22    The Evolved Brain

The species of fossils, minerals, plants, animals, which are found in the Waters, and near the surface of the Earth, are still more intricately diversified; and if we regard the different manners of their production, their mutual influence in altering, destroying, supporting one another, the orders of their succession seem to admit of an almost infinite variety. … To introduce order and coherence into the mind’s conception of this seeming chaos of dissimilar and disjointed appearances … it became necessary to suppose, first, That all the strange objects of which it consisted were made up out of a few, with which the mind was extremely familiar: and secondly, That all their qualities, operations, and rules of succession, were no more than different diversifications of those to which it had long been accustomed.

—Adam Smith (1723–1790)¹

I open this chapter with a quote from an essay by Adam Smith, author of The Wealth of Nations. When it was written is not clear; it was published posthumously in 1795. Why Smith did not publish the essay in his lifetime is not known. He did not destroy it, as he destroyed most of his unpublished papers, but he might have worried about the theological implications. The parallels with his thinking on economics are as obvious as those with evolutionary theory, and he was a close friend of the geologist James Hutton, who was certainly wary of religious sensitivities. Hutton transformed our understanding of the earth by deciphering the messages carried by rocks. What he saw told that the geological features of our world have been formed over immense time by the continual operation of diverse forces, acting from above by continual erosion and from below by intense heat and pressure; that the present appearance of our world reflects processes that appear to have been continuing indefinitely.

The hypothalamus is sometimes described as primitive, but it might be better described as highly evolved. The hypothalamus and its hormones have their evolutionary roots in Urbilateria, wormlike marine organisms that are the last common ancestor of vertebrates, flies, and worms. In Urbilateria, peptide-secreting cells probably responded to cues from the ancient marine environment. In the mammalian hypothalamus too, many neurons respond to environmental signals: some to osmotic pressure, some to sodium, some to glucose, some to fatty acids and other nutrients, and some to temperature.

Oxytocin is a sequence of nine amino acids and it differs in only two of these from vasopressin. Other similar peptides are present in many invertebrates, including locupressin in insects, conopressin in gastropods, cephalotocin in cephalopods, and annetocin in annelids. The original ancestor of these peptides appears to have emerged at about the time of evolution of a symmetrical body plan. Generally invertebrates have just one such peptide. The only known exceptions are the octopus and the cuttlefish, which have both an oxytocin-like peptide (octopressin) and a vasopressin-like peptide (cephalotocin).² In the octopus, octopressin is involved in reproduction, cardiac circulation, and feeding: it can induce contractions in muscles of the penis, oviduct, rectum, and anterior aorta, tissues that are innervated by octopressin neurons. It also regulates the electrolyte composition of the hemolymph (the invertebrate equivalent of blood) and urine. Cephalotocin has none of these effects but is secreted into the hemolymph, so it might act as a circulating hormone.

Fish first appeared about 530 million years ago during the Cambrian explosion, an apparently sudden emergence of many diverse species with a profusion of body forms. The earliest fossils are of jawless fish, like modern lampreys. Lampreys have only one peptide that is like oxytocin and vasopressin, vasotocin, which differs from vasopressin by just one amino acid in its sequence of nine amino acids. The first jawed fish appeared about 440 million years ago and included cartilaginous fish and bony fish. All cartilaginous fishes have vasotocin and also an oxytocin-like peptide. The Pacific ratfish has oxytocin itself, but six other homologs have been identified in different species: aspargtocin, valitocin, asvatocin, phasvatocin, phasitocin, and glumitocin. Bony fish have vasotocin and an oxytocin-like peptide, isotocin; they evolved into ray-finned fish, which constitute nearly all of the more than 30,000 species of modern fishes, and into lobe-finned fish, living examples of which are coelacanths and lungfish. All four-limbed vertebrates (tetrapods), including amphibians, mammals, reptiles, and birds, evolved from lobe-finned fish and all have at least one homolog of oxytocin (usually isotocin, mesotocin, or oxytocin) and one of vasopressin (vasopressin or vasotocin).

It is not just the peptides that are highly conserved through evolution. In vertebrates, the peptide genes are also highly conserved in both their structure and sequence. The cDNA for conopressin in pond snails codes for a precursor molecule that is very like the oxytocin/vasopressin precursor, and the vasotocin genes from cyclostomes, teleosts, and chicken all have a three-exon structure like that of the vasopressin gene in mammals.

In ray-finned fishes, vasotocin is involved in osmoregulation and isotocin in regulating ionic concentration. It therefore seems that, in the aquatic vertebrates where vasotocin and isotocin first appeared as distinct hormones, both were involved in salt and water balance. They may also have been involved in reproduction, the timing of which is, generally, tied to environmental conditions. Isotocin also seems to have extensive effects within the fish brain: Gil Levkowitz at the Weizmann Institute in Israel estimates that of the 300 isotocin neurons in the brain of an adult zebrafish, only about 30 project to the pituitary—the rest innervate extensive areas of the brain and, as in mammals, modulate social behavior and fear responses.³

With the emergence of land vertebrates, isotocin evolved into oxytocin through an intermediary stage of mesotocin; mesotocin differs from isotocin by just one amino acid and oxytocin from mesotocin by another. Vasotocin evolved into vasopressin; again, this change involves just a single amino acid substitution. Land mammals secrete vasopressin to concentrate the urine, and in some, including rodents, oxytocin contributes to body fluid and electrolyte balance by promoting salt excretion. These observations in species separated by several hundred million years suggest that there has been strong conservation not only of the genes but also of some functions of oxytocin and vasopressin-like peptides.

Thus vasopressin and oxytocin arose through duplication of the vasotocin gene in a species of jawless fish that lived about 400 million years ago; a separate duplication event probably gave rise to octopressin and cephalotocin in octopus and cuttlefish. This “degenerate” duplication in jawless fish allowed the neuroendocrine systems to diverge, refining and elaborating distinctive roles. In the guppy, a viviparous teleost, isotocin is involved in the induction of parturition, so this role of oxytocin that we might have imagined to be particularly mammalian has an ancient origin.

While the duplication of a peptide gene is a necessary prerequisite for the evolution of two functional systems, much more needs explanation. For two descendants of a peptide to acquire differentiated functions, different receptors must arise through which their functions can be exercised, because the actions of peptides cannot be anatomically confined to a very localized region—once released, they persist in the extracellular fluid and can diffuse or be conveyed by the flows of that fluid, sometimes to distant sites. Secondly, the two peptides must be regulated differently, and this requires that they be housed in different cells.

Gene duplication is the main way in which new genes arise. When a gene is duplicated, one copy can usually maintain the original function, leaving the other free to mutate. The duplication is not necessarily harmful, because how much protein a gene will produce is regulated. Overproduction of a secreted product will generally be compensated for by downregulation of the relevant receptors. Organisms are thus very tolerant of different levels of gene expression, and after a gene is duplicated, mutations in one copy will not necessarily lead directly to any loss of function, even if the peptide product is overproduced. When a mutation prevents one copy from encoding a functional protein it may become a “pseudogene,” a functionless remnant of the duplication event, prone to accumulating further mutations.⁴ Occasionally, however, a mutation will be beneficial and one copy will acquire a new function and become a new gene. The human genome contains about 20,000 pseudogenes, and most of these probably have no function; they seem to be evolving without any selection constraints. These pseudogenes are evidence of an evolutionary history of extensive gene duplication and are part of the “junk DNA” that litters the human genome.

There are four known receptors for oxytocin and vasopressin. Vasopressin acts in the kidney tubule to regulate the water permeability of the renal collecting ducts at the V2 receptor. Other actions of vasopressin, in the brain, pituitary, and on blood vessels, are mediated by V1a and V1b receptors, while oxytocin acts on the mammary gland and uterus at oxytocin receptors. Orthologs of these four receptors (i.e., proteins in different species that are so similar in structure, sequence, and function that they can be considered to be the same) have been described in all vertebrates investigated so far.

The four receptors are very similar, suggesting that they originated from the same ancestral gene. During the Cambrian explosion there were two episodes of whole genome duplication in vertebrates, and it is probably these that gave rise to this set of receptors.⁵ V2 receptors are different, and probably arose from an earlier gene duplication. Lampreys, which have only one member of the vasopressin/oxytocin family (vasotocin), have both V2-type receptors and V1-type receptors.⁶ Thus, when the vasotocin gene was duplicated in early vertebrate evolution, there were already two families of receptors present that could allow the functions of descendant peptides to diverge.

For their functions to diverge, vasopressin and oxytocin also had to be expressed in different neurons. When a gene is initially duplicated, the two copies will normally be expressed in the same cells. Exactly where a gene is expressed in the body is determined by regulatory elements in the DNA that are usually close to the protein-coding region of the gene, so those elements are likely to be duplicated along with the protein-coding sequence. Identifying these regulatory elements in the rat genome is hard because of the large amount of junk DNA: the rat genome has 3 billion bases, but only a few of these are in protein-coding sequences. A small proportion of the genome encodes functional RNA molecules, and some sequences control gene expression and determine the structure of the chromosomes, but about 90% of the genome consists of repetitive, mutationally degraded material with no apparent function. This includes the pseudogenes, but much more common are transposable elements. These sequences have been called “parasitic” or “selfish” because of their capacity to multiply while (in most cases) serving no useful function.

The human genome contains a similar amount of junk DNA as does the rat genome. It includes about a million copies of one particular parasitic sequence of about 300 bases: these “Alu elements” comprise about a tenth of the genome and have been implicated in many hereditary diseases, including hemophilia and breast cancer.⁷ By contrast, the pufferfish has just 390 million bases with very few repetitive elements, yet about as many genes as mammals (20,000–25,000). It might be imagined that the difference in size between the pufferfish and human genomes simply reflects the difference in complexity of the organisms. This doesn’t hold up. While the human genome has 8 times more DNA than that of a pufferfish, it has 40 times less than that of a lungfish. More than 200 salamander genomes have been analyzed so far, and all are between 4 and 35 times larger than the human genome. The pufferfish is remarkable because, for reasons still obscure, it has managed to either shed junk DNA or avoid being encumbered by it in the first place.

In the fish preoptic area, isotocin and vasotocin are expressed in separate neurons. In Bristol, David Murphy and his colleagues produced transgenic rats by inserting 40,000 bases of pufferfish DNA that included the isotocin gene. In these rats isotocin was expressed only in oxytocin neurons, and, in response to dehydration, expression of both isotocin and oxytocin was stimulated in a similar way.⁸

From these experiments we can conclude a lot. Every cell type has its own molecular “password,” a combination of a few genes that determine its identity. Genes with regulatory elements that recognize this password will then also be expressed in those cells. When a gene is duplicated, the two copies are normally expressed in the same cells because the regulatory elements that determine where it will be expressed are generally close to the gene and hence are also duplicated. Because the fish isotocin gene recognizes the mammalian oxytocin cell, that oxytocin cell must have the same password as isotocin cells in fish. This implies that the password arose early in vertebrate evolution and has been tightly conserved through subsequent evolution. Equally, the regulatory elements of oxytocin-like genes must also have appeared early in vertebrate evolution.

For a few cell types we can spell out the molecular password precisely. In zebrafish embryos, differential expression of two transcription factors, nk2.1 and pax6, separates the developing forebrain into two. The nk2.1+ region gives rise to the hypothalamus and preoptic area, and, within this, vasotocin neurons are determined by the expression of a tissue-specific microRNA, miR-7, and two other transcription factors, rx3 and orthopedia (otp). Thus the molecular password (miR-7+, nk2.1+, rx+, otp+) defines the cell type (vasotocinergic extraocular photoreceptors), and the same password defines the same cell type in the invertebrate annelid worm Platynereis dumerilii. In the dorsal preoptic area of zebrafish, vasotocin cells mingle with cells that express isotocin, and both of these cell types project to the posterior pituitary gland. Two transcriptional regulators, orthopedia b and simple-minded 1, are required for expression of vasotocin and isotocin in this region.⁹

This vasotocin system is plastic and can be influenced by many factors, including behavioral factors such as social hierarchy.¹⁰ In dominant individuals, the preoptic area has between one and three pairs of large vasotocin neurons, whereas subordinate individuals have between seven and eleven pairs of small vasotocin neurons in a slightly different location. In fish, vasotocin regulates electrolyte balance as vasopressin does in mammals and, again like vasopressin in mammals, it also influences social behavior. In the bluehead wrasse, a sex-changing fish with both territorial and nonterritorial males, vasotocin increases aggression in nonterritorial males but decreases it in territorial males.

So just as vasotocin in fish is homologous to vasopressin in mammals, the vasotocin cells of fish are homologous to the vasopressin cells of mammals, and are descended from the vasotocin cells of invertebrates. So how did the oxytocin cells diverge from vasopressin cells to become a distinct cell type?

It might be relevant that all magnocellular vasopressin cells also express some oxytocin, though mostly very little, while all magnocellular oxytocin cells express some vasopressin, though again mostly very little.¹¹ It’s hard to believe that this coexpression is functionally meaningful. These neurons make massive amounts of their principal products: the machinery for synthesizing peptides is exceptionally active, because it must make enough to provide biologically effective concentrations in the systemic circulation. But very many other peptides are expressed at low levels in these neurons. Some of these “coexisting” peptides—like dynorphin—do have important roles, but why so many others are expressed at low amounts is not clear. Perhaps the cost of evolving ways of repressing such expression completely is not worth the modest cost of some promiscuous but biologically irrelevant expression. Not everything produced by a cell necessarily matters; gene expression is, like everything in a cell, noisy and messy.

However, a few neurons express both peptides at about the same, intermediate level, and the proportion that do so increases in conditions of sustained demand. Again it seems unlikely that this ambivalence has any functional significance, but it is nevertheless interesting that it should happen at all. Perhaps related to this is the observation that, although phasic firing is a characteristic of vasopressin cells, since the earliest studies of Wakerley and Lincoln we have known that a few phasic neurons display milk-ejection bursts during suckling. Thus, some neurons have phenotypic characteristics of both oxytocin cells and vasopressin cells. Are these the neurons that express intermediate amounts of both oxytocin and vasopressin? We don’t know. These observations indicate that vasopressin cells and oxytocin cells have evolved ways to suppress the expression of the alternate peptide, and presumably, with that, to suppress the expression of things that determine the alternate neuronal phenotype. Oxytocin and vasopressin cells have many things in common but also have important differences: vasopressin cells express vasopressin receptors and oxytocin cells express oxytocin receptors, for example, and while both express dynorphin, vasopressin cells do so much more strongly. The channels that determine their intrinsic electrical properties are expressed in both clans, but at different average levels (though with considerable heterogeneity within each clan).

That a few neurons have an intermediate phenotype suggests that perhaps the developmental process of differentiation between oxytocin and vasopressin cells is itself a bistable dynamical system, that the fate of bipotential progenitor neurons is tipped one way or another by factors in the environment of the developing neurons. In other words, perhaps the same mechanism that induces some neurons to express high levels of oxytocin also represses the expression of vasopressin, and vice versa. By this reasoning the few ambivalent neurons might be a few that remain in an unstable equilibrium of cell fate determination. A similar circumstance seems to hold for neurons in the arcuate nucleus: all of the α-MSH cells appear to make some AgRP and NPY, but usually at very low levels, while those that make large amounts of NPY and AgRP all appear to produce some small amounts of α-MSH.¹²

The earliest neurons combined properties that we have thought of as separate properties of endocrine cells and neurons. They used a diversity of signaling mechanisms, made both peptides and neurotransmitters, and were endowed with a wide range of specialized senses. They had not a single role to which they were committed, but multiple behavioral and physiological functions. As these ancestor neurons proliferated in descendant species, populations differentiated not primarily by gaining properties but by losing properties. Paradoxically, we have become more intelligent as our neurons have become less complex.

Nevertheless, the neurons of the hypothalamus retain the multifunctionality of their distant ancestors, and their multitude of sensory abilities. Magnocellular oxytocin neurons regulate milk ejection, parturition, and sodium excretion by what they secrete into the blood, and there is no conflict between these roles: the uterus expresses abundant receptors to oxytocin only at term pregnancy, and the mammary gland only in lactation; the mammary gland “sees” only the pulses of secretion that occur at reflex milk ejection, while the kidney sees low concentrations, and what matters there is the secretion evoked by the background chattering of oxytocin neurons. The oxytocin neurons also govern reproductive and appetitive behaviors, and these are governed reciprocally, not by the oxytocin that is released into the blood but by oxytocin released from dendrites. Vasopressin neurons in the retina are sensitive to light; those in the supraoptic nucleus both to osmotic pressure and to temperature¹³—vasopressin is released in hot conditions to preserve body water in the face of evaporative loss. Both oxytocin and vasopressin neurons are sensitive to multiple chemical cues from the internal environment—they have receptors for glucocorticoids and gonadal steroids, and for leptin, prolactin, and insulin, as well as for many of the peptides released from the brain itself. Similar things are true of the other hypothalamic clans.

James Hutton was not a great writer, and his ideas gained influence only when re-presented by his friend the mathematician John Playfair. But he wrote one perfect line, summarizing his conclusion that the appearance of the earth’s rocks suggested a long history of continual and continuing change: “The result, therefore, of our present enquiry is, that we find no vestige of a beginning,—no prospect of an end.”¹⁴

Hutton’s line encapsulates what we see when we peer into the human genome. We know there was a beginning, but every complex animal has a genome of similar complexity to all others. The genomes display evidence of continual change, but no evidence of progression. We have a brain a million times more complex than that of a zebrafish, but we do not have a million times as many genes; we have about the same number, and they are essentially the same genes. While we are good at being humans, zebrafish are good at being zebrafish: we are not more evolved, only differently evolved. So what makes us human, and more clever and resourceful than zebrafish if we believe ourselves to be so? We must look to understand our brains less as the product of new and better rules, but more as the product of repeated iteration of the same rules of development as are implemented in less complex animals. By this reasoning, intelligence and other higher functions are not the product of new genes and new mechanisms, but emergent phenomena that arise from complexity.

Notes

1.  Smith, Adam (1795) The principles which lead and direct philosophical enquiries; illustrated by the history of the ancient physics. In Essays on Philosophical Subjects. Dublin, 134

2.  Henry J, Cornet V, Bernay B, Zatylny-Gaudin C (2013) Identification and expression of two oxytocin/vasopressin-related peptides in the cuttlefish Sepia officinalis. Peptides 46:159–166.

3.  Braida D, Donzelli A, Martucci R, Capurro V, Busnelli M, Chini B, Sala M (2012) Neurohypophyseal hormones manipulation modulate social and anxiety-related behavior in zebrafish. Psychopharmacology (Berlin) 220:319–330.

4.  Palazzo AF, Gregory TR (2014) The case for junk DNA. PLoS Genetics 10(5):e1004351.

5.  Paré P, Paixão-Côrtes VR, Tovo-Rodrigues L, Vargas-Pinilla P, Viscardi LH, Salzano FM, Henkes LE, Bortolini MC (2016) Oxytocin and arginine vasopressin receptor evolution: implications for adaptive novelties in placental mammals. Genetics and Molecular Biology 39:646–657.

6.  Mayasich SA, Clarke BL (2016) The emergence of the vasopressin and oxytocin hormone receptor gene family lineage: clues from the characterization of vasotocin receptors in the sea lamprey (Petromyzon marinus). General and Comparative Endocrinology 226:88–101.

7.  Kazazian HH Jr, Moran JV (1998) The impact of L1 retrotransposons on the human genome. Nature Genetics 19:19–24.

8.  Murphy D, Si-Hoe SL, Brenner S, Venkatesh B (1998) Something fishy in the rat brain: molecular genetics of the hypothalamo-neurohypophysial system. Bioessays 20:741–749.

9.  Eaton JL, Holmqvist B, Glasgow E (2008) Ontogeny of vasotocin-expressing cells in zebrafish: selective requirement for the transcriptional regulators orthopedia and single-minded 1 in the preoptic area. Developmental Dynamics 237:995–1005.

10.  Larson ET, O’Malley DM, Melloni RH Jr (2006) Aggression and vasotocin are associated with dominant-subordinate relationships in zebrafish. Behavioral Brain Research 167: 94–102.

11.  Gainer H (2012) Cell-type specific expression of oxytocin and vasopressin genes: an experimental odyssey. Journal of Neuroendocrinology 24:528–538.

12.  Lam BYH, Cimino I, Polex-Wolf J, Nicole Kohnke S, Rimmington D, Iyemere V, Heeley N et al. (2017) Heterogeneity of hypothalamic pro-opiomelanocortin-expressing neurons revealed by single-cell RNA sequencing. Molecular Metabolism 6:383–392.

13.  Zaelzer C, Hua P, Prager-Khoutorsky M, Ciura S, Voisin DL, Liedtke W, Bourque CW (2015) ΔN-TRPV1: A molecular co-detector of body temperature and osmotic stress. Cell Reports 13:23–30.

14.  Hutton J (1788) Theory of the earth. Transactions of the Royal Society of Edinburgh 1:304.