPA as an end product and use it as a neurotransmitter or neuromodulator, similar to what has been observed for monoenzymatic THsynthesizing cells in the laboratory rat brain. ß 2011 Elsevier B.V. All rights reserved.

Keywords: Catecholamine Prairie vole Dopamine L-DOPA Monogamy Olfaction

1. Introduction Prairie voles (Microtus ochrogaster) are unusual mammals because their social organization includes hallmarks of monogamy including pairbonding after mating, biparental care of offspring, and alloparenting of younger siblings by non-reproducing adults (Carter et al., 1995; Young et al., 2011). Neurochemicals necessary for the display of these social behaviors in prairie voles include the neuropeptides vasopressin and oxytocin, as well as the neurotransmitter dopamine (DA) (Carter et al., 2008; Phelps et al., 2010; Ross and Young, 2009; Young et al., 2011). The essential role for DA in prairie vole social bonding is evident by the ability of peripheral or intra-accumbens administration of a D2 receptor antagonist to

* Corresponding author. Tel.: +1 517 353 8675; fax: +1 517 432 2744. E-mail address: [email protected] (J.S. Lonstein). 1 Present address: Department of Biology, Mercer University, Macon, GA 31207, USA. 0891-0618/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2011.10.006

block partner preferences in mated prairie voles, while D2 receptor agonism forges social preferences even without the need for mating (Aragona et al., 2003; Gingrich et al., 2000; Wang et al., 1999). Furthermore, stimulating the second messenger systems downstream of the D1 receptor inhibits partner preferences while downregulating them (such as that seen after D2 agonism) promotes social bonding (Aragona et al., 2006; Aragona and Wang, 2007). With regards to parenting, DA receptor blockade with the mixed D1/D2 receptor antagonist haloperidol suppresses pup licking and general contact with the litter in both sexes of prairie voles, although the neural sites of action for this effect remain unknown (Lonstein, 2002). Almost all studies of DA’s effects on social behaviors in prairie voles have focused on the mesocorticolimbic system (Young et al., 2011). Known and putative DA-synthesizing cells are found throughout the vertebrate forebrain (Smeets and Gonza´lez, 2000), however, and project to brain areas essential for socioreproductive functions (e.g., Cheung et al., 1998; Li et al., 1993; Lindvall and Stenevi, 1978; Miller and Lonstein, 2009; Northcutt

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and Lonstein, 2011; Palkovits et al., 1977). In this context, it is particularly interesting that in male prairie voles the densely interconnected principal nucleus of the bed nucleus of the stria terminalis (pBST) and posterodorsal medial amygdala (MeApd) each contain hundreds of neurons intensely immunoreactive for tyrosine hydroxylase (TH), the enzyme catalyzing the conversion of tyrosine to the dihydroxyphenylalanine (DOPA) necessary for catecholamine synthesis (Northcutt et al., 2007; Northcutt and Lonstein, 2009, 2011). These TH-immunoreactive (TH-ir) cells are not immunoreactive for dopamine beta-hydroxylase (DBH) (Northcutt et al., 2007) so we have proposed they may instead be dopaminergic (DAergic). In addition, because the numerous other adult rodents that have been examined have few or no TH-ir cells in these sites, we have proposed these cells contribute to male prairie voles’ monogamous behaviors (Northcutt et al., 2007; Northcutt and Lonstein, 2009, 2011). This may occur through pBST and MeApd integration of olfactory and hormonal information, which is known to be necessary for many socioreproductive behaviors in mammals (Coolen and Wood, 1998; Newman, 1999; Numan and Insel, 2003; Wood and Swann, 2005). In support, we previously found that general social contact maintains basal immediate-early gene (IEG) expression in TH-ir cells of the male prairie vole pBST and MeApd and that mating to ejaculation is particularly potent (compared to other social stimuli including pups) in further increasing this IEG expression (Northcutt and Lonstein, 2009). Many of the TH-ir cells in the male prairie vole pBST and MeApd project to the medial preoptic area (Northcutt and Lonstein, 2011), creating a unique TH-containing neural network among sites essential for social discrimination, copulation, parenting, and aggression (see Newman, 1999). Although it is intriguing to suggest that TH-ir cells projecting from the male prairie vole pBST and MeApd to the MPO comprise a newly discovered DAergic system necessary for monogamous behaviors in this species, it is unknown if these TH-ir cells contain the full complement of enzymes necessary to produce DA. Indeed, many TH-ir cells in the brains of other mammals do not synthesize DA or other catecholamines (Bjo¨rklund and Lindvall, 1984) because they do not contain the enzyme aromatic L-amino acid decarboxylase (AADC) necessary to convert LDOPA to DA. The distribution of these TH-positive/AADC-negative cells is generally similar across species (Jaeger et al., 1984a,b; Karasawa et al., 2007; Kitahama et al., 1988, 1990a,b; Skagerberg et al., 1988) and, instead of DA, these cells are thought to produce LDOPA as an end product and utilize it as a neurotransmitter and neuromodulator (see Misu and Goshima, 2006; Ugrumov, 2009). To better understand the phenotype and possible function of the tremendous number of TH-ir cells in the male prairie vole pBST and MeApd, we here used dual-label immunohistochemistry to examine the overlap between the distribution of TH-ir cells and AADC-ir cells in these brain sites. We also examined other brain sites that more traditionally contain TH-ir cells, including the anteroventral preoptic area (AVPV), medial preoptic area (MPO), arcuate nucleus (ARH), zona incerta (ZI), substantia nigra (SN) and ventral tegmental area (VTA). Lastly, we describe numerous populations of AADC-containing cells distributed widely outside these TH-synthesizing regions of the brain. 2. Methods 2.1. Subjects Subjects were adult male and female prairie voles (M. ochrogaster) born and raised in our colony at Michigan State University, which originated with voles captured in Urbana, IL and last outbred in 2000 with voles from Illinois. Voles were housed in a 14:10 light:dark cycle with an ambient temperature of 21 8C. After weaning at 20 days old, subjects were housed in mixed-sex groups of littermates in clear plastic cages (48  28  16 cm) containing wood chips, wood shavings, and a covering of hay. Voles were later rehoused at least 10 days before sacrifice into

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cages containing only their same-sex siblings. Water and a food mixture containing cracked corn, whole oats, sunflower seeds, and rabbit chow (Tekland rodent diet No. 2031; Harlan, Madison, WI) in a 1:1:2:2 ratio were provided ad libitum. All procedures were performed in accordance with the Institutional Animal Care and Use Committee at Michigan State University and the EU Directive 2010/63/EU for animal experiments. 2.2. Tissue collection and immunohistochemistry Voles were overdosed with sodium pentobarbital and perfused through the heart with 100 ml 0.9% saline, followed by 100 ml 4% paraformaldehyde in 0.1 M sodium phosphate buffer (NaPB; pH = 7.6). Brains were removed and postfixed overnight in 4% paraformaldehyde, and then submerged in 20% sucrose in NaPB for at least two days before the forebrain and midbrain of each were cut into 35 mm sections on a freezing microtome. TH and AADC dual-immunohistochemistry was performed on every other section throughout the brain using methods standard in our laboratory (Cavanaugh and Lonstein, 2010; Northcutt et al., 2007; Northcutt and Lonstein, 2011). The freefloating sections were rinsed three times with 0.1 M Trisma-buffered saline (TBS; pH = 7.4) between each incubation. Sections were incubated in 0.1% sodium borohydride in TBS for 15 min, incubated in 1% hydrogen peroxide in 0.3% Triton X100 in TBS for 10 min, blocked with 20% normal goat serum (NGS) in 0.3% Triton X100 in TBS for 30 min, and incubated overnight at room temperature with rabbit anti-AADC polyclonal antiserum (1:1000, AB1569; Millipore, Temecula, CA). The next day, sections were rinsed in TBS then incubated in goat anti-rabbit biotinylated secondary antiserum in 2% NGS and 0.3% Triton X-100 in TBS for 1 h at room temp (1:500; Vector Laboratories) and incubated in avidin–biotin complex (Vectastain Elite; Vector Laboratories, Burlingame, CA) in TBS for 1 h. Immunoreactivity was visualized with 0.05% 3,30 -diaminobenzidine (Sigma) and 0.01% hydrogen peroxide in TBS, which produced a light brown reaction product. After rinsing, sections were re-blocked with 20% NGS and 0.3% Triton X-100 in TBS, and incubated overnight in a mouse anti-TH primary monoclonal antiserum (1:2000, MAB318; Chemicon, Temecula, CA) in 2% NGS and 0.3% Triton X-100 in TBS at room temperature. Sections were then incubated in a goat anti-mouse secondary antiserum (1:500; Vector Laboratories) in 2% NGS and 0.3% Triton X-100 in TBS for 1 h, followed by 1 h in avidin–biotin complex in TBS. TH was visualized using Vector SG (Vector Laboratories), which provided a light blue label. Omission of the primary or secondary antisera abolished specific immunohistochemical labeling. After tissue processing, sections were mounted onto slides, dehydrated, and coverslipped. We previously demonstrated using Western blot analysis that the TH antiserum used here detects a single band at the expected size in the prairie vole forebrain and midbrain, and this band is at the same location as that found in the laboratory rat forebrain and midbrain (Northcutt and Lonstein, 2011). A recent Western blot analysis performed in our laboratory using the AADC antiserum from the present study verified a band at the expected size (55 kD) in the forebrain and midbrain of both prairie voles and laboratory rats, as well as a very heavy band in both species at 110 kD that is likely the dimerized AADC protein found in central nervous system tissue (Kubrusly et al., 2008; Siow and Dakshinamurti, 1990; Zhu and Juorio, 1995). Control blots that omitted either the primary or secondary antisera did not contain any immunoreactive bands. 2.3. Microscope analysis Slides were coded so the single observer (EIA) was blind to subject sex. Brain sections containing eight TH-rich regions of interest (pBST, MeApd, AVPV, MPO, ARH, ZI, SN and VTA) were examined bilaterally with a Nikon E400 light microscope at up to 200 magnification with the aid of a reticle in one ocular lens. The areas of analysis were standardized for each brain site, remained consistent for each subject, and covered the area where TH-ir cells are found within each site of interest. The area of analysis for each forebrain site was similar to what we have done in our previous work studying TH-ir cells in the prairie vole brain (Cavanaugh and Lonstein, 2010; Lansing and Lonstein, 2006; Northcutt et al., 2007; Northcutt and Lonstein, 2011) and involved three sections in the series through the pBST, four sections through the MeApd, three sections through the AVPV, three sections through the MPO, and four sections each through the ARH and ZI (Fig. 1). The VTA was analyzed from four sections and SN from three sections corresponding to approximately Plate 38 from Swanson’s (1998) atlas of the rat brain (Fig. 1). The location and number of cells in each section that contained TH immunoreactivity (contained blue reaction product), AADC immunoreactivity (contained brown reaction product), and both immunoreactive products were recorded and totaled across sections. Cells were considered double-labeled only if their cytoplasm contained unmistakably overlapping blue and brown immunoreactivity. Three to six voles per sex were examined for each brain site (see Table 1), although females are mentioned below only for the two sites where sex differences were detected (pBST, MeApd). Some additional brain regions were examined because they contain cells immunoreactive for AADC (but not TH) in other small mammals (Jaeger et al., 1984a,b; Kitahama et al., 1988, 1990a,b), or we noticed large populations of AADC-ir cells that had not been previously described in any animal. These sites were outside our primary interest in TH-rich brain areas, but because there has never been any examination of AADC expression in the prairie vole brain,

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Fig. 1. Schematic representation of areas of the (A) pBST, AVPV, and MPO, (B) ARH and ZI, (C) MeApd, and (D) VTA and SN where cells containing TH-ir and AADC-ir were quantified (indicated by black boxes). Areas are shown unilaterally but were analyzed bilaterally. aco = anterior commissure, AQ = cerebral aqueduct, cpd = cerebral peduncle, fr = fasciculus retroflexus, fx = fornix, int = internal capsule, ME = median eminence, ml = medial lemniscus, mtt = mammillothalamic tract, och = optic chiasm, opt = optic tract, st = stria terminalis, VL = lateral ventricle, V3 = third ventricle. Figure modified from Swanson (1998).

Table 1 Mean number ( SEM) of cells in the prairie vole brain singly labeled with TH immunoreactivity, singly labeled with AADC immunoreactivity, labeled with both immunoreactive products, and the percentage of all TH-immunoreactive cells that also contain AADC immunoreactivity quantified bilaterally from 3 or 4 brain sections per site (see Section 2 for details). No sex differences were found outside the pBST and MeApd, so data shown for other sites are collapsed across sex. Site

n

# TH-ir only cells

# AADC-ir only cells

# Dual-labeled cells

% all TH-ir cells also AADC-ir

pBST

3 (male) 3 (female) 3 (male) 3 (female) 6 6 10 10 12 10

122  12 40  13 115  31 14  10 210  9 28  4 27  6 116  13 98  15 125  13

66  15 24  5 90  5 22  1 0a 66  12 82  13 137  25 162  29 149  35

0 0 0 0 0a 52  11 44  5 143  11 101  15 99  13

0 0 0 0 0 62  5 64  5 56  5 52  5 44  3

MeApd AVPV POA ARH ZI VTA SN a

Group average < 1 cell.

we provide a descriptive analysis of AADC-ir cells in these sites. To further confirm the distribution of AADC-ir cells, single-label AADC immunohistochemistry was performed as described in Section 2.2 on an alternate series of brain sections obtained from three male and three female prairie voles from our colony. No atlas of the prairie vole brain exists so another set of alternate brain sections from three male and three female prairie voles were Nissl-stained to help confirm anatomical boundaries with reference to Swanson’s (1998) atlas of the rat brain and Morin and Wood’s (2001) atlas of the hamster brain.

3. Results 3.1. AADC expression in brain regions containing TH-ir cells As we previously reported, a very large number of TH-ir cells existed in the pBST of male prairie voles, while relatively few TH-ir cells were found in the pBST of females (Cavanaugh and Lonstein,

2010; Northcutt et al., 2007; Northcutt and Lonstein, 2008, 2011). AADC-ir cells were also found in the pBST of both sexes of prairie vole, although males had almost three times as many AADC-ir cells in their pBST than did females (Table 1). Cells lightly immunoreactive for AADC were intermingled with TH-ir cells, but no cells contained both TH-ir and AADC-ir. A particularly dense accumulation of cells intensely immunoreactive for AADC could be seen adjacent to the fornix (medial to most TH-ir cells in the prairie vole rostral pBST and our area of quantification) (Fig. 2). Visual examination of sections in the pBST caudal to our area of quantification revealed large population of AADC-ir cells between the fornix and stria terminalis, but almost all were dorsal to the THir cells at this level and no dual-labeled cells were found. The MeApd also contained cells immunoreactive for TH or AADC, and similar to the pBST, both types of cell were much greater

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Fig. 2. Photomicrograph of cells containing immunoreactivity for TH (blue cytoplasmic labeling) or immunoreactivity for AADC (brown cytoplasmic labeling) in the pBST of a representative male prairie vole. No dual-labeled cells were found in the pBST. aco = anterior commissure; fx = fornix; LV = lateral ventricle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

in number in males compared to females. Also similar to the pBST, no dual-labeled cells were found in the MeApd. AADC-ir cells were more likely to exist dorsomedial to where TH-ir cells are found in the male prairie vole MeApd, although we noticed that quite a few AADC-ir cells were also found ventromedial to the TH-ir population (Fig. 3; Table 1). As we previously reported, the AVPV of both sexes of prairie vole contained a similarly large number of TH-ir cells (Lansing and Lonstein, 2006). Surprisingly, however, we found very few AADC-ir cells in the AVPV (some subjects had none) and each vole had either have very few or no cells immunoreactive for both antigens. In contrast, there were many AADC-ir cells in the nearby MPO and approximately half of TH-ir cells in the MPO contained AADC-ir (Fig. 4; Table 1).

Fig. 4. Photomicrographs of cells containing immunoreactivity for TH (blue cytoplasmic labeling) and AADC (brown cytoplasmic labeling) in the (A) AVPV and (B) MPO of representative male prairie voles. Some cells containing immunoreactivity for both TH and AADC in panel B are highlighted in black boxes and shown under higher magnification in panels C and D; note overlapping blue and brown immunoreactive products in these cells. 3 V = third ventricle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Photomicrograph of cells containing immunoreactivity for TH (blue cytoplasmic labeling) or immunoreactivity for AADC (brown cytoplasmic labeling) in the MeApd of a representative male prairie vole. No dual-labeled cells were found in the MeApd. opt = optic tract above. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

More than 60% of all TH-ir cells in the ARH contained AADCir, although it was apparent that dual-labeling was more abundant in the dorsal ARH compared to the ventral ARH (Fig. 5). A large number of ARH cells single-labeled for AADC was also observed. A similar percentage of all TH-ir cells in the ZI contained AADC-ir, although single-labeled cells of both

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for TH could be observed per section in both sites, particularly in the lateral SN at some levels, which greatly contributed to the lower overall percentage of double-labeled cells). Many cells containing only AADC-ir were also readily identified in both sites (Fig. 7; Table 1). 3.2. AADC expression in brain regions not containing large populations of TH-ir cells

Fig. 5. Photomicrograph of cells containing immunoreactivity for TH (blue cytoplasmic labeling, small arrows), AADC (brown cytoplasmic labeling), or both TH and AADC (large arrows) in the ARH of a representative male prairie vole. 3V = third ventricle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

An enormous number of AADC-ir cells were found in the prairie vole forebrain and midbrain outside the regions containing TH-ir cells. Jaeger et al. (1984b) described 11 such populations of these ‘‘D’’ cells in the rat midbrain and forebrain. Very similar to their results, AADC-ir cells could be found in the prairie vole dorsal periaqueductal gray (corresponding to the rat D4), pretectal region (D5), lateral habenula (D6; Fig. 8a), dorsal thalamus (D7; Fig. 8a), ventral premammillary region (D8; Fig. 8b), lateral ZI (D10), lateral and dorsomedial hypothalamus (D11 and D12), and suprachiasmatic nucleus (D13). Quite a few scattered AADC-ir cells also existed in each section through the medial nucleus accumbens and at the same rostrocaudal level in the superficial layers of the cerebral cortex, including the cingulate and somatosensory cortices (Fig. 9). Very few or no AADC-ir cells existed in each vole’s primary motor cortex and only a small number were seen in the piriform cortex. Moving caudally, the lateral preoptic area contained a handful of AADC-ir cells although a striking population containing hundreds of AADC-ir cells was found in the medial septum. This dense immunoreactivity extended into the nearby choroid plexus and lining of the lateral ventricles (Fig. 10). Another large population of AADC-ir cells was observed in the lateral midbrain surrounding the medial geniculate nucleus and extending ventromedially to the SN (Fig. 11). Lastly, hundreds of AADC-ir cells were seen through the dorsal and median raphe nuclei, likely corresponding to the B5–B8 serotonin cells groups (Dahlstrom and Fuxe, 1964). 4. Discussion

Fig. 6. Photomicrograph of cells containing immunoreactivity for TH (blue cytoplasmic labeling), AADC (brown cytoplasmic labeling), or both TH and AADC (both blue and black labeling) in the ZI of a representative male prairie vole. Some of the many cells containing only TH-ir at the border of the ZI are indicated by black arrows. fx = fornix. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

phenotypes were especially numerous at the ventral and lateral borders of the ZI (Fig. 6; Table 1). In the VTA, a little more than half of all TH-ir cells also contained AADC-ir (Table 1), and a smaller percentage (44%) of TH-ir cells in the SN were dual labeled (Table 1). Dozens of cells single-labeled

4.1. AADC and TH immunoreactivity in the pBST and MeApd The monogamous behaviors displayed by male prairie voles are relatively unique among mammals (Kleiman, 1977) and are driven by species-specific brain characteristics (Young et al., 2011). Many studies have revealed differences between monogamous and nonmonogamous mammals in neurochemical receptor distribution and density, but not in the source or release of the neurochemicals themselves. For example, the distribution and number of OT- or AVP-synthesizing cells and fibers do not differ between male prairie voles and promiscuous montane voles, but the density of OT

Fig. 7. Photomicrographs of cells containing immunoreactivity for TH (blue cytoplasmic labeling), AADC (brown cytoplasmic labeling) in the VTA and SN of a representative male prairie vole. Panel B is higher magnification of SN cells within black box on the background panel, with arrows showing some of the many SN cells containing only TH immunoreactivity. ml = medial lemniscus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 10. Photomicrograph of cells containing immunoreactivity for AADC (brown cytoplasmic labeling) in the medial septum of a representative male prairie vole. fx = fornix. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8. Photomicrographs of cells containing immunoreactivity for AADC (brown cytoplasmic labeling) in the (A) lateral habenula and paraventricular thalamus and (B) ventral premammillary region of a representative male prairie vole. 3Vm = third ventricle, mammillary recess; Lhb = lateral habenula; PVT = paraventricular nucleus of the thalamus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

and V1a binding in many brain sites does differ among vole species (Insel and Shapiro, 1992; Insel et al., 1994). Prairie voles also differ from promiscuous voles in their cortical and striatal DA receptor (Aragona et al., 2006; Smeltzer et al., 2006) and extended amygdalar estrogen receptor a expression (Cushing and WynneEdwards, 2006). Given that differences in the brains of monogamous and polygamous voles have only been found postsynaptically, our earlier finding that unmanipulated adult male prairie voles differed dramatically from promiscuous male meadow voles

(as well as male laboratory hamsters and rats) in the number of TH-ir cells in the pBST and MeApd was quite unexpected (Northcutt et al., 2007). Because many of the TH-ir cells in the prairie vole pBST and MeApd express estrogen and androgen receptors (Cavanaugh and Lonstein, 2010), increase TH synthesis in response to gonadal hormones (Cavanaugh and Lonstein, 2010; Northcutt et al., 2007), and in males show increased IEG expression particularly in response to mating with ejaculation compared to other social stimuli (Northcutt and Lonstein, 2009), we hypothesized that these TH-ir cells were involved in mating and subsequent formation or maintenance of socially monogamous pairbonds. Our recent analysis of the neuroanatomical projections of these TH-ir cells in prairie voles was consistent with this suggestion because almost half of all TH-ir cells in their pBST and MeApd project to a single site – the MPO (Northcutt and Lonstein, 2011). The role of the MPO in male prairie vole behavior has not been studied in detail, but its IEG expression significantly increases after

Fig. 9. Photomicrographs of cells containing immunoreactivity for AADC (brown cytoplasmic labeling) in the cerebral cortex of a representative male prairie vole. Panel B shows larger image of AADC-ir cells contained in black square in Panel A. cc = corpus callosum. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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example is that the arcuate nucleus of male Syrian hamsters (Mesocricetus auratus) housed in short photoperiods has approximately half the number of AADC-ir cells of their long photoperiod counterparts (Krajnak and Nunez, 1996), and this may be associated with changes in DA production and pituitary hormone release during the breeding period. How age and photoperiod influence AADC expression in TH-ir cells of the male prairie vole extended olfactory amygdala remain to be investigated, but we have found that TH-ir cells in their pBST and MeApd still do not contain AADC-ir when animals are examined either a few days after mating or when living in established pairbonds (Northcutt and Lonstein, unpublished observations). Instead of producing DA, the hundreds of TH-ir cells in the male prairie vole pBST and MeApd may produce other neurochemicals as their end product. L-DOPA itself acts as a neurotransmitter and neuromodulator when released from cells not containing AADC (Misu and Goshima, 2006; Ugrumov, 2009). A specific receptor for LDOPA has yet to be identified but L-DOPA does induce depolarization and glutamate release in a calcium-dependent manner from striatal neurons (Goshima et al., 1993). It also acts on presynaptic badrenergic receptors to influence DA release in the striatum and potentiates postsynaptic D2 receptor function in the striatum (Goshima et al., 1991; Hume et al., 1995; Misu et al., 1986). In other neural networks, L-DOPA affects release of norepinephrine, acetylcholine, and glutamate, as well as postsynaptic responses to GABA (Goshima et al., 1986, 1991, 1993; Honjo et al., 1999; Ueda et al., 1995). L-DOPA released by TH-synthesizing terminals may be taken up by neighboring AADC-synthesizing neurons to produce DA (Ugrumov et al., 2002), which may occur for the many TH-ir cells in the male prairie vole pBST and MeApd that terminate in the MPO (Northcutt and Lonstein, 2011) so that the cells can cooperatively produce DA. Analysis of DOPA immunoreactivity or extracellular DOPA in the prairie vole brain will help evaluate these possibilities, but it is clear that TH-ir cells in their pBST and MeApd can potentially influence the MPO and other brain sites via non-DAergic mechanisms that in other rodents directly affect both postsynaptic receptor activity and presynaptic neurotransmitter release. Fig. 11. Photomicrograph of cells containing immunoreactivity for AADC (brown cytoplasmic labeling) adjacent to the MGN of a representative male prairie vole. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

1–2 h of cohabitation or copulation with an unfamiliar female (Cushing et al., 2003; Lim and Young, 2004). This increase still exists 48 h later when pairbonds become more firmly established (Wang et al., 1997), and even weeks later it can rise again when males interact with their bonded mate (Gobrogge et al., 2007). DA release in the MPO facilitates sexual behavior in male rodents (Hull and Dominguez, 2007) and in monogamous species may be involved in the social memory necessary for partner preferences and pairbonding (Millan et al., 2007; Popik and van Ree, 1991). We previously found that none of the TH-ir cells in the prairie vole pBST and MeApd are immunoreactive for DBH (Northcutt et al., 2007), so are not noradrenergic, and based on that result it was possible they provided DAergic input to the MPO necessary for males’ social behaviors. However, the present study revealed that no TH-ir cells in the pBST or MeApd contained AADC-ir. It could be that AADC exists in the these cells at somal levels too low to be detectable using immunohistochemistry or perhaps within terminals of the TH-ir cells. Nonetheless, it is probably unlikely they provide DAergic innervation to anywhere in the brain under the physiological conditions studied here, but still may express AADC and produce DA under other conditions. For example, very few cells in the ARH of embryonic and early neonatal rats express both TH and AADC, but the number of bi-enzymatic neurons increases dramatically as the animals age (Ershov et al., 2002a,b). Another

4.2. AADC-ir in TH-ir cells of other brain regions In contrast to the pBST and MeApd, many or most TH-ir cells in traditional DA-synthesizing regions of the brain were found to contain AADC-ir. A large number of TH-ir cells in the prairie vole ARH were dual-labeled, particularly in the dorsal ARH, which is consistent with studies of the location of TH-ir, AADC-ir and DA-ir cells within the ARH of adult rats (Ershov et al., 2002a,b; Meister et al., 1988; Okamura et al., 1988; Zoli et al., 1993), guinea pigs (Smits et al., 1990), and primates (Karasawa et al., 2007; Kitahama et al., 1998). In the prairie vole ZI, notably more TH/AADC cells were found medially than laterally, which is also consistent with that observed in laboratory rats (Skagerberg et al., 1988; Jaeger et al., 1984b). However, Skagerberg et al. (1988) reported ‘‘almost total’’ or ‘‘complete’’ colocalization of AADC within TH-ir cells of the ZI, whereas we detected considerably lower colocalization in our prairie voles (56%), which could be related to methodological differences, including the colchicine treatment used by Skagerberg and colleagues that can inadvertently affect many neurochemical systems (Corte´s et al., 1990). We also found what appears on the surface to be a surprising number of cells in the VTA and particularly the SN that were singlelabeled with TH-ir. Comparing our observations in prairie voles with those made on other species is difficult without direct quantification in most studies, and although Jaeger et al. (1984a) stated that the majority of TH-ir cells in the rat SN contained AADCir, their Fig. 2 indicates many more TH-ir cells than AADC-ir cells at some levels in the SN (and in the VTA). Ikemoto et al. (1998)

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reported a ‘‘significant number’’ of TH-only cells especially at the margins of the human VTA, in addition to some in the SN, consistent with the finding that some human VTA neurons produce L-DOPA rather than DA (Komori et al., 1993). Monoenzymatic TH-ir cells have also been observed in the marmoset SN (Karasawa et al., 2007). In contrast, Kitahama and colleagues have reported that virtually all TH-ir cells in the human SN, and the cat SN and VTA, are immunoreactive for AADC (Kitahama et al., 1990a,b, 2009). The AVPV is a notable exception to the other sites we examined because it contained very few AADC-ir cells, and as a consequence, almost no TH+/AADC+ cells were detected. This differs from laboratory rats and guinea pigs, whose AVPV contains a number of AADC-ir cells that also express TH (Jaeger et al., 1984b; Lemoine et al., 2005), although the cells’ DA content cannot be detected without colchicine (Mons et al., 1990; Smits et al., 1990). The hamster AVPV contains individual AADC-ir cells and TH-ir cells (Vincent, 1988; Vincent and Hope, 1990) but it is unclear if these populations overlap. The AVPV of colchicine-treated cats holds many AADC cells, TH-ir cells, and DA-ir cells (Kitahama et al., 1988, 1990a,b). Interestingly, of the small number of mammals examined, prairie voles are most similar to male common marmosets (Callithrix jacchus), which also appear to have no AADC-ir cells in their AVPV (Karasawa et al., 2007) and are also often socially monogamous (Goldizen, 1988). In some species, the AVPV contains the rostral A14 DA cell group (Bjo¨rklund et al., 1975). DA released from these cells is thought to regulate pituitary gonadotropin release in laboratory rats and other mammals (Simerly et al., 1985). There is a sex difference in these TH-ir cells, such that female rats have 3–4-fold more TH-ir cells in their AVPV than do males (Simerly et al., 1985). We previously found that the prairie vole AVPV is unusual because both sexes have a similarly high number of TH-ir cells (Lansing and Lonstein, 2006). This lack of a sex difference in TH-ir cells and relative absence of AADC-ir cells in the prairie vole AVPV may be related to the fact that female prairie voles are induced ovulators, rather than spontaneous ovulators such as rats (Carter et al., 1980). Female cats are also induced ovulators (Brown, 2011), though, and do have AADC-ir, AADC-ir/TH-ir, and DA-ir cells in their AVPV (Kitahama et al., 1988, 1990a,b). Further, common marmosets that have no AADC-ir cells in their AVPV are not induced ovulators (Harlow et al., 1984). Similar to the pBST and MeApd discussed above, in the absence of the ability to independently synthesize DA, most TH-ir cells of the prairie vole AVPV may synthesize L-DOPA for use as a neurotransmitter or neuromodulator. 4.3. AADC-ir cells outside traditional catecholamine-synthesizing brain regions This study is the first to describe the presence of AADC-ir cells in putative catecholaminergic and non-catecholaminergic brain regions in the cooperative breeding and socially monogamous prairie vole. Although not intended to provide an exhaustive comparison, our results indicated many similarities – and a few differences – between prairie voles and laboratory rats, cats and primates in the distribution of cell groups containing only AADC-ir. Jaeger et al. (1984b) detailed 14 large populations of AADC-ir cells outside the traditional monoamine-synthesizing brain regions in laboratory rats, with 11 of these ‘‘D’’ cell groups in the forebrain and midbrain. Similar to their findings, we also observed clusters of AADC-ir cells in the periaqueductal gray (corresponding to the D4 group of laboratory rats), pretectal region (D5), lateral habenula (D6), dorsal thalamus (D7), ventral premammillary region (D8), lateral ZI (D10), lateral hypothalamus (D11), dorsomedial hypothalamus (D12), and suprachiasmatic nucleus (D13). Differences we observed between prairie voles and adult laboratory rats included: (1) the relative absence of AADC-ir cells in the prairie

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vole dorsocaudal ARH (D9 group in rats), (2) presence of numerous AADC-ir cells in the superficial layers of the prairie vole cortex, lateral preoptic area, medial nucleus accumbens, and surrounding the medial geniculate nucleus, and (3) a tremendous number of AADC-ir cells in the prairie vole medial septum and choroid plexus. AADC-ir cells in the lateral preoptic area, in or near the medial nucleus accumbens, and adjacent to the medial geniculate nucleus have not been seen in rats, but have been reported in cats (Kitahama et al., 1988, 1990a,b). The presence of AADC in the rat choroid plexus has previously been suggested (Jancso´, 1974). AADC-ir cells have been described in the human cingulate cortex (Ikemoto et al., 1999) and deep layers of the cerebral cortex of juvenile (but not adult) mice (Komori et al., 1991), but as far as we are aware, AADC-ir cells in the superficial layers of the lateral cortex and medial septum may be relatively unique to prairie voles. The cerebral cortex of voles is not well studied, but electrophysiological mappings shows a relatively expanded representation of the perioral region in the rostral somatosensory cortex (Campi et al., 2007). AADC-ir cells in and near the prairie vole somatosensory cortex may be associated with their high propensity for physical contact, the establishment and maintenance of pairbonds (DeVries and Carter, 1999), and the ability of tactile contact with mates to facilitate parenting behaviors (Simoncelli et al., 2010). Any functional significance of AADC-ir cells in the medial septum is unknown, but vasopressin or its V1a receptor antagonist infused at the border of the medial and lateral septum produce opposite effects on parental and pairbonding behaviors in male prairie voles (Liu et al., 2001; Wang et al., 1994), and relevant interactions between septal vasopressin fibers and AADC-containing cells in prairie voles may eventually be demonstrated (e.g., Ishida et al., 2002; Jaeger et al., 1983; Kontostavlaki et al., 2006). As is true for any species, despite the fact that seemingly thousands of AADC-ir cells lie outside TH-synthesizing brain regions in prairie voles, these cells can still influence brain monoamine function. First, AADC decarboxylizes amino acids to produce diffusible or releasable biogenic amines that influence DA and noradrenergic activity in the striatum and elsewhere in the brain (Berry, 2004). Second, in the presence of exogenous L-DOPA, AADC-containing cells in many areas of the brain (including the cat dorsal BST and MeA; Kitahama et al., 2007) produce detectable amounts of DA (Lidbrink et al., 1974; Karasawa et al., 1994; Mura et al., 1995; Kitahama et al., 2007; Ugrumov et al., 2002). Lastly, as mentioned above with regard to monoenzymatic cells in the pBST and MeApd, even under natural conditions AADC-containing cells anywhere in the prairie vole brain could cooperate with apposing TH-containing somata and terminals to synthesize physiologically relevant levels of DA (Ugrumov et al., 2002) and this may occur when additional DAergic output is required or when traditional monoaminergic cells are compromised (Ershov et al., 2005). Ethical statement The work described herein was carried out in accordance with the EU Directive 2010/63/EU for animal experiments and the Uniform Requirements for manuscripts submitted to Biomedical journals. Conflicts of interest The authors declare no conflicts of interest. Contribution All authors have materially participated such that all three of us contributed to the experiment conceptualization and design, Drs. Ahmed and Northcutt carried out different aspects of running the

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project, collecting the data and analyzing the results, and all three authors contributed to writing and preparing this manuscript. All authors have approved the final article. Acknowledgements The authors would like to thank Mr. Marcus Weera for his technical assistance with numerous aspects of this project. This research was supported in part by NSF grant #0515070 to JSL. References Aragona, B.J., Wang, Z., 2007. Opposing regulation of pair bond formation by cAMP signaling within the nucleus accumbens shell. J. Neurosci. 27 (48), 13352–13356. Aragona, B.J., Liu, Y., Curtis, J.T., Stephan, F.K., Wang, Z., 2003. A critical role for nucleus accumbens dopamine in partner-preference formation in male prairie voles. J. Neurosci. 23, 3483–3490. Aragona, B.J., Liu, Y., Yu, Y.J., Curtis, J.T., Detwiler, J.M., Insel, T.R., Wang, Z., 2006. Nucleus accumbens dopamine differentially mediates the formation and maintenance of monogamous pair bonds. Nat. Neurosci. 9, 133–139. Berry, M.D., 2004. 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