Heather K. Caldwell

Vasopressin 1a receptor knockout mice have a subtle olfactory deficit but normal aggression

Two receptors for vasopressin (Avp) are expressed in the brain, the Avp 1a receptor (Avpr1a) and the Avp 1b receptor (Avpr1b). To investigate the role of Avpr1a in behaviors in mice more extensively, we generated a line of mice lacking a functional Avpr1a (knockout, Avpr1a−/−). We first performed a baseline phenotypic screen of the Avpr1a knockouts followed by a more detailed analysis of their circadian rhythms and olfactory function. When free‐running in constant darkness, the Avpr1a−/− mice have a longer circadian tau than the wild types. There are also subtle olfactory deficits in Avpr1a−/− mice as measured in an olfactory habituation/dishabituation test and in the discrimination of female urine from male urine using an operant testing paradigm. An extensive body of research has shown that manipulation of the Avpr1a alters behavior, including aggression and social recognition. Therefore, we expected profound behavioral deficits in mice lacking the Avpr1a gene. Contrary to our expectations, social aggression, anxiety‐like behavior and social recognition are unaffected in this line of Avpr1a knockout mice. These data suggest either that the Avpr1a is not as critical as we thought for social behavior in mice or, more likely, that the neural circuitry underlying aggression and other social behaviors compensates for the life‐long loss of the Avpr1a. However, the olfactory deficits observed in the Avpr1a−/− mice suggest that Avp and Avpr1a drugs may affect behavior, in part, by modulation of chemosensory systems.

The vasopressin 1b receptor and the neural regulation of social behavior

To date, much of the work in rodents implicating vasopressin (Avp) in the regulation of social behavior has focused on its action via the Avp 1a receptor (Avpr1a). However, there is mounting evidence that the Avp 1b receptor (Avpr1b) also plays a significant role in Avps modulation of social behavior. The Avpr1b is heavily expressed on the anterior pituitary cortiocotrophs where it acts as an important modulator of the endocrine stress response. In the brain, the Avpr1b is prominent in the CA2 region of the hippocampus, but can also be found in areas such as the paraventricular nucleus of the hypothalamus and the olfactory bulb. Studies that have employed genetic knockouts or pharmacological manipulation of the Avpr1b point to the importance of central Avpr1b in the modulation of social behavior. However, there continues to be a knowledge gap in our understanding of where in the brain this is occurring, as well as how and if the central actions of Avp acting via the Avpr1b interact with the stress axis. In this review we focus on the genetic and pharmacological studies that have implicated the Avpr1b in the neural regulation of social behaviors, including social forms of aggressive behavior, social memory, and social motivation. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.

Lesions to the CA2 region of the hippocampus impair social memory in mice

The function of the CA2 region of the hippocampus is poorly understood. Although the CA1 and CA3 regions have been extensively studied, for years the CA2 region has primarily been viewed as a linking area between the two. However, the CA2 region is known to have distinct neurochemical and structural features that are different from the other parts of the hippocampus and in recent years it has been suggested that the CA2 region may play a role in the formation and/or recall of olfactory‐based memories needed for normal social behavior. Although this hypothesis has been supported by hippocampal lesion studies that have included the CA2 region, no studies have attempted to specifically lesion the CA2 region of the hippocampus in mice to determine the effects on social recognition memory and olfaction. To fill this knowledge gap, we sought to perform excitotoxic N‐methyl‐D‐aspartate lesions of the CA2 region and determine the effects on social recognition memory. We predicted that lesions of the CA2 region would impair social recognition memory. We then went on to test olfaction in CA2‐lesioned mice, as social memory requires a functional olfactory system. Consistent with our prediction, we found that CA2‐lesioned animals had impaired social recognition. These findings are significant because they confirmed that the CA2 region of the hippocampus is a part of the neural circuitry that regulates social recognition memory, which may have implications for our understanding of the neural regulation of social behavior across species.

The role of the vasopressin 1b receptor in aggression and other social behaviours.

While the importance of vasopressin (Avp) in the neuroendocrine regulation of behaviour is clear, most of Avps effects on behaviour have been linked to its action via its 1a receptor (Avpr1a) subtype. There is, however, emerging evidence and cross-species consensus that the vasopressin 1b receptor (Avpr1b) is also important in mediating the effects of Avp on behaviour. The Avpr1b is highly expressed in the anterior pituitary where it is thought to play a role in the neuroendocrine response to stress. The Avpr1b is also prominently expressed in the pyramidal cells of the CA2 hippocampal area. Interestingly, in mice, Avpr1b mRNA within the pyramidal neurons of the CA2 field is unaffected by restraint stress or adrenalectomy. Avpr1b knockout mice (--) have provided strong, consistent evidence that the Avpr1b plays a critical role in the regulation of social behaviour. Avpr1b(-/-) mice display reduced levels of social forms of aggression, reduced social motivation and impaired social memory (including the Bruce effect). Avpr1b(-/-) mice, however, have normal main olfactory ability, spatial memory and defensive and predatory behaviours. Mice lacking a functional accessory olfactory system display many of these same behavioural deficits, suggesting that Avpr1b(-/-) mice may have a deficit in the processing, perception and/or integration of olfactory stimuli detected by the accessory olfactory system. We suggest that the role of the Avpr1b is to couple socially relevant accessory olfactory cues with the appropriate behavioural response. Furthermore, given its prominence in the CA2 field of the hippocampus, we hypothesize that Avpr1b may be important for the formation or recall of memories that have an olfactory-based social component.

Normal Maternal Behavior, but Increased Pup Mortality, in Conditional Oxytocin Receptor Knockout Females

Oxytocin (Oxt) and the Oxt receptor (Oxtr) are implicated in the onset of maternal behavior in a variety of species. Recently, we developed two Oxtr knockout lines: a total body knockout (Oxtr-/-) and a conditional Oxtr knockout (OxtrFB/FB) in which the Oxtr is lacking only in regions of the forebrain, allowing knockout females to potentially nurse and care for their biological offspring. In the current study, we assessed maternal behavior of postpartum OxtrFB/FB females toward their own pups and maternal behavior of virgin Oxtr-/- females toward foster pups and compared knockouts of both lines to wildtype (Oxtr+/+) littermates. We found that both Oxtr-/- and OxtrFB/FB females appear to have largely normal maternal behaviors. However, with first litters, approximately 40% of the OxtrFB/FB knockout dams experienced high pup mortality, compared to fewer than 10% of the Oxtr+/+ dams. We then went on to test whether or not this phenotype occurred in subsequent litters or when the dams were exposed to an environmental disturbance. We found that regardless of the degree of external disturbance, OxtrFB/FB females lost more pups on their first and second litters compared to wildtype females. Possible reasons for higher pup mortality in OxtrFB/FB females are discussed.

Impairments in the initiation of maternal behavior in oxytocin receptor knockout mice.

Oxytocin (Oxt) acting through its single receptor subtype, the Oxtr, is important for the coordination of physiology and behavior associated with parturition and maternal care. Knockout mouse models have been helpful in exploring the contributions of Oxt to maternal behavior, including total body Oxt knockout (Oxt −/−) mice, forebrain conditional Oxtr knockout (Oxtr FB/FB) mice, and total body Oxtr knockout (Oxtr −/−) mice. Since Oxtr −/− mice are unable to lactate, maternal behavior has only been examined in virgin females, or in dams within a few hours of parturition, and there have been no studies that have examined their anxiety-like and depression-like behavior following parturition. To improve our understanding of how the absence of Oxt signaling affects maternal behavior, mood and anxiety, we designed a study using Oxtr −/− mice that separated nursing behavior from other aspects of maternal care, such as licking and grooming by thelectomizing (i.e. removing the nipples) of Oxtr +/+ mice and sham-thelectomizing Oxtr −/− mice, and pairing both genotypes with a wet nurse. We then measured pup abandonment, maternal behavior, and postpartum anxiety-like and depression-like behaviors. We hypothesized that genetic disruption of the Oxtr would impact maternal care, mood and anxiety. Specifically, we predicted that Oxtr −/− dams would have impaired maternal care and increased anxiety-like and depression-like behaviors in the postpartum period. We found that Oxtr −/− dams had significantly higher levels of pup abandonment compared to controls, which is consistent with previous work in Oxtr FB/FB mice. Interestingly, Oxtr −/− dams that initiated maternal care did not differ from wildtype controls in measures of maternal behavior. We also did not find any evidence of altered anxiety-like or depressive-like behavior in the postpartum period of Oxtr −/− dams. Thus, our data suggest that Oxt lowers the threshold for the initiation of maternal behavior.

Behavioural studies using temporal and spatial inactivation of the oxytocin receptor

Oxytocin (Oxt), synthesized in magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei for transport to and release from the posterior pituitary, is released during parturition and is essential for lactation. Lesser amounts of Oxt are made by smaller cells of the PVN and a few other forebrain nuclei and released into the central nervous system (CNS) to influence various other behaviours. In both the periphery and CNS, Oxt actions are transduced by the oxytocin receptor (Oxtr). Previously, it has been reported that Oxt(-/-) (knockout, KO) mice show a failure of milk ejection and thus are incapable of rearing their offspring. Unexpectedly, these mice have largely normal reproductive and maternal behaviours, perhaps due to compensatory mechanisms through activation of the Oxtr by vasopressin or through development. To examine the specific roles of the Oxtr during development and in particular brain areas, we created conditional Oxtr(-/-) mice in which we could control the spatial and temporal inactivation of the Oxtr. We flanked the neomycin-resistance selectable marker in an Oxtr intron with FRT sites to enable its removal using FLP recombinase. Coding sequence within exons 2 and 3 was flanked by two loxP sites enabling subsequent inactivation of the gene by targeted expression of Cre recombinase. The first Oxtr KO lines we created have either total or relatively specific forebrain elimination. The latter was achieved by crossing the conditional Oxtr line with a transgenic line in which the Camk2a promoter drives expression of Cre recombinase to significant levels beginning 21-28 days after birth, thus eliminating potential compensation for a deleted Oxtr gene during early development. This Cre-expressing line also significantly spares the main olfactory bulb reducing the potential confound of an olfactory deficit. We have investigated various behaviours, most notably social recognition, in both Oxtr KO strains (Oxtr(-/-) and Oxtr(FB/FB)).

Oxytocin, Vasopressin, and the Motivational Forces that Drive Social Behaviors

The motivation to engage in social behaviors is influenced by past experience and internal state, but also depends on the behavior of other animals. Across species, the oxytocin (Oxt) and vasopressin (Avp) systems have consistently been linked to the modulation of motivated social behaviors. However, how they interact with other systems, such as the mesolimbic dopamine system, remains understudied. Further, while the neurobiological mechanisms that regulate prosocial/cooperative behaviors have been extensively examined, far less is understood about competitive behaviors, particularly in females. In this chapter, we highlight the specific contributions of Oxt and Avp to several cooperative and competitive behaviors and discuss their relevance to the concept of social motivation across species, including humans. Further, we discuss the implications for neuropsychiatric diseases and suggest future areas of investigation.

Heightened aggressive behavior in mice with lifelong versus postweaning knockout of the oxytocin receptor.

Previous work implicating the neuropeptide oxytocin (Oxt) in the neural regulation of aggression in males has been limited. However, there are reports of heightened aggression in Oxt knockout and Oxt receptor (Oxtr) knockout male mice when they are born to null mutant mothers; suggesting that intrauterine exposure to Oxt may be important to normal aggression in adulthood. To explore this, we examined aggression in two lines of Oxtr mice, a total knockout (Oxtr-/-), in which the Oxtr gene is absent from the time of conception, and a predominantly forebrain specific knockout (Oxtr FB/FB), in which the Oxtr gene is not excised until approximately 21-28days postnatally. Aggression was measured in males from both lines, as well as control littermates, using a resident-intruder behavioral test. Consistent with previous reports, male Oxtr-/- mice had elevated levels of aggression relative to controls. Oxtr FB/FB mice on the other hand displayed levels of aggression similar to control animals. In addition, following a resident-intruder test, Oxtr+/+ mice that displayed aggression had less c-fos immunoreactivity in the ventral portion of the lateral septum than those that did not. Further, Oxtr-/- mice had increased c-fos immunoreactivity in the medial amygdala relative to controls. These data suggest that Oxt may play an important role during development in the organization of the neural circuits that underlie aggressive behavior in adulthood, with its absence resulting in heightened aggression.

Localization and quantification of 5-hydroxytryptophan and serotonin in the central nervous systems of Tritonia and Aplysia

Serotonin (5‐hydroxytryptamine, 5‐HT) plays a central role in several behaviors in marine molluscs and other species. In an effort to better understand the regulation of 5‐HT synthesis, we used high performance liquid chromatography (HPLC) with electrochemical detection and immunohistochemistry to measure and map the distribution of the immediate precursor of 5‐HT, 5‐hydroxytryptophan (5‐HTP), in two model opisthobranch molluscs, the nudibranch Tritonia diomedea and the anaspid Aplysia californica. HPLC measurements showed that 5‐HTP is present at approximately the same level as the 5‐HT metabolite, 5‐hydroxyindolacetic acid (5‐HIAA) but is more than 100 times lower in concentration than either 5‐HT or dopamine in the same tissue. Specific 5‐HTP immunoreactivity was colocalized with serotonin in both species. The overall intensity of 5‐HTP immunoreactivity in individual ganglia agreed with HPLC measurements for those ganglia. The intensity of 5‐HTP immunolabeling varied between cell types and was correlated with the intensity of 5‐HT immunolabeling. In particular, differences in staining intensity were consistently seen among the three dorsal swim interneurons of the Tritonia swim central pattern generator circuit. Some nonserotonergic neurons also displayed low levels of 5‐HTP immunolabeling that were above background levels. Together, these results support the notion that production of 5‐HTP is a rate‐limiting step in serotonin synthesis and suggest that there may be additional regulation that allows 5‐HTP to accumulate to varying levels. J. Comp. Neurol. 437:91–105, 2001.