Laboratory of Neurobiology and Behavior
Donald W. Pfaff
Laboratory of Neurobiology and Behavior
Donald Pfaff’s forthcoming book will be published in May 2017 by Harvard University Press.
Some of the lab's work can be summarized in four steps. First, we worked on the localization of hormone target neurons in the brain and discovered estrogen-binding neurons and androgen-binding neurons in a limbic/hypothalamic system. The discovery initially was made in rat brains, but our work on fish CNS through monkey CNS showed it to be a general vertebrate system. We followed up the histochemical findings to demonstrate consequences of hormone binding for electrophysiological activity and neuronal growth. Secondly, we then worked out the first neural circuit for a vertebrate behavior, the estrogen-dependent lordosis behavior, a social behavior. The lordosis behavior circuit proved that it is possible to explain how mechanisms for a vertebrate social behavior work. Third, we found hormone-dependent genes in the brain. Their induction by estrogenic hormones has temporal, spatial and gender specificities appropriate to reproductive behavior. Fourth, in turn, the products of some of these hormone-dependent genes are required for hormone-dependent lordosis behavior.
Taken together, these four findings showed that specific neurochemical reactions in specific parts of the brain physically determine a specific mammalian behavior.
Some of the Lab's New Work
- Genetic and hormonal influences on brain arousal – both sexual and generalized arousal (GA) in mice. For example, we follow mouse responsivity during the light-to-dark (low-to-high arousal) transition, measuring behavioral arousal with 20 millisecond resolution. Is this a physically defined phase transition? What mathematical function are these animals following? Why is there a sex difference?
- The “master cells” for GA are certain large medullary reticular neurons (nucleus gigantocellularis, NGC). We have determined the transcriptome of the specific set of NGC neurons with axons projecting to the thalamus (for activating the cortex). We believe these neurons are responsible for “waking up” the brain from zero states: e.g. coma, deep sleep, deep anesthesia.
- Exactly when and where are NGC neurons born and how do they migrate to their final functional positions? These experiments will determine the transcription factor (hox gene) spatiotemporal patterning that produces the NGC neurons we need to understand.
- The genes for the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are expressed in these NGC neurons. Since GR and MR are ligand-activated transcription factors, effects of stresses on these neurons can be studied at the transcriptional and epigenetic levels.
- We have preliminary data indicating that at least some NGC neurons are outside the blood-brain-barrier. If we can replicate that evidence then NGC neurons would be unusually susceptible to blood-borne influences.
- Molecular, biophysical and behavioral studies of “generalized arousal” transmitter actions on nerve cells. These include opioid peptides, histamine and norepinephrine. In particular, we are testing the hypothesis that the functions of delayed rectifier potassium channels are crucial for high levels of NGC excitability and therefore, perhaps, at the base of entry into consciousness.
- Use of viral vectors to alter gene expression in NGC neurons and measure the behavioral consequences: for GA and for specific motivated behaviors.
- Follow up microarray studies of steroid hormone effects on gene expression in specific brain regions of the adult; and of sex differences in the developing brain. Follow-up at RNA and protein levels. The findings will lead to predictions for epigenetic studies.
Molecular Mechanisms for Behavior
We use molecular techniques to analyze: 1) how the mammalian brain manages specific natural behaviors; and 2) hormonal and genetic influences on generalized brain arousal. Some of this work can be done in nerve cell lines, but it is really necessary to study nerve cells in their normal synaptic context to see how, in the governance of behavior, the brain's special connectivity uses the types of molecular mechanisms seen in other tissues.
As noted above, we have expanded the focus of our lab to discover molecular and physiological events necessary for achieving basal states of consciousness – “waking up” from the zero states: coma, deep sleep, deep anesthesia. We expect that some of the mechanisms we discover will prove to be universal among mammals.
First, advantageous for molecular studies, hormone effects on nerve cells build upon some of the best examples of eukaryotic transcription control. Steroid sex hormones and stress hormones have massive developmental effects, and in the adult brain they control a variety of natural behaviors. During development, for example, sex hormone actions around the time of birth determine behavioral sex differences, and early stress hormone exposure influences later responses to stress.
We have shown significant effects of estrogens on the transcription of the genes for the progesterone receptor, for oxytocin and the oxytocin receptor and for the opioid peptide enkephalin. These hormone effects occur in specific parts of the brain and are required for normal reproductive behavior. They are strong in females but not males, again correlated with behavioral results. These transcriptional effects are due to the binding of estrogen receptors to their cognate DNA sequences, "estrogen response elements," in the promoters of the progesterone receptor and enkephalin genes. Other transcriptional systems that are hormone sensitive in the brain include the genes for the delta opioid receptor, the alpha adrenergic receptor, GnRH (LHRH) and the GnRH receptor. Microarrays have added many more candidate genes.
Interestingly, other transcription factors can interact with the estrogen receptor in the brain to influence estrogenic effects on gene expression and behavior. That is, thyroid hormone receptors, themselves transcriptionally active, can interfere with estrogen-dependent transcription and behavior. In doing so, thyroid hormones and their receptors bring reproductive controls into concordance with environmental signals, particularly environmental temperature.
Second, with respect to generalized brain arousal, Inna Tabansky’s work in collaboration with the Friedman lab has yielded the complete transcriptome specific to those giant medullary reticular neurons that have projections to the thalamus, crucial for CNS arousal and probably crucial for maintaining the basal, elementary states of consciousness.
The explosion in the number of interesting genetically altered mice is giving us new insights into the relationship between mammalian gene expression and behavior. Using estrogen receptor knockout mice, we showed that the effect of a specific gene on a specific behavior can depend upon the gender in which that gene is expressed as well as upon exactly when and where it is expressed. Functional genomics applied to these problems employ biophysical and behavioral assays in knockout mice. For us, that includes patch clamping of identified cells in mouse brain, with their molecular signatures determined by RT/PCR. In turn, the combination of mouse gene knockouts with neuropharmacology and genetic editing technology offers the chance to open a new era, understanding mechanisms connecting gene expression to mammalian behavior.