by Donald Pfaff
The simplest theory would be that such interconnectedness could magnify effects of hormones on strings of neurons important for mating behavior. For example, if estrogen exposure multiplies physiological activity by some constant k in neurons named a, b, and c, and if neuron a projects to neuron b which in turn projects to neuron c, then it seems possible that system output following estrogen is multiplied as follows: ka × kb × kc. Of course, large numbers of other such theories are possible, and they can be tested with modern electrophysiological and optogenetic techniques.
In summary, this tremendous amount of work told us how endocrine signaling systems impact the brain. In Chapters 2 through 4 we will go on with physiological and genomic techniques to work out how hormone-dependent neurons work through circuitry to produce an entire vertebrate behavior. That is, we understand how the nervous system works by taking a specific behavioral function and solving it as an analytic problem.
Principle inferred: The brain needs to be continually informed about the state of the body. For the purpose of successful reproduction, that is accomplished by systems of specific hormone receptor proteins in neurons in selected parts of the brain. The system of hormone-binding neurons that I discovered appears to be universal among vertebrates.
In the mammalian brain, specific nuclear receptors for estrogens offer a launching point, one of three, for working out the first neural circuit for a vertebrate behavior, as described in Chapter 2. And these nuclear receptors, being ligand-activated transcription factors, allowed us to apply the techniques of molecular biology to our developing field, as described in Chapter 3.
Further Reading
Arnold, A., F. Nottebohm, and D. W. Pfaff. 1976. “Hormone-Concentrating Cells in Vocal Control and Other Areas of the Brain of the Zebra Finch (Poephila guttata).” Journal of Comparative Neurology 165: 487–512.
Barfield, R. J., G. Ronay, and D. W. Pfaff. 1978. “Autoradiographic Localization of Androgen-Concentrating Cells in the Brain of the Male Domestic Fowl.” Neuroendocrinology 26: 297–311.
Davis, P. G., M. S. Krieger, R. J. Barfield, B. S. McEwen, and D. W. Pfaff. 1982. “The Site of Action of Intrahypothalamic Estrogen Implants in Feminine Sexual Behavior: An Autoradiographic Analysis.” Endocrinology 111: 1581–1586.
Davis, P. G., B. McEwen, and D. W. Pfaff. 1979. “Localized Behavioral Effects of Tritiated Estradiol Implants in the Ventromedial Hypothalamus of Female Rats.” Endocrinology 104: 898–903.
Davis, R. E., J. I. Morrell, and D. W. Pfaff. 1977. “Autoradiography Localization of Sex Steroid-Concentrating Cells in the Brain of the Teleost Macropodus opercularis (Osteichthyes: Belonttidae).” General and Comparative Endocrinology 33: 496–505.
Halpern, M., J. I. Morrell, and D. W. Pfaff. 1982. “Cellular 3H-Estradiol and 3H-Testosterone Localization in the Brains of Garter Snakes: An Autoradiographic Study.” General and Comparative Endocrinology 46: 211–224.
Harlan, R. E., B. D. Shivers, L.-M. Kow, and D. W. Pfaff. 1982. “Intrahypothalamic Colchicine Infusions Disrupt Lordotic Responsiveness in Estrogen-Treated Female Rats.” Brain Research 238: 153–167.
______. 1983. “Estrogenic Maintenance of Lordotic Responsiveness: Requirement for Hypothalamic Action Potentials.” Brain Research 268: 67–78.
Kelley, D. B., I. Lieberburg, B. S. McEwen, and D. W. Pfaff. 1978. “Autoradiographic and Biochemical Studies of Steroid Hormone-Concentrating Cells in the Brain of Rana pipiens.” Brain Research 140: 287–305.
Kelley, D. B., J. I. Morrell, and D. W. Pfaff. 1975. “Autoradiographic Localization of Hormone-Concentrating Cells in the Brain of an Amphibian, Xenopus laevis. I. Testosterone.” Journal of Comparative Neurology 164: 47–62.
Krieger, M. S., J. I. Morrell, and D. W. Pfaff. 1976. “Autoradiographic Localization of Estradiol-Concentrating Cells in the Female Hamster Brain.” Neuroendocrinology 22: 193–205.
Kringelbach, M. L. 2016. “Limbic Forebrain: The Functional Neuroanatomy of Emotion and Hedonic Processing.” In Neuroscience in the 21st Century. 2nd edition. Edited by D. W. Pfaff and N. D. Volkow. New York: Springer, 1335–1363.
Lauber, A. H., C. V. Mobbs, M. Muramatsu, and D. W. Pfaff. 1991. “Estrogen Receptor mRNA Expression in Rat Hypothalamus as a Function of Genetic Sex and Estrogen Dose.” Endocrinology 129 (6): 3180–3186.
Lauber, A. H., G. J. Romano, C. V. Mobbs, and D. W. Pfaff. 1990. “Estradiol Regulation of Estrogen Receptor Messenger Ribonucleic Acid in Rat Mediobasal Hypothalamus: An in Situ Hybridization Study.” Journal of Neuroendocrinology 2 (5): 605–611.
Meisel, R., G. Dohanich, B. McEwen, and D. W. Pfaff. 1987. “Antagonism of Sexual Behavior in Female Rats by Ventromedial Hypothalamic Implants of Antiestrogen.” Neuroendocrinology 45: 201–207.
Morrell, J. I., A. Ballin, and D. W. Pfaff. 1977. “Autoradiographic Demonstration of the Pattern of 3H-Estradiol Concentrating Cells in the Brain of a Carnivore, the Mink, Mustela vison.” Anatomical Record 189 (4): 609–624.
Morrell, J. I., D. Crews, A. Ballin, A. Morgentaler, and D. W. Pfaff. 1979. “3H-Estradiol, 3H-Testosterone and 3H-Dihydrotestosterone Localization in the Brain of the Lizard Anolis carolinensis: An Autoradiographic Study.” Journal of Comparative Neurology 188: 201–224.
Morrell, J. I., D. B. Kelley, and D. W. Pfaff. 1975. “Autoradiographic Localization of Hormone-Concentrating Cells in the Brain of an Amphibian, Xenopus laevis. II. Estradiol.” Journal of Comparative Neurology 164: 63–78.
Morrell, J. I., M. S. Krieger, and D. W. Pfaff. 1986. “Quantitative Autoradiographic Analysis of Estradiol Retention by Cells in the Preoptic Area, Hypothalamus and Amygdala.” Experimental Brain Research 62: 343–354.
Morrell, J. I., and D. W. Pfaff. 1978. “A Neuroendocrine Approach to Brain Function: Localization of Sex Steroid Concentrating Cells in Vertebrate Brains.” American Zoologist 18: 447–460.
______. 1982. “Characterization of Estrogen-Concentrating Hypothalamic Neurons by Their Axonal Projections.” Science 217: 1273–1276.
Pfaff, D. W. 1965. “Cerebral Implantation and Autoradiographic Studies of Sex Hormones.” In Sex Research: New Developments. Edited by J. Money. New York: Holt, Rinehart & Winston, 219–234.
______. 1968. “Autoradiographic Localization of Radioactivity in Rat Brain after Injection of Tritiated Sex Hormones.” Science 161: 1355–1356.
______. 1970a. “Mating Behavior of Hypophysectomized Rats.” Journal of Comparative and Physiological Psychology 72: 45–50.
______. 1970b. “Nature of Sex Hormone Effects on Rat Sex Behavior: Specificity of Effects and Individual Patterns of Response.” Journal of Comparative and Physiological Psychology 73: 349–358.
Pfaff, D. W., and L. C. A. Conrad. 1978. “Hypothalamic Neuroanatomy: Steroid Hormone Binding and Patterns of Axonal Projections.” In International Review of Cytology, vol. 54. Edited by G. Bourne. New York: Academic, 245–265.
Pfaff, D. W., J. Gerlach, B. S. McEwen, M. Ferin, P. Carmel, and E. Zimmerman. 1976. “Autoradiographic Localization of Hormone-Concentrating Cells in the Brain of the Female Rhesus Monkey.” Journal of Comparative Neurology 170: 279–294.
Pfaff, D. W., and M. Keiner. 1973. “Atlas of Estradiol-Concentrating Cells in the Central Nervous System of the Female Rat.” Journal of Comparative Neurology 151: 121–158.
Rhodes, C. H., J. I. Morrell, and D. W. Pfaff. 1981. “Distribution of Estrogen-Concentrating, Neurophysin-Containing Magnocellular Neurons in the Rat Hypothalamus as Demonstrated by a Technique Combining Steroid Autoradiography and Immunohistology in the Same Tissue.” Neuroendocrinology 33: 18–23.
Rothfeld, J. M., R. E. Harlan, B. D. Shivers, and D. W. Pfaff. 1986. “Reversible Disruption of Lordosis via Midbrain Infusions of Procaine and Tetrodotoxin.” Pharmacology Biochemistry and Behavior 25: 857–863.
Swanson, L. W. 2013. “Basic Principles of Nervous System Organization.” In Neuroscience in the 21st Century, vol. 3. Edited by D. W. Pfaff. New York: Springer, 3: 1255–1288.
Tabansky, I., J. N. H. Stern, and
D. W. Pfaff. 2015. “Implications of Epigenetic Variability within a Cell Population for ‘Cell Type’ Classification.” Frontiers in Behavioral Neuroscience 9: 342.
Wallis K., S. Dudazy, M. van Hogerlinden, K. Nordström, J. Mittag, and B. Vennström. 2010. “The Thyroid Hormone Receptor α1 Protein Is Expressed in Embryonic Postmitotic Neurons and Persists in Most Adult Neurons.” Molecular Endocrinology 24: 1904–1916.
Zigmond, R. E., R. A. Detrick, and D. W. Pfaff. 1980. “An Autoradiographic Study of the Localization of Androgen Concentrating Cells in the Chaffinch.” Brain Research 182: 369–381.
Zigmond, R. E., F. Nottebohm, and D. W. Pfaff. 1973. “Androgen-Concentrating Cells in the Midbrain of a Songbird.” Science 179: 1005–1007.
2
DISCOVERING THE NEURAL CIRCUIT FOR A VERTEBRATE BEHAVIOR ESSENTIAL TO REPRODUCTION
Problem: No one had put together the mechanisms for a complete vertebrate behavior. It had been thought that for ease of methodology and conceptual clarity, simpler animals such as Aplysia and fruit flies must be used to study the neural basis of behavior. For a mammalian behavior, we cover the subject from ion channels through neuroanatomy through electrophysiology to the logic of the social behavior itself.
You cannot state seriously that you have the mechanisms for a behavior unless you have figured out the entire neural circuit. So that is what we did. From the work discussed in Chapter 1, we had the information about how hormones impact the brain. In Chapters 3 and 4 we will be able to link these hormones to gene expression and genomic influences on behavior. But now, in Chapter 2, we are looking for a manifestation of lordosis behavior mechanisms in physical terms.
Lordosis behavior involves the vertebral dorsiflexion by the female in response to the male’s mount, a behavior essential for fertilization and therefore reproduction. Working out the entire neural circuit for producing lordosis took the combined efforts of many smart young scientists over a period of many years. Throughout this chapter I will recognize their contributions.
So there were three points of entry that allowed discovery of the first neural circuit for producing a vertebrate behavior: First: the neurons expressing the hormone receptors whose action in hypothalamic neurons facilitate the behavior (Chapter 1). Second: the cutaneous stimuli (from mounting by the male) that triggers the behavior. Third: working our way in backward from the muscles that execute the behavior, through the motor neurons, and backward up the motor control pathways. A large number of analytic experiments revealed a spinal–midbrain–spinal loop whose activity is regulated by an estrogen-dependent output from the hypothalamus. The estrogen-dependent output relies on hormone-regulated gene expression (Chapter 3).
The flow of the sections in this chapter will take us from the hypothalamus out to the midbrain, at the top of the spinal–midbrain circuit we discovered. In subsequent sections we start from the relevant sensory surface, work our way up ascending pathways to the midbrain (to meet the hypothalamic outflow), and then come back down descending pathways to the relevant motor neurons. Unless otherwise stated, female animals were treated with estrogen whenever appropriate to the experimental aim.
Hypothalamic Outflow Adds the Hormone Dependence
Neuroanatomy of Ventromedial and Other Hypothalamic Neurons’ Projections
From the point of view of lordosis behavior control, projections from the ventromedial nucleus of the hypothalamus (VMH) were the most important to determine. As will be noted later, in lesions of VMH abolish lordosis, stimulation of VMH triggers lordosis and estrogen exposure in VMH is necessary and sufficient for lordosis (Chapter 1). The task of VMH projection discovery fell to the Rockefeller graduate student Lily C. A. Conrad, a young neuroanatomist so skilled that I told her, “You could be the next century’s Ramón y Cajal!” Instead, after receiving her Ph.D. she went to medical school. Lily and I used autoradiographic techniques with long exposure times to chart all the efferents of VMH (Conrad and Pfaff 1976a).
Ascending VMH axons swept through the anterior hypothalamus and preoptic area into the diagonal band and septum. Projections were prominent in the preoptic area itself and the lateral ventral septum. Some fibers ran into the stria terminalis and its bed nucleus. Most terminations were ipsilateral to the isotope’s microinjection in VMH, but scattered contralateral projections were also detected.
Descending projections, most important for lordosis behavior, formed two systems. One group of labeled axons ran laterally and posteriorly, distributing through the zona incerta and fields of Forel to terminate in the midbrain reticular formation lateral to the mesencephalic central grey. These axons formed a sheet-shaped projection that twisted ninety degrees as it curved posteriorly to the midbrain. Some fibers, following the supraoptic commissure, made it into the amygdala.
The second group ran medially, turning through the posterior hypothalamus and following a periventricular route to the central grey itself. These fibers were scattered in bundles posterior to VMH as they swept past premammillary regions, projecting to ventral and dorsal premammillary nuclei on the way. Then, from a supramammillary projection they turned dorsally and projected to all nuclei of the mesencephalic central grey.
Postdoctoral researcher Monica Krieger added more detail to this description of VMH efferents by microinjecting the tritiated leucine more discretely into different components of this behaviorally crucial hypothalamic nucleus (Krieger, Conrad, and Pfaff 1979). For example, axons emanating from the ventrolateral subdivision of VMH, the neuronal group with most intense estrogen binding, emphasized projections to the medial preoptic area, axons sneaking laterally beneath the cerebral peduncle to arrive at the medial amygdala, and ascending periventricular projections that would innervate the most rostral pole of the dorsolateral central grey. No axons made it all the way to the pons, a fact that helps define the midbrain module of the lordosis behavior circuit.
In addition, as covered in Chapter 1, Joan Morrell’s combination of retrograde tracing using the fluorescent dye primuline with estrogen binding defined by steroid autoradiography proved that estrogen-concentrating VMH neurons really do project to the mesencephalic central grey and surrounding dorsal midbrain (Morrell and Pfaff 1982).
These descending projections from VMH would later be shown physiologically to be the essential link from the hypothalamic module to the midbrain module of the newly discovered lordosis behavior neural circuit.
ULTRASTRUCTURE
Having defined VMH projections that would be shown as the output of the hypothalamic module of the lordosis circuit, we wanted to take a look at the nature of the relevant synapses in the midbrain central grey (MCG) (Chung, Pfaff, and Cohen 1990a). Following electrolytic lesions in the VMH, we saw a type of degenerative pattern in the central grey known as “watery degeneration”: both in the presynaptic processes and processes (identified by the presence of a postsynaptic density) there was a swollen appearance, electron lucent. Dendrites were devoid of microtubules. In animals surviving longer after the VMH lesion (eight days), presynaptic vesicles were clumped and misshapen, there are abnormally large and dark mitochondria, and the postsynaptic processes are surrounded by reactive glia.
We followed up that work with chemical (kainic acid and N-methyl aspartate) lesions of the VMH to make sure that the foregoing results were not due to fibers of passage (Chung, Pfaff, and Cohen 1990b). Once again, in the MCG, we could easily see axonal, presynaptic and postsynaptic degeneration. Presynaptic endings were shrunken and dense, and contained clumped synaptic vesicles and abnormally large dark mitochondria. Postsynaptic processes were swollen and showed watery degeneration. Dendrites were devoid of microtubules. Midbrain central grey synapses were engulfed by glial processes. These experiments relieved us by showing the central grey fine structural phenomena were really due to VMH projections, not to fibers of passage.
As a side point, I note that communication between the hypothalamic module of the female reproductive behavioral neural circuit and the midbrain module issue is due to
descending fibers from the VMH, not ascending fibers from the MCG. That is, Jerry Eberhart, during his year in the laboratory, saw many ascending projections from the MCG, but they did not go to the VMH. Another side point: Lily Conrad’s subsequent results dealt with efferents from the medial preoptic area and gave strong neuroanatomical support to what I said in Chapter 1 (Conrad and Pfaff 1976b). Neurons in regions with estrogen-binding cells tend to project to others that do the same.
Electrophysiology. Yasuo Sakuma’s plane arrived from Japan on a Monday night, and he started his first experiment at 7:30 AM on Tuesday. Armed with and an M.D. and a Ph.D. (in neurophysiology) from the Yokohama City University School of Medicine, he was the most brilliant protégé of one of the founders of modern neuroendocrinology, Masami Kawakami. I was not surprised at the quality and volume of Yasuo’s accomplishments at Rockefeller nor have I been surprised at his subsequent, highly successful career as a professor at Tokyo’s Nippon Medical School (and now president of a school for allied medical sciences). I was not surprised because, before Yasuo came to New York, I had made a lecture trip to Japan, and, walking in and around Yasuo’s electrophysiology rig, I thought, “This is a guy who really knows what he is doing.”
Yasuo used microelectrode recordings to characterize the properties of lordosis-critical neurons with axons to the MCG. These VMH neurons were identified by “back firing” them from the MCG, that is, by recording antidromic potentials in the VMH neuronal cell body having stimulated their axon in the MCG. Further confirmation of the crucial VMH / MCG connection was shown when an orthodromic spike emanating from the VMH cell body canceled the antidromic spike coming from the MCG (Sakuma and Pfaff 1981, 1982). Latencies for the antidromic spike to reach the cell body varied widely, from 1.4 to 41.5 milliseconds. Amazingly, a few VMH neurons with projections to MCG were also identified, using the same technique, as projecting to the amygdala. These various electrophysiological defined connections were not altered by estrogen. However, it can be said that there were detailed differences between males and females. The latencies of the VMH spikes to the antidromic stimulation were significantly different—females were faster—and one entire latency class was absent in the male. The apparent orderliness of flow of hypothalamic and preoptic axons to the midbrain was already noted in Chapter 1.