Researchers One Step Closer To Holy Grail Of Neurobiology

"If you ask someone on the street what arousal means, that person might have an intuitive concept of arousal in terms of sexual excitement, alertness or an emotional response such as fear," says Pfaff, professor and head of the Laboratory of Neurobiology and Behavior at Rockefeller. "But, if you ask, 'Exactly what does arousal mean scientifically,' it's been very hard for scientists to pin down."

Scientists who study arousal historically were divided into two camps: those who consider arousal to be a single, "monolithic" physiological function, and those who believe that arousal does not exist as a whole, but is a collection of small specific abilities.

In the PNAS paper, Pfaff and colleagues present a mathematical equation that for the first time unifies these two disparate schools of thought and combines generalized arousal with various specific forms of arousal, such as sex, hunger and fear. They also show that experiments can be designed to measure arousal in laboratory mice, which ultimately will provide genetic answers to the question "what is arousal?"

In humans, deficits in arousal contribute to such cognitive problems as attention deficit hyperactivity disorder, autism and Alzheimer's disease. Erosion of arousal also may account for some of the mental difficulties that people face as they age. Understanding general arousal may help scientists develop pharmacological methods to enhance alertness during the day and sleep at night. Analyzing the mechanisms of arousal may lead to a more precise anesthesiology.

The operational definition of arousal proposed by the Rockefeller researchers states that an aroused animal or human will be more sensitive to sensory stimuli, be physically more active; and react more emotionally. According to Pfaff, this definition allows for interaction among arousal states. In other words, changes in sexual arousal could dampen or increase response to pain, or vice versa.

"The overall state of arousal at any given moment is going to be a function both of the person's global, or generalized, arousal state and any individual arousal states that might be present," says Pfaff.

Using the equation, the researchers were able to perform what they call a "principal components analysis" of a series of behavioral experiments with laboratory mice that measured the animal's response to sensory stimuli, motor activity and emotional reactivity. They found that a "generalized arousal function" underlies about one-third of all the data in arousal-related tasks.

"The existence of a generalized arousal function is not exclusive of several specific forms of arousal," says Pfaff. "For example, if you scare the heck out of me, I'm more likely to react strongly if I'm generally aroused anyway, whereas if I'm asleep, I'm just a bit sluggish in my response."

To understand how changes in the expression of specific genes can influence arousal, the scientists studied changes in arousal of two different types of genetically altered mice: one mouse lacked the gene for the estrogen receptor-alpha, while the other was missing the gene for the estrogen receptor-beta.

"Estrogens, sex behavior and sexual arousal are useful as a 'bridge' to the discovery of fundamental arousal mechanisms," says Pfaff. "Because estrogens strongly drive female sex behaviors, we were interested in determining if the genes encoding the estrogen receptors alpha and beta could be involved in a more global brain function."

The experiments were set up to study female mice sleeping in their home cages as they do during the light phase of the daily light/dark cycle. According to Pfaff, studying the animal in its home cage during the light cycle ensures that the mouse will not be exploring its cage. This approach removes biases due to fear and anxiety and provides a zero baseline. A computer automatically measured the responses to carefully presented sensory stimuli.

The sensory stimuli administered were vestibular, in which the mouse's home cage was vertically raised; tactile, in which a mice was exposed to a brief air puff strong enough to deflect the hair on its back; olfactory, in which the mouse's response to various odors was measured; and auditory, a five second burst of loud white noise.

The key to getting arousal into a modern genetic and physiologic framework, Pfaff says, is to have physical responses, which are the proof of arousal.

"In addition to receiving sensory stimuli, we have the animal moving on a running wheel," Pfaff says. "That's also part of the operational definition. So what we're trying to do is turn the most fundamental behavioral concept into a set of responses measured physically and quantitatively."

From responses to stimuli and the running wheel experiments, Pfaff and his colleagues found that the sensory alertness and the motor activity of mice lacking the estrogen receptor-alpha was significantly less than genetically normal control mice. Surprisingly, there was no significant change in motor activity between mice lacking the beta estrogen receptor and control mice.

With respect to the findings of the estrogen receptor-alpha knockout mice, Pfaff says, "It was surprising to us that a gene for a sex hormone receptor would have such a global affect on arousal."

Pfaff's co-authors are J. Garey, Ph.D., A. Goodwillie, M. Morgan, Ph.D., and S. Ogawa, Ph.D., at Rockefeller; J.-A. Gustaffson, M.D., at the Karolinska Institute, Sweden; O. Smithies, Ph.D., at University of North Carolina, Chapel Hill; and K.S. Korach, Ph.D., at National Institute on Environmental Health Sciences, Research Park Triangle, NC.

This research was supported in part by the National Institute of Child Health and Human Development and a National Institute of Mental Health Training Grant.