How the Vertebrate Brain Regulates Behavior

by Donald Pfaff

  Estrogenic Facilitation of Ventromedial Hypothalamic Neurons’ Electrical Activity

  A long series of electrophysiological experiments led by my long-time colleague Lee-Ming Kow led to the following conclusion: VMH neuronal responses to virtually every neurotransmitter effective for triggering VMH firing would, in turn, be amplified by estrogen treatment. Lee had gotten his Ph.D. in biophysics at the California Institute of Technology (“Cal Tech”) and wanted to come to my laboratory for his postdoctoral work; and his wife, the best dentist on the planet, was pleased to come to New York City as well. Fortunately for a generation of young scientists in my laboratory, Lee simply did not want to do what a typical American professor does—he has been pleased to stay in my laboratory as a pure neurobiological scholar. The reference list of this chapter hardly could do justice to his scientific productivity.

  The easiest way to summarize parts of Lee’s massive accomplishments will be to handle them neurochemical system by system. First cholinergic, then noradrenergic, then histamine, then glutamate. To describe the explanatory power of the results most clearly, I would like to use the idea of a logical syllogism, that is, reasoning in this form: “If (a) John is a scientist and (b) all scientists are good, then (c) John is good.”

  ACETYLCHOLINE

  Lee’s electrophysiology was motivated by others’ behavioral results. Gary Dohanich, a professor at Tulane University, and Lynn Clemens at Michigan State had shown that agonists that stimulate the muscarinic type of cholinergic receptor would increase lordosis behavior and, conversely, that antagonists would decrease it. Laura Kaufman, in our laboratory, restricted cholinergic agonist application to the VMH and got the same result: increased female reproductive behavior.

  How does that work? Tom Rainbow, working with Bruce McEwen, showed that estrogens increase muscarinic receptor levels in VMH. Previously, Lee-Ming Kow and I had reported two results: that acetylcholine increases VMH electrical activity and that estrogen administration increases VMH responses to acetylcholine (Kow and Pfaff 1985, 1995).

  To summarize this work, (a) estrogens heighten lordosis behavior, and (b) estrogens also increase muscarinic receptors and electrophysiological responses to acetylcholine in VMH neurons. Therefore, it follows that (c) one way in which estrogens facilitate reproductive behavior is to increase acetylcholine receptors and electrical responses to acetylcholine by VMH neurons.

  NOREPINEPHRINE

  Bundles of noradrenergic fibers, ascending to the hypothalamus from their sources, the locus coeruleus and medullary cell groups A1 and A2, innervate the VMH. The late Robert Moss at the University of Texas reported that damage to the ventral bundle as well as the administration of noradrenergic α1 receptor blockers decrease lordosis, whereas receptor agonists applied specifically to the VMH increase it.

  How does that work? Anne Etgen, a professor at the Albert Einstein School of Medicine, showed that estrogens increase the amount of noradrenergic α1b receptors in hypothalamus. In addition, Victoria Luine, at Hunter College, reported that estrogens decrease the activity of monoaminoxidase, an enzyme that degrades norepinephrine. In parallel, when recording from individual neurons in the VMH, Lee-Ming Kow and I had two results: that an α1 receptor agonist would increase electrical activity, and the estrogens would increase the magnitude of responses to norepinephrine (Kow and Pfaff 1987, 1995; Kow, Weesner, and Pfaff 1992; and with patch clamp, Lee et al. 2008).

  Thus, to summarize, (a) estrogens heighten lordosis behavior, and (b) estrogens also increase noradrenergic α1b receptors and electrophysiological responses to noradrenaline in VMH neurons. Therefore, it follows that (c) one way in which estrogens facilitate reproductive behavior is to increase adrenergic receptors and electrical responses to noradrenaline by VMH neurons.

  HISTAMINE

  Stephano and Donoso had shown that bathing the hypothalamus in histamine (HA) would increase lordosis behavior, so we wanted to use electrophysiological techniques to measure responses of VMH neurons to HA. Years ago our laboratory was blessed with the arrival of Jin Zhou, M.D., who had placed first in her class at the medical school in Beijing, People’s Republic of China. Jin used patch-clamp techniques to demonstrate that VMH neurons had excitatory responses to HA and that estrogens would potentiate HA actions in a way that would explain the positive effects of HA on lordosis (Zhou et al. 2007). Christophe Dupré went farther to explore the ionic basis of the HA effect (Dupré et al. 2010). Lee-Ming Kow would use the patch-clamp recording of VMH neurons with a variety of ion substitution and pharmacological maneuvers to explore in greater detail exactly how HA depolarizes these neurons in a way that would foster lordosis behavior (Kow et al. 2016).

  First, as Christophe Dupré had reported, H1 receptors are involved. Second, neither sodium currents nor calcium currents were necessary. In detail, Chris’s results showed that HA acting through H1 receptors depolarizes these neurons. Further, acute administration of estradiol, an estrogen necessary for lordosis behavior to occur, heightens this effect. Hyperpolarization, which tends to decrease excitability and enhance inhibition, was not affected by acute estradiol or mediated by H1 receptors but was mediated by the other HA receptor subtypes H2 and H3. Sampling of messenger RNA (mRNA) from individual VMN neurons showed colocalization of expression of H1 receptor mRNA with estrogen receptor α (ER-α) mRNA but also revealed ER colocalization with the other HA receptor subtypes and colocalization of different subtypes with each other. The latter finding provides the molecular basis for complex push-pull regulation of VMN neuronal excitability by HA. Thus, in the simplest causal route, HA, acting on VMN neurons through H1 receptors, provides a mechanism by which elevated states of generalized central nervous system arousal can foster a specific estrogen-dependent, aroused, sexual behavior.

  Ion channels. To explore potassium (K+) ion channels using a blocking approach, cesium (Cs+, 2 mM), and 4-aminopyridine (5 mM), which block fast-acting channels such as the A-current, were added to the channel blocker tetraethylammonium. With this combination, HA depolarization was essentially abolished. This result shows that K+ currents are essential for mediating HA depolarization.

  Then, to determine whether K+ currents are also sufficient to support HA depolarization, VMH neurons were tested in a “K+-only” environment, where K+ was the only permeable cation in both internal and external solutions. HA depolarized VMH neurons, demonstrating that K+ was sufficient. Thus, K+ currents are both necessary and sufficient for HA depolarization. A reasonable inference is that depolarization caused by HA is due mainly to the inhibition of K+ currents.

  Moreover, estrogens can rapidly potentiate depolarization induced by HA, applied either through the bath or by pico-spritzing. And hormonal specificity is further confirmed by the experiments using the estrogen agonists PPT [(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol] for ER-α and DPN [2,3-bis-(4-hydroxy-phenyl)-propionitrile] for ER-β. PPT acted like estradiol to potentiate depolarization in VMH neurons, whereas DPN had no significant effect. I inferred that the rapid potentiating action of estradiol was mediated via ER-α.

  To summarize, (a) estrogens heighten lordosis behavior, and (b) estrogens also increase electrophysiological responses to HA depolarizations in VMH neurons. Therefore, it follows that (c) one way in which estrogens facilitate reproductive behavior is to increase excitatory responses to HA by VMH neurons.

  GLUTAMATE AGONIST

  NMDA. As the most common excitatory neurotransmitter in the brain, it would be expected that the glutamate receptor agonist N-methyl-D-aspartate (NMDA) would depolarize VMH neurons. Indeed, Lee-Ming Kow demonstrated that NMDA, whenever effective, evoked only depolarizations in these neurons. NMDA’s evocation of depolarization also had five characteristics: 1) reduced the latency to action potential, 2) increased the action potential number, 3) increased relative depolarization, 4) increased the depolarization rate, and 5) lowered the action potential threshold (Kow et al. 2016). Estradiol can rapidly potentiate the depolarization induced directly by bath-applied NMDA
by pico-spritzed NMDA applied at an appropriate holding potential.

  Under these circumstances, microinjections of NMDA should be able to increase lordosis behavior, but the initial data on this subject were mixed and complex. The most confusing aspects of NMDA would include the extremely rapid desensitization of its receptors and its ability to affect nearby inhibitory neurons.

  Finally, Jose Bueno, a Spanish neurologist in my laboratory for a year, showed that estrogenic treatment can increase the spontaneous activity of a subset of VMH neurons, those with extremely low firing rates. These neurons, likewise, would be expected to facilitate lordosis behavior.

  Estrogen-Dependent Hypothalamic Outflow Regulation of Lordosis Behavior

  Early in Yasuo Sakuma’s work at Rockefeller, we decided to find out which of the estrogen-binding cell groups is necessary and sufficient for lordosis behavior. As noted previously, estrogen administration limited to the VMH permits lordosis behavior and antiestrogen administration limited to the VMH prevents lordosis behavior.

  To accompany those findings, Yasuo Sakuma electrically stimulated the VMH (Pfaff and Sakuma 1979b) and electrically destroyed the VMH (Pfaff and Sakuma 1979a). In the first study, electrical stimulation of the VMH significantly increased lordosis in response to the appropriate cutaneous stimuli (Figure 2.1). This effect was significant in that electrical stimulation in the preoptic area, another estrogen-binding cell group, had exactly the opposite effect. The stimulus currents were low (12.5 microamps), the optimal stimulus frequencies were low (between ten and thirty pulses per second), and the time course of behavioral facilitation featured latencies as little as 15 minutes with peak behavior occurring at about 1 hour. Pretreatment with estrogen was absolutely necessary for the behavioral effect of stimulation, and the best lordosis responses to electrical stimulation came from the part of the VMH that has a high concentration of estrogen-binding neurons.

  Second, what about destroying VMH neurons? Bilateral lesions led to a decline of lordosis to near-zero levels within forty-eight hours, and then partial recovery after about three weeks. Lesions never abolished all of the VMH. At Emory University, Ann Clark and David Edwards extended this type of work using not only electrolytic lesions but also knife cuts of outgoing VMH fibers. Using these approaches they showed massive decreases in the percentage of time lordosis occurred in response to the male (e.g., 94 to 16 percent, and 88 to 7 percent). Also, the lesions and knife cuts severely reduced the precopulatory courtship behaviors by the female.

  Figure 2.1. Top: Electrical stimulation of neurons in the ventromedial nucleus of the hypothalamus (VMH) potentiated lordosis behavior, the duration of the effect depending on the length of stimulation. Bottom: Estrogen pretreatment potentiated the effect of VMH stimulation (Stim) on behavior compared with the unstimulated control (Pre) as a function of estradiol dose. (Adapted from Pfaff and Sakuma 1979b.)

  Kirk Manogue, a Rockefeller graduate student working with Lee-Ming Kow and myself, showed that knife cuts interrupting ascending and descending fibers at the intercollicular level could eliminate lordosis. Kirk followed that up by using knife cuts that interrupt those VMH fibers that converge with the ascending part of the lordosis circuit at the MCG (Manogue, Kow, and Pfaff 1980). If both the pathways that sweep lateral and dorsally as they head to the central grey and the medial, periventricular pathway were cut, lordosis behavior was abolished. Blocking only the medial pathway did not work.

  Kirk interrupted the lateral pathway using two different methods. Parasagittal transections at the level of the VMH showed that the lateral pathway was not required for lordosis if and only if the medial pathway to the central grey was intact. Selective parasagittal cuts farther posterior showed that among the lateral trajectories toward the central grey no particular subset was crucial for the behavior. As long as a sufficient percentage of the fibers remained unharmed, lordosis behavior could occur. The main features of the results lent themselves to the interpretation that the VMH provides a tonic estrogen-dependent facilitation of supraspinal mechanisms essential for producing lordosis, mechanisms located from the midbrain down to the medullary reticular formation.

  David Edwards and Jill Pfeifle at Emory University replicated and extended these results. Their extensive parasagittal knife cuts lateral to VMH abolished lordosis, as ours had. Then they used clever combinations of hypothalamic and midbrain cuts to show that the sweeping trajectory of VMH axons going toward MCG, earlier described by Lilly Conrad’s neuroanatomy studies, was the fiber trajectory required for normal lordosis behavior.

  What about the MCG itself? We used combinations of electrical stimulation and electrolytic lesions applied through the same electrodes to piece together the story (Sakuma and Pfaff 1979a,b). Electrical stimulation almost tripled lordosis performance (Figure 2.2). The maximum effect was evident in the very first behavioral test after the beginning of stimulation, at about 5 minutes, and lasted as long as the stimulation was on. Lesions markedly interfered with lordosis, studied in three ways. First, they abolished lordosis, but after some hours behavior returned to about one-third its prelesion level. Second, lesions abolished the effect of electrical stimulation on the behavior, both applied through the same electrodes. Third, and most important, central grey lesions blocked the facilitatory effects of VMH stimulation, demonstrating the role of the central grey in linking estrogen-dependent signals to the rest of the lordosis behavior circuit. Lesion effects were specific in that destruction too far dorsal, posterior, or lateral were not effective. Pfeifle and Edwards followed our work with a different kind of behavioral assay and reached the same conclusion: lesions that interrupted fibers descending from the central grey toward the hindbrain reduced lordosis performance to zero.

  Figure 2.2. Neurons in the midbrain central grey, having received estrogen-dependent signals from the ventromedial hypothalamus, facilitate lordosis behavior. Top: Stimulation of neurons in the midbrain central grey rapidly elevated lordosis behavior in the female rat. Greater electrical pulse amplitude was associated with higher levels of lordosis. Bottom: Estrogen pretreatment potentiated the ability of central grey (CG) electrical stimulation to facilitate lordosis, compared with the unstimulated control (Pre), as a function of estradiol dose. (Adapted from Sakuma and Pfaff 1979a.)

  Thus, MCG neurons, especially those along the lateral border which are clearly competent to receive hypothalamic inputs as shown by Sakuma’s other studies, can facilitate lordosis; and their destruction severely reduces lordosis behavior.

  How do central grey neurons exert their behavioral influence? This analysis will be discussed later.

  Sensory Inputs: How the Behavior Is Initiated

  Only somatosensory stimuli are involved. Vision, audition, olfaction, and taste are not necessary.

  Cathy Lewis, who worked in my laboratory for several years before she took up an academic career in chemistry, used frame-by-frame film analyses set against a history of ethological descriptions of reproductive behavior to determine the sensory stimuli necessary and sufficient for lordosis behavior (Pfaff and Lewis 1974). The entire chain of hormone-dependent behaviors will be covered in Chapter 7, but suffice it to say here that a competent male rat or mouse is almost always following the female, approaching from the rear. When he mounts, his forepaws contact her flanks, rump, and tail base. Then his initial pelvic thrusts will press against the skin of her perineum. The female’s vertebral dorsiflexion, the behavior essential for fertilization, starts about the time of the first thrust.

  The exact points of contact between the male and female were charted by coating the ventral surface of male rats with a dye. The flanks just in front of the female’s rear legs, the rump, the tail base, and the perineum of the female were most intensely marked (Pfaff, Montgomery, and Lewis 1977). Somatosensory, cutaneous inputs from these regions are most obviously involved. Other sensory modalities do not seem to be necessary.

  Subcutaneous injections of the local anesthetic procaine into these regions of the female’s s
kin virtually eliminate lordosis responses. However, the highest doses of estrogen could reduce the magnitude of the procaine effect. There was a hormone dose / sensory input trade-off. Likewise, surgical denervation of the female’s skin areas contacted by the male, most obviously the severing of the pudendal nerve (innervating the perineum) markedly reduced lordosis behavior (Kow and Pfaff 1976). Thus, cutaneous stimulation of the skin regions contacted by the male is necessary and sufficient for lordosis.

  Quantitative determination of cutaneous stimulus parameters that are sufficient showed that simple hair deflection is not sufficient but that light pressure on the female’s skin in the range of 50 to 450 millibars would lead to the behavior reliably (Kow, Montgomery, and Pfaff 1979). Increasing the dose of estrogen increased the probability of lordosis behavior to a given pressure of stimulus.