13 Whispered Secrets and Public Announcements
It is impossible that the whisper of a faction should prevail against the voice of a nation.
—Lord John Russell (1792–1878)1
As explained in the last chapter, a single nerve ending in the pituitary gland typically contains about 200 peptide-containing vesicles. In conditions of intense physiological stimulation, about one of these vesicles is released every 30 minutes, during which time perhaps 6,000 spikes will have invaded that ending. These nerve endings have often been likened to synaptic endings in the brain, which often contain some large, peptide-containing dense-core vesicles as well as much more abundant small synaptic vesicles that contain classical neurotransmitters. Certainly they look similar, and the molecular mechanisms by which dense-core vesicles are released are very like those by which synaptic vesicles are released. For these reasons neuropeptides are described in many journal articles and textbooks as another class of neurotransmitters, as though, like neurotransmitters, they carry a message specifically from one neuron to another. This description is, to a first approximation, a lie.2
More than 100 different peptides are released in the brain. To glimpse the complexity, consider just one small nucleus in the hypothalamus, the arcuate nucleus. This is one of many nuclei in the hypothalamus—others mentioned in this book are the supraoptic, suprachiasmatic, paraventricular, periventricular, and ventromedial nuclei, the lateral hypothalamus, and the rostral hypothalamus—others include the dorsomedial, mammillary, and supramammillary nuclei and the preoptic area.
The arcuate nucleus is at the base of the hypothalamus adjacent to the median eminence. It hosts three populations of neuroendocrine neurons: one secretes GHRH, which controls growth hormone secretion. Another secretes dopamine to regulate prolactin secretion; these fire in synchronous bursts every minute, and also express a peptide, met-enkephalin. Another population of dopamine neurons innervates the intermediate lobe of the pituitary to regulate α-MSH secretion into the blood. Other neurons control feeding: one population makes NPY and AgRP, both of which stimulate feeding, while another makes three peptides: α-MSH,which, in the brain, stimulates sexual behavior and inhibits feeding; the opioid peptide beta-endorphin; and CART (cocaine- and amphetamine-related transcript), which also inhibits feeding. Yet another neuronal population makes three more peptides kisspeptin, neurokinin B, and dynorphin, packaged in three different populations of vesicles. These regulate the pulsatile secretion of GnRH. Yet another makes somatostatin, and there is some evidence that another makes ghrelin. This exuberance is not unusual, and the list is far from exhaustive even for the arcuate nucleus. Most nuclei in the hypothalamus contain many clans of neurons that perform different physiological functions and express a diversity of peptides in addition to classical neurotransmitters. Many of these clans express several different peptides, in relative amounts that seem to vary from cell to cell. Just as all vasopressin cells express some oxytocin and all oxytocin cells express some vasopressin, it seems that all of the α-MSH cells make a small amount of NPY and all of the NPY neurons make a small amount of α-MSH. Neurons are messy things.
It is a widespread presumption that neuropeptides are an extended class of neurotransmitters: that they act at synapses, that their effects are tightly localized, and that their important effects, like those of classical neurotransmitters, are to alter the excitability of postsynaptic cells. This presumption, in all its facets, is wrong.
Vasopressin is the most abundant peptide in the brain. Most is made in the magnocellular neurons, but it is also made by neurons that regulate the secretion of ACTH from the anterior pituitary: this vasopressin is secreted together with another peptide, corticotropin-releasing hormone, into the blood vessels that supply the anterior pituitary. Vasopressin is also abundant in the suprachiasmatic nucleus, which controls daily rhythms of behavior, physiology, and hormone secretion, and these rhythms are entrained by light information that is carried by yet another population of vasopressin neurons, in the retina. Centrally projecting vasopressin neurons of the paraventricular nucleus regulate blood pressure and body temperature. Vasopressin neurons are also present in the olfactory bulb, anterior olfactory nucleus and piriform cortex, where they regulate social recognition by olfactory cues. In all of these neurons, vasopressin is packaged in large dense-core vesicles. But each of the nerve endings in the pituitary contains far more of these vesicles than has been seen in any synaptic ending in the brain. In the brain, no synapses contain 200 large dense-core vesicles or anything like that many; they contain a handful at most. Even if any synapse releases dense-core vesicles at the same rate as they are released at nerve endings in the pituitary, what sense can we make of a signal that is released on average, once for every 6,000 spikes?
Neuropeptides are fundamentally different from classical neurotransmitters. At a classical synapse, each spike typically releases (on average) one synaptic vesicle—often none, sometimes two or three; most synapses are not terribly reliable. One synaptic vesicle contains about 5,000 molecules of a neurotransmitter such as glutamate, and this is released into a narrow synaptic cleft, acts on receptors on the postsynaptic site, and is rapidly removed by transporters to be recycled. Everything is over in a few milliseconds. In the synaptic cleft, the concentration of neurotransmitter reaches very high levels, and the receptors at which it acts require these high concentrations. Peptide vesicles carry a much larger cargo (about 85,000 molecules), and their receptors are sensitive to concentrations a thousandfold lower than receptors for neurotransmitters. Peptides are broken down slowly, with half-lives that are generally a few minutes—at least 10,000 times longer than those of neurotransmitters.
We have to understand peptides as being released not from single neurons but from populations, and released in a coordinated way. Consider the magnocellular oxytocin neurons that generate milk-ejection bursts during suckling. The bursts occur every ten minutes or so for perhaps 12 hours each day, and are synchronized among 8,000 neurons. Each neuron has two dendrites: if a burst releases just 5 vesicles from each of these, then 80,000 will be released. Over a day, the average release rate from each cell would be just 1 vesicle per minute, and the total daily release would be less than a tenth of the total dendritic content.
What is released in the brain is degraded within brain tissue by aminopeptidases, and what survives arrives in the cerebrospinal fluid (CSF), from where it is cleared into the circulation. Concentrations of oxytocin are much higher there than in the blood, but only some of the oxytocin released in the brain reaches the CSF without being degraded. We can get closer to the true release rate by measuring neurophysin. Neurophysin is co-released with oxytocin in equimolar amounts, but is not significantly degraded in the brain, and for every molecule of oxytocin in CSF there are about 50 of the associated neurophysin. The rat cerebral ventricles contain about 7 pg of oxytocin—4 billion molecules—and about 200 billion molecules of neurophysin. Neurophysin is cleared from the CSF with a half-life of about 40 minutes, so we can calculate that, on average, about a million vesicles enter the CSF every 40 minutes—one every 20 seconds from each oxytocin neuron.
Clearly therefore, the notion that 10 vesicles are released from each oxytocin neuron during a milk-ejection burst is a conservative estimate. This, from 8,000 neurons, will deliver about 7 billion molecules into the hypothalamus, enough to raise the average concentration of oxytocin to 100 pM in a fluid volume of 0.1 ml. In the brain, the extracellular space is about a fifth of the tissue volume, so 0.1 ml is about the volume of extracellular fluid in a tissue volume of 0.5 ml. This is close to the total tissue volume of the rat forebrain.
Now, I am not saying that the oxytocin released in a milk-ejection burst floods the forebrain, only that enough is released to do so. Although it seems there is more than enough oxytocin in dendrites to deliver a widespread “hormonal” signal in the brain, we must consider what concentration is in fact effective at neurons, how far oxytocin released in the hypothalamus reaches, and where and how it is inactivated.
The first question, what concentration is effective, is surprisingly difficult to answer. Pharmacologists describe the actions of drugs by measuring how some response of a particular cell type varies with the applied concentration of that drug, and by measuring binding—how much of a drug is bound to receptors in a given tissue when different concentrations are applied. Generally, these measurements are reduced to single numbers, representing the concentration that will achieve a half-maximal effect (EC50), and the concentration that will achieve half-maximal binding (the binding affinity). These are only loosely related, because the effect achieved when a ligand binds to a receptor will depend on the properties of the cell and on the particular ligand. Peptides can have many different effects, and for each the EC50 may be different.
The human oxytocin receptor has an affinity for oxytocin of 0.28 nM,3 meaning that oxytocin will bind about half of the oxytocin receptors that are exposed to this concentration. Affinity is a measure of probability—the probability that a receptor will be activated by a ligand at a given concentration. For any peptide, there are two ways of achieving a greater effect—you can either increase the concentration of the peptide, or increase the density of receptors: the effects are equivalent. Thus you can’t predict what will be a physiologically effective concentration without considering both the density of receptor expression and the efficacy of the peptide—how many receptors must be activated to achieve a given effect.
This means that, while the concentrations of vasopressin and oxytocin in CSF are higher than are needed to activate peripheral tissues, we cannot be sure they are sufficient to activate neurons in the brain. Different cells in different brain regions might require much lower or much higher concentrations.
The second question, about how peptides disperse in the brain, is even more complicated. The extracellular fluid of the brain is not static, it is in constant motion. The traditional view has been that CSF is produced by the choroid plexuses in the lateral ventricles, and drains into the lymphatic space and subarachnoid space to be absorbed into the blood. More recent views suggest a much more complex picture, with continuous bidirectional fluid exchange at the blood-brain barrier. Movement of extracellular fluid is still more complex, and involves bulk flow alongside blood vessels and fiber tracts.4 At present, we do not have a clear understanding of the direction and speed of flow of extracellular fluid in different brain regions.
The third question, about where and how peptides are inactivated also has no simple answer. Peptides are degraded by enzymes—some (like the opioid peptide β-endorphin, produced by some of the arcuate neurons) are apparently not degraded at all in the brain, while others have a half-life of just a few minutes. Oxytocin is degraded mainly by placental leucine aminopeptidase, also known as oxytocinase. Oxytocinase is expressed by neurons in a regionally specific manner and is mainly anchored to the membranes of those neurons. It seems inevitable that oxytocin released in a milk-ejection burst will flood the hypothalamus and adjacent regions, but exactly where it reaches is hard to predict; there’s too much that we don’t know.
Oxytocin neurons during a milk-ejection burst fire about 100 spikes in two seconds, and I have suggested that this releases just 10 vesicles of oxytocin in the brain. A classical neuron firing this way would release about 100 synaptic vesicles from each of 10,000 axon terminals; because many terminals end on the same cells, perhaps 1,000 neurons will be affected. But the oxytocin cell is not alone, and when all are active together, their collective signal will act throughout large areas of the brain. The anatomical connectivity becomes irrelevant: all that counts is where the receptors are expressed and in what amounts. Whereas neurotransmitters are whispered secrets that pass from one neuron to another at a very specific time and place, peptides are public announcements, broadcast to whole populations.
What is true of oxytocin and vasopressin, that their effects are not localized to synapses, has to be true of all peptides in the brain. Any neuron that contains any large dense-core vesicles—and most neurons in the brain do—can release them only very infrequently, and will release these vesicles not consistently from a few sites, but sparsely from many different sites—mainly from the varicosities or swellings that stud most axons in the brain. Even if one vesicle might affect just a few neurons close to the site at which it is released, the different sites at which these vesicles are released will affect different groups of neurons and do so erratically. But when many neurons of a peptide-secreting population are activated together, the net effect will be a local, hormone-like release of peptide that indiscriminately affects all neurons that express the necessary receptors in the particular brain region that they innervate, with long-lasting effects.
Peptides operate on multiple scales: they have feedback effects on the cells of origin that modulate activity patterning, and local effects on neighboring cells to coordinate the behavior of a population; and the hormone-like release of peptides from cell populations can have organizational effects on distant targets. It’s a mode of communication quite different from neurotransmitter release. Oxytocin, as we have seen, by its priming actions, can affect how oxytocin cells communicate with each other. How common such priming actions are we don’t know. But all peptides can affect gene expression and can alter the behavior of neurons by changing what receptors they express and what they secrete. These actions of peptides together underlie what we might see as a reprogramming of communication in the brain.
Notes