Contributed By
Margaret McCarthy
University of Maryland
Phone: 410-706-2655
Fax: 410-706-8341
e-mail: mmccarth@umabnet.ab.umd.edu
The following was presented at the Breast Development Physiology and Cancer Conference which was held at the National Institutes of Health; June 26-28, 1997.
Slide 1 Breast development in the absence of appropriate behavior towards offspring to allow suckling would be of little consequence. In animal models, the onset of maternal behavior towards young is not a spontaneous event but rather requires hormonal induction. Systems have evolved such that many of the same hormones involved in breast development and lactation also act in the brain to coordinate peripheral physiological events with central control of behavior. Estrogen and progesterone exposure during pregnancy sensitize the neural substrate to the subsequent actions of oxytocin and prolactin which act in the brain to induce maternal behavior. The neural circuitry controlling maternal behavior has been well mapped and evidence suggests that expression of immediate early genes by neurons in this circuit is a critical mediator of the behavioral response. Preliminary research suggests that many of the same hormonal factors are involved in the maternal behavior of primates, including humans.
The most frequently used animal model for the hormonal control of maternal behavior has been the laboratory rat. Empirical tests generally consist of placing newborn pups at some distance from the nest site of a test female and then quantifying the latency to retrieval of the pups to the nest, grooming of the pups and crouching over them in a lactational posture. The combination of all these responses is taken as full maternal behavior. Latencies to show a full response are typically on the order of days and females are tested with fresh pups each day. Data are often expressed as the percent of females showing maternal responding.
The profile of plasma levels of estradiol and progesterone across the course of pregnancy is presented. Years of research by investigators such as Jay Rosenblatt, Harold Siegel and others have established that the precipitous decline of progesterone against a background of increasing levels of estradiol is a critical component of the onset of maternal behavior in the rat. During the normal course of pregnancy, the decline in progesterone begins one to two days prior to parturition.
In order for steroids to be influencing behavior they must be acting in the brain. Pictured here is a diagram of the preoptic area, a subdivision of the hypothalamus. The black circles represent neurons shown to contain estrogen receptor with the use of in vivo receptor autoradiography and 3H-estradiol. The seminal work of Michael Numan has established that the preoptic area is the major brain area regulating the onset of maternal behavior and the work of Susan Fahrbach and Don Pfaff has demonstrated that estrogen action in the preoptic area is both necessary and sufficient for the hormonal induction of maternal behavior.
When steroid hormones bind to their cogent receptors they act as transcription factors and thereby do not influence brain functioning directly. The currency of the brain is neurotransmission. The neurohypophyseal hormone oxytocin is released from the posterior pituitary into the circulation where it induces milk letdown and uterine contraction. However, the discovery that there is also a dense network of intracerebral oxytocinergic projections and that oxytocin can influence neuronal excitability, opened the possibility for an direct effect of oxytocin on behavior.
The first reports of oxytocin-induction of maternal behavior were from Cort Pedersen at University of North Carolina. These initial reports were subsequently replicated and extended by Susan Farhbach at the Rockefeller University. Note from the title shown here that the females were treated with estrogen before oxytocin exposure.
Determining the site of action of oxytocin requires knowing where the receptors are localized in the brain. One of the notable aspects of oxytocin receptor distribution in the brain is its tremendous variability between species. This is an autoradiogram of 125I OVTA (a specific oxytocin antagonist) in the mouse brain, which has a high level of binding in the septum. The pattern is markedly different in the rat brain, versus the vole, versus the hamster, versus the primate. The functional significance of this species variability is unknown, yet the evidence suggests that behaviorally, oxytocin exerts remarkably consistent effects across species, promoting a wide range of affiliative behaviors such as maternal behavior, sexual behavior, pair bonding etc.
This slide shows data from Susan Fahrbach and demonstrates the potent effect of exogenously administered oxytocin on reducing the latency for the expression of maternal behavior in estrogen-primed rats.
Susan carried this work one step further by demonstrating the importance of endogenous oxytocin to the normal expression of the behavior. Using either an antibody to oxytocin or an antagonist to the oxytocin receptor, the onset of maternal behavior in hormonally-primed females was significantly delayed. There was no influence of an antagonist to vasopressin, further demonstrating the specificity of oxytocin in regulating this behavior.
However, the initial reports of oxytocin inducing maternal behavior in the rat were not without some controversy. Different labs were having difficulty replicating the findings and issues such as the purity of the oxytocin, the strain of rat used and the testing paradigm were all considered. Susan Fahrbach again made a major contribution to the field when she noted that some investigators tested their subjects in their home cage whereas others moved the subject to a testing arena some time before the beginning of the behavioral test. Rats are adverse to novel environments and the movement to a testing arena would be predicted to induce a stress response. Susan directly tested the influence of the testing conditions by treating females with oxytocin and testing in their home cage or a novel environment. As you can see here, the oxytocin was only effective when the animals were tested in a novel environment, suggesting there was an interaction between the stress response and the action of oxytocin on maternal behavior.
Around the time these studies were completed, there was great excitement in the field about the influence of oxytocin on a wide variety of behaviors. A conference sponsored by the New York Academy of Sciences in 1992 brought together a collection of researchers working on the behavioral effects of oxytocin and its role in maternal, sexual and social behaviors. The cover painting is by an American impressionist, Mary Cassatt.
Since that time it has become clear that oxytocin influences many behaviors in numerous species. In general these are behaviors associated with affiliation, but there are also influences on learning and memory, feeding, anxiety and depression. Many of these behaviors are also influenced by estrogen and evidence suggests that estrogen induction of oxytocin receptor in brain is a major contributor to steroid hormone influences on behavior.
Since there was a great deal of enthusiasm for the importance of oxytocin in regulation of behavior, an obvious next step seemed to be to knock it out. Two different groups developed oxytocin knock out mice and there was great consternation when it was reported, as seen here, that oxytocin was not required for maternal behavior in mice.
HOWEVER, in my opinion this is an unfair conclusion. First of all, mice are not little rats, and making general statements about hormonal control of behavior based on a mouse seems risky at best. Second, the laboratory mouse isn't really much of a mouse at all! Pictured here is a wild house mouse, the type that lives in your basement or perhaps your cupboard if your unlucky, and next to it is your stereotypical laboratory mouse, a Swiss Webster in this case. As nicely shown here, there is a basic behavioral difference between these two animals. What you can't see here, is that the reason mice have never been used in studies of maternal behavior is because laboratory mice are SPONTANEOUSLY maternal, there is no induction to study. This is no doubt the result of decades of selective breeding in which any female that did not show a high degree of maternal behavior was immediately removed from the colony. The wild mouse, on the other hand, does show some semblance of normal adaptive behavior.
Shown here is the response of virgin female and pregnant female wild house mice to a pup placed into their cage for 30 min. These females were the offspring of animals caught in the field and bred in the laboratory. Not surprisingly, wild mice breed quite nicely under laboratory conditions. The response is scored as Infanticide, Parent or Unhandled. As you can see, approximately 60% of the virgin females will kill a pup placed in their cage, and greater than 90% of the pregnant females will kill pups.
Now this is not because the females are stressed by the laboratory conditions or any other aberrancy. This is an evolutionarly adaptive strategy in which females competing for limited resources simply eliminate the competition if given the chance. One hundred percent of the previously infanticidal pregnant females showed full maternal behavior to their own young when they were born as little as 24 hrs after the previous behavioral test. When their own young are weaned, a substantial portion of these females will return to killing unfamiliar pups placed into their cage.
The next question is whether there is any hormonal control of this behavior. Previously infanticidal females were treated with oxytocin and re-tested with a new pup. There was a significant reduction in the frequency of killing behavior, as well as a significant induction of maternal behavior by the pregnant females, although many of the virgin and pregnant females treated with oxytocin simply left the pup untouched during this second test.
In the previous behavioral tests the oxytocin had been given peripherally, so it could not be said with confidence that it was acting on the brain. Giving an intracerebroventricular (ICV) injection to a mouse is a bit tricky (another advantage of rats) but it can be done as is show here. This is an injection into a laboratory mouse, which is briefly immobilized with inhalant anesthetic, and the same thing can be done on a wild mouse.
Shown here is the results of an ICV injection of either saline vehicle or oxytocin to previously infanticidal female mice. As you can seen there is a significant reduction in the frequency of killing behavior in the oxytocin infused females, confirming that this neurohormone is in fact acting in the brain.
Up to now I have been focusing on the role of oxytocin in the onset of maternal behavior, but of equal importance appears to be prolactin. Initially dismissed as insignificant, the story of prolactin has been doggedly pursued and carefully developed by Robert Bridges. Shown here is the title page of one of the first reports demonstrating the infusions of prolactin into the brain does in fact induce short-latency maternal behavior in estrogen-primed rats.
One of the more fascinating aspects of this story has been the realization that apparently the endogenous ligand is not prolactin itself, but rather the placental lactogens produced by the developing fetus.
There are two rat placental lactogens, I and II, and shown here is that they are both equally effective at significantly reducing the latency for expression of various components of maternal behavior, including both retrieval and crouching. These placental lactagens appear to be capable of readily crossing the blood brain barrier and have been found in high concentrations in the CSF of pregnant women. An important difference between the actions of oxytocin and prolactin (or placental lactogens acting at the prolactin receptor) is that the latter requires at least 5 days of continuous exposure, where a single infusion of oxytocin is effective. Therefore it appears that two different mechanisms are at work to insure the appropriate expression of maternal behavior. One is mediated by the fetus during pregnancy and the other is associated with the oxytocin release that occurs at parturition.
This diagram demonstrates the effective sites for prolactin or placental lactogen at inducing maternal behavior in the preoptic area, again confirming the importance of this brain region.
The last topic I wanted to discuss is the role of immediate early genes, in particular the fos family of proteins. In neurons, depolarization or activation, is often accompanied by the induction of immediate early gene expression. For behavioral neuroscientists, the immunocytochemical detection of the c-fos protein has become a valuable tool for detailed mapping of neural circuitries regulating complex behavioral responses. This technique has been put to great use by Michael Numan to further map the pathways mediating maternal behavior. Shown here is a diagram of neurons in the preoptic area that were positive for c-fos protein in a female that exhibited maternal behavior (top) and a female that did not exhibit maternal behavior (bottom). As you can see there are substantially more neurons activated in the preoptic area of females that show maternal behavior.
This slide is from an unrelated study and is only for purposes of showing what a c-fos positive neuron looks like. Shown on the bottom panel, the black dots are actually neuronal nuclei in which c-fos protein has been detected by immunocytochemistry.
Most of what has been presented so far involved the hormonal induction of maternal behavior. However, it has long been known that female rats can also be induced to behave maternally if they are simply exposed to a rat pup day after day after day. This process is called "sensitization" and whether the hormonal and neural basis of the induction of the behavior in this way is the same has long been a topic of investigation.
Using the same technique as for the hormonal induction of the behavior, Numan also looked at females that either were or were not maternal after a period of sensitization. Shown on the left are the females that were maternal, versus those on the right which were not. What is immediately obvious is that the same neural circuitry appears to be activated in females induced to be maternal by sensitization as those induced hormonally. As an aside, I should mention that wild house mice females exposed to pups day after day do not sensitize, they will continue to show high rates of killing behavior for as long as you continue to provide them with pups.
The significance of immediate early genes took a new turn when this report was made from Greenberg that mice with a null mutation for the fos B gene did not show maternal behavior. This was a surprising finding and one perhaps still open to interpretation. Nonetheless, it suggests that specific expression of immediate early genes in the neural circuitry regulating maternal behavior is not only a good marker, but is causally involved in the expression of this behavior as well. This opens up new avenues of research into the molecular basis of maternal behavior and will no doubt spur novel and exciting research into understanding this complex response.
Finally, as I have tried to demonstrate, generalizing even from rats to mice is fraught with difficulties. Trying to further draw conclusions regarding the hormonal basis of maternal behavior in humans may seem completely out of reach. However, given the frequent news reports of women failing to care for their newborn infants, i.e. a lack of the onset of maternal behavior, this is not a health problem of trivial significance. Certainly there are cultural and social influences involved in these tragic instances of infanticide, but there are also likely to be biological ones as well.
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last update: June 1998