During human evolution, the size of the brain increased, especially in a particular part called the neocortex. The neocortex enables us to speak, dream and think.
In search of the causes underlying neocortex expansion, researchers at the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, together with colleagues at the University Hospital Carl Gustav Carus Dresden, previously identified a number of molecular players.
These players typically act cell-intrinsically in the so-called basal progenitors, the stem cells in the developing neocortex with a pivotal role in its expansion.
The researchers now report an additional, novel role of the happiness neurotransmitter serotonin which is known to function in the brain to mediate satisfaction, self-confidence and optimism – to act cell-extrinsically as a growth factor for basal progenitors in the developing human, but not mouse, neocortex.
Due to this new function, placenta-derived serotonin likely contributed to the evolutionary expansion of the human neocortex.
The research team of Wieland Huttner at the Max Planck Institute of Molecular Cell Biology and Genetics, who is one of the institute’s founding directors, has investigated the cause of the evolutionary expansion of the human neocortex in many studies.
A new study from his lab focuses on the role of the neurotransmitter serotonin in this process. Serotonin is often called the happiness neurotransmitter because it transmits messages between nerve cells that contribute to well-being and happiness.
However, a potential role of such neurotransmitters during brain development has not yet been explored in detail.
In the developing embryo, the placenta produces serotonin, which then reaches the brain via the blood circulation. This is true for humans as well as mice. Yet, the function of this placenta-derived serotonin in the developing brain has been unknown.
The postdoctoral researcher Lei Xing in the Huttner group had studied neurotransmitters during his doctoral work in Canada. When he started his research project in Dresden after that, he was curious to investigate their role in the developing brain.
Lei Xing says, “I exploited datasets generated by the group in the past and found that the serotonin receptor HTR2A was expressed in fetal human, but not embryonic mouse, neocortex. Serotonin needs to bind to this receptor in order to activate downstream signaling.
I asked myself if this receptor could be one of the keys to the question of why humans have a bigger brain.” To explore this, the researchers induced the production of the HTR2A receptor in embryonic mouse neocortex.
“Indeed, we found that serotonin, by activating this receptor, caused a chain of reactions that resulted in the production of more basal progenitors in the developing brain. More basal progenitors can then increase the production of cortical neurons, which paves the way to a bigger brain,” continues Lei Xing.
Significance for brain development and evolution
“In conclusion, our study uncovers a novel role of serotonin as a growth factor for basal progenitors in highly developed brains, notably human. Our data implicate serotonin in the expansion of the neocortex during development and human evolution,” summarizes Wieland Huttner, who supervised the study.
He continues: “Abnormal signaling of serotonin and a disturbed expression or mutation of its receptor HTR2A have been observed in various neurodevelopmental and psychiatric disorders, such as Down syndrome, attention deficit hyperactivity disorder and autism.
Our findings may help explain how malfunctions of serotonin and its receptor during fetal brain development can lead to congenital disorders and may suggest novel approaches for therapeutic avenues.”
Brain 5-HT is a neurotransmitter playing a key role in modulating neuronal circuit development and activities. The serotonergic neurons, through their extensive axonal network, are able to reach and influence nearly all the Central Nervous System (CNS) areas.
As a consequence, 5-HT regulates a plethora of functions such as sleep and circadian rhythms, mood, memory and reward, emotional behavior, nociception and sensory processing, autonomic responses, and motor activity .
Our current understanding of the development, evolution, and function of 5-HT neurotransmission is derived from different model organisms, spanning from invertebrates to vertebrates . It is noteworthy that in all species, the serotonergic network is highly plastic, showing changes in its anatomical organization all through the life of the organisms.
5-HT metabolic pathways, reuptake, and degradation are broadly conserved among multicellular organisms . 5-HT is synthesized from the amino acid tryptophan, which is an essential dietary supplement. Tryptophan is hydroxylated to 5-hydroxytryptophan (5-HTP) by the tryptophan-hydroxylase (TPH) – the rate limiting enzyme for 5-HT biosynthesis. 5-HTP, in turn, is converted in 5-HT by the aromatic L-amino acid decarboxylase.
The enzyme TPH has two distinct isoforms encoded by two genes: the Tph1 is expressed in peripheral tissues and pineal gland, while the Tph2 is selectively expressed in the CNS and in the enteric neurons of the gut . Studies on TPH -knockout (KO) mice confirmed that the synthesis of 5-HT in the brain is driven by TPH2, whereas the synthesis of 5-HT in peripheral organs is driven by TPH1 .
Since 5-HT is unable to cross the blood–brain barrier, at least in adult life, the central and the peripheral serotonergic systems are independently regulated. The synaptic effects of 5-HT are mainly terminated by its reuptake into 5-HT nerve terminals mediated by the 5-HT transporter.
The vast array of brain functions exerted by 5-HT neurotransmission in the CNS is made more complex by the interaction of the 5-HT system with many other classical neurotransmitter systems.
Through the activation of serotonergic receptors located on cholinergic, dopaminergic, GABAergic or glutamatergic neurons, 5-HT exerts its effects modulating the neurotransmitter release of these neurons [5,6].
In addition, cotransmission -here defined as the release of more than one classical neurotransmitter by the same neuron—occurs also in 5-HT neurons. Among the cotransmitters released by 5-HT neurons, glutamate , and possibly other amino acids  were identified. The regulation and functional effects of this neuronal cotransmission are still poorly understood and are the object of intense investigation .
Role of Serotonin in Morphological Remodeling of CNS Circuits
In the mammalian brain, 5-HT neurons are among the earliest neurons to be specified during development . They are located in the hindbrain and are grouped in nine raphe nuclei, designated as B1–B9 .
Although they are relatively few (about 30,000 in the mouse and 300,000 in humans), they give rise to extensive rostral and caudal axonal projections to the entire CNS, representing the most widely distributed neuronal network in the brain .
In addition to its well-established role as a neurotransmitter, 5-HT exerts morphogenic actions on the brain, influencing several neurodevelopmental processes such as neurogenesis, cell migration, axon guidance, dendritogenesis, synaptogenesis and brain wiring .
Besides the endogenous 5-HT, the brain of the fetus also receives it from the placenta of the mother. Thus, the placenta represents a crucial micro-environment during neurodevelopment, orchestrating a series of complex maternal-fetal interactions.
The contribution of this interplay is essential for the correct development of the CNS and for long-term brain functions . Therefore, maternal insults to placental microenvironment may alter embryonic brain development, resulting in prenatal priming of neurodevelopmental disorders .
For instance, in mice it has been shown that maternal inflammation results in an upregulation of tryptophan conversion to 5-HT within the placenta, leading to altered serotonergic axonal growth in the fetal forebrain.
These results indicate that the level of 5-HT during embryogenesis is critical for proper brain circuit wiring, and open a new perspective for understanding the early origins of neurodevelopmental disorders [16,17,18].
The importance of a correct 5-HT level in the brain has been demonstrated by numerous studies on mice models. When the genes involved in 5-HT uptake or degradation are knocked out, the increased 5-HT levels in the brain lead to the altered topographical development of the somatosensory cortex and incorrect cortical interneuron migration [19,20].
On the other hand, the transient disruption of 5-HT signaling, during a restricted period of pre- or postnatal development, using pharmacological (selective serotonin reuptake inhibitor exposure) animal models, leads to long-term behavioral abnormalities, such as increased anxiety in adulthood [21,22].
These animals do not show gross morphological alterations in the CNS suggesting that the lack of cerebral 5-HT may only affect the fine tuning of specific serotonergic circuits. This hypothesis has been recently confirmed using a mouse model in which the enhanced green fluorescent protein is knocked into the Tph2 locus, resulting in lack of brain 5-HT, and allowing the detection of serotonergic system through enhanced fluorescence, independently of 5-HT immunoreactivity.
In these mice, the serotonergic innervation was apparently normal in cortex and striatum. On the other hand, mutant adult mice showed a dramatic reduction of serotonergic axon terminal arborization in the diencephalic areas, and a marked serotonergic hyperinnervation in the nucleus accumbens and in the hippocampus .
These results demonstrate that brain 5-HT plays a key role in regulating the wiring of the serotonergic system during brain development. Interestingly, the transient silencing of 5-HT transporter expression in neonatal thalamic neurons affects somatosensory barrel architecture through the selective alteration of dendritic structure and trajectory of late postnatal interneuron development in the mouse cortex .
Altogether, these findings indicate that perturbing 5-HT levels during critical periods of early development influences later neuronal development through alteration of CNS connectivity that may persist into the adulthood [17,25,26]. Interestingly, recent evidence demonstrated that changes in 5-HT homeostasis affect axonal branch complexity, not only during development but also in adult life .
In adult TPH2-conditional KO mice it was shown that the administration of the serotonin precursor 5-hydroxytryptophan was able to re-establish the 5-HT signaling and to rescue defects in serotonergic system organization .
Interestingly, in recent elegant experiments that combined chemogenetics and fMRI, it was demonstrated that, in adult mice, the endogenous stimulation of 5-HT-producing neurons does not affect global brain activity but selectively activates specific cortical and subcortical areas. By contrast, the pharmacological increase of 5-HT levels determined widespread fMRI deactivation, possibly reflecting the mixed contribution of central and perivascular constrictive effects .
On the whole, findings from genetic mouse models confirm that the level of 5-HT during brain ontogeny is critical for proper CNS circuit wiring, and suggest that alterations in 5-HT signaling during brain development have profound implications for behavior and mental health across the life span. Indeed, a plethora of genetic and pharmacological studies have linked defects of brain 5-HT signaling with psychiatric and neurodevelopmental disorders, such as major depression, anxiety, schizophrenia, obsessive compulsive disorder and Autism Spectrum Disorders (ASD) [17,29,30].
In addition, it is becoming increasingly clear that 5-HT has a crucial role also in the maintenance of mature neuronal circuitry in the brain, opening novel perspectives in rescuing defects of CNS connectivity in the adult. For instance, the potential of 5-HT neurons to remodel their morphology during the entire life is indicated by the well-known capability of 5-HT axons of the adult to regenerate and sprout after lesions [26,31].
However, understanding the cellular and molecular mechanisms underlying the effects of 5-HT during brain development, maintenance and dysfunction is challenging, in part due to the existence of at least 14 subtypes of receptors (5-HTRs) grouped in seven distinct classes (from 5-HT1R to 5-HT7R). All 5-HT receptors are broadly distributed in the brain where they display a highly dynamic developmental and region-selective expression pattern and trigger different signaling pathways.
The 5-HT receptors are typical G-protein-coupled-receptors with seven transmembrane domains, with the exception of the 5-HT3 receptor, which is a ligand-gated ion channel .
Role of the 5-HT7R in Shaping Neuronal Circuits
The 5-HT7R, the last discovered member of the 5-HTR family [33,34], has always been the subject of intense investigation, due to its high expression in functionally relevant regions of the brain [35,36]. Accordingly, several recent data have elucidated its role in a wide range of physiological functions in the mammalian CNS and also in peripheral organs .
Interestingly, emerging findings indicate that 5-HT7R is involved in brain plasticity, being one of the players contributing not only to shape brain networks during development but also to remodel neuronal wiring in the mature brain, thus controlling higher cognitive functions (see Section 2.2 and Section 2.3).
Therefore, this receptor is currently considered as potential target for the treatment of several neuropsychiatric and neurodevelopmental disorders, (as discussed in Section 3), also in view of the fact that its ligands have a wide range of neuropharmacological effects [38,39].
In the mammalian CNS, the 5-HT7R is mainly expressed in the spinal cord, thalamus, hypothalamus, hippocampus, prefrontal cortex, striatal complex, amygdala and in the Purkinje neurons of the cerebellum [40,41]. This wide distribution reflects the numerous functions in which the receptor is involved, such as circadian rhythms, sleep-wake cycle, thermoregulation, learning and memory processing, and nociception .
In mammals, this receptor exhibits a number of functional splice variants due to the presence of introns in the 5-HT7R gene and to alternative splicing. The splice variants of the receptor, named 5-HT7(a), (b), (c) in rodents, and 5-HT7(a), (b), (d) in humans [36,42,43], do not show significant differences in localization, ligand binding affinities, and activation of adenylate cyclase . To date, the only functional difference between the splice variants is that the human 5-HT7(d) isoform displays a different pattern of receptor internalization compared to the other isoforms .
The 5-HT7R is a G protein-coupled receptor, that activates at least two different signaling pathways. The classical pathway relies on the activation of Gαs and the consequent stimulation of adenylate cyclase, leading to an increase in cyclic adenosine monophosphate (cAMP).
The latter activates protein kinase A (PKA), that in turn phosphorylates various proteins such as the mitogen-activated protein kinase and extracellular signal-regulated kinases (ERK) .
Another 5-HT7R pathway depends on the activation of Gα12, that in turn triggers stimulation of Rho GTPases, Cdc42 and RhoA; these intracellular signaling proteins, critical for the regulation of cytoskeleton organization, lead to morphological modifications of fibroblasts and neurons .
5-HT7R signaling also involves changes in intracellular Ca2+ concentration and Ca2+/calmodulin pathways [46,47], as well as PKA independent mechanisms which include exchange protein directly activated by cAMP (EPAC) signaling .
5-HT receptor signaling has been recently shown to also depend on their oligomerization. In particular the 5-HT7R can form homodimers, as well as heterodimers with 5-HT1AR .
The latter, when is in a monomeric conformation, causes a decrease in cAMP concentration through activation of the Gi. Heterodimerization with 5-HT7R inhibits the 5-HT1AR cAMP signaling pathway, while homodimerization of both receptors do not influence the respective cAMP pathways. These findings suggest that oligomerization of G-protein-coupled-receptors may have profound functional consequences on their downstream signaling, thus triggering cellular and developmental-specific regulatory effects.
Role of the 5-HT7R in Shaping Neuronal Circuits during Development
The influence of the 5-HT7R on neuronal morphology has stimulated interest in studying its potential role in the establishment and maintenance of brain connectivity and in synaptic plasticity. The availability of selective agonists and antagonists, as well as that of genetically modified mice lacking the 5-HT7R, has shed light on the physio-pathological role of this receptor [39,50,51].
By using rodents’ primary cultures of hippocampal neurons and various 5-HT7R agonists in combination with selective antagonists, it was consistently shown that the pharmacological stimulation of the endogenous 5-HT7R promotes a pronounced extension of neurite length [48,52,53].
The morphogenic effects of 5-HT7R stimulation have also been demonstrated in cultured neurons from additional embryonic forebrain areas, such as the striatum and the cortex [54,55] (Figure 1). Neurite elongation was shown to rely on de novo protein synthesis and multiple signaling systems, such as ERK, Cdk5, the RhoGTPase Cdc42 and mTOR. These pathways converge to promote the reorganization of the neuronal cytoskeleton through qualitative and quantitative changes of selected proteins, such as microtubule-associated proteins and cofilin [54,56].
In hippocampal neurons, it has been demonstrated that 5-HT7R finely modulates the NMDA receptors activity [57,58]. Furthermore, 5-HT7R activation increases phosphorylation of the GluA1 AMPA receptor subunit and AMPA receptor-mediated neurotransmission in the hippocampus [59,60]. Consistent with these findings, 5-HT7R-KO mice display reduced LTP in the hippocampus .
Chronic stimulation of the 5-HT7R/Gα12 signaling pathway promotes dendritic spine formation, enhances basal neuronal excitability, and modulates LTP in organotypic slices preparation from the hippocampus of juvenile mice. Interestingly, 5-HT7R stimulation does not affect neuronal morphology, synaptogenesis, and synaptic plasticity in hippocampal slices from adult animals, probably due to decreased hippocampal expression of the 5-HT7R during later postnatal stages .
It has been recently hypothesized that this decline could be due to the simultaneous upregulation of the microRNA (miR)-29a in the developing hippocampus. Indeed 5-HT7R mRNA is downregulated by the miR-29a in cultured hippocampal neurons, and miR-29a overexpression impairs the 5-HT7R-dependent neurite elongation .
Neuronal remodeling is highly influenced by the extracellular matrix. Accordingly, it has been shown that the physical interaction between the 5-HT7R and the hyaluronan receptor CD44, a main component of the extracellular matrix, plays a crucial role in synaptic remodeling.
Briefly, stimulation of the 5-HT7R increases the activity of the metalloproteinase MMP-9, which, in turn, cleaves the extracellular domain of CD44. This signaling cascade promotes detachment from the extracellular matrix, thus triggering dendritic spine elongation in the hippocampal neurons of the mice .
In accordance with the influence of the 5-HT7R signaling pathways in remodeling developing forebrain neuron morphology, it was shown that prolonged stimulation of this receptor and the downstream activation of Cdk5 and Cdc42 increased the density of filopodia-like dendritic spines and synaptogenesis in cultured striatal and cortical neurons .
The crucial role of 5-HT7R in shaping developing synapses (Figure 1) was confirmed by the pharmacological inactivation of the receptor as well as through the analysis of early postnatal neurons isolated from 5-HT7R-deficient mice. It is noteworthy that, when 5-HT7R was blocked pharmacologically, and in 5-HT7R-KO neurons, the number of dendritic spines decreased, suggesting that constitutive receptor activity is critically involved in dendritic spinogenesis. From this point of view, a detailed analysis of dendritic spine shape and density in the brain of 5-HT7R-KO mice at various ages would be crucial to assess the physiological effects of this receptor on neuronal cytoarchitecture.
The involvement of 5-HT7R in spinogenesis and synaptogenesis—together with the demonstration that its activation is able to stimulate protein synthesis-dependent neurite elongation, as well as axonal elongation [54,56]—suggests the intriguing possibility that the activation of this receptor may be linked to the axonal and synaptic system of protein synthesis. The local system of protein synthesis has been demonstrated to play a crucial role in synaptic plasticity—although its regulatory mechanisms are only partially understood [66,67,68]—and 5-HT7R and its related pathways are good candidates to be part of this system.
Role of the 5-HT7R in Remodeling Neuronal Circuits in Adults
Neuronal circuits remain able to reorganize in response to experience well into adulthood, continuing to exhibit robust plasticity along the entire life . Consistently, the action of 5-HT7R on the modulation of neuronal plasticity is not restricted to embryonic and early postnatal development, but can also occur in later developmental stages and in adulthood (Figure 1).
Interestingly, it was shown that selective pharmacological stimulation of 5-HT7R during adolescence determines its persistent upregulation in adult rat forebrain areas . Likewise, it has been hypothesized that 5-HT7R may underlie the persistent structural rearrangements of the brain reward pathways occurring during postnatal development, following exposure to methylphenidate, the elective drug for the treatment of Attention Deficit Hyperactivity Disorder .
Accordingly, stimulation of the 5-HT7R in adolescent rats leads to increased dendritic arborization in the nucleus accumbens—a limbic area involved in reward—as well as increased functional connectivity in different forebrain networks likely to be involved in anxiety-related behavior .
Changes in dendritic spine formation, turnover and shape occur during the entire life span in response to stimuli that trigger long-term alterations in synaptic efficacy, such as LTP and LTD [73,74,75]. Consistently, it has been shown that the activation of 5-HT7R in hippocampal slices from wild type mice (as well as in Fragile X Syndrome mice, see next paragraph) reverses LTD mediated by metabotropic glutamate receptors (mGluR-LTD), a form of plasticity playing a crucial role in cognition and in behavioral flexibility .
Moreover, the acute in vivo administration of a selective 5-HTR7 agonist improved cognitive performance in mice . These results are consistent with the hypothesis that long-term changes of synaptic plasticity, which are a substrate of learning and memory formation, lead to neural network rewiring (Figure 1). Accordingly, the 5-HT7R-KO mice exhibit reduced hippocampal LTP, and specific impairments in contextual learning, seeking behavior and allocentric spatial memory [61,77].
Interestingly, the expression level of 5-HT7R in the hippocampal CA3 region, an area of the brain involved in allocentric navigation, decreases with age , suggesting that the spatial memory deficits associated with aging could be attributed to decreased 5-HT7R activity in this region of the brain. Conversely, another group reported that hippocampal expression of 5-HT7R does not change with age, but exhibits 24 h rhythms .
This observation should be taken into account in the interpretation of previous findings, as well as in planning future experiments. Several other studies have produced contradictory results related to the involvement of 5-HT7R in memory and attention-related processes [80,81], probably due to experimental differences (animal strain, behavioral tests, compounds and doses, route of administration, etc.). In conclusion, although the role of this receptor on cognitive functions needs to be fully elucidated, it is clear that it modulates various aspects of learning and memory processes.
Interestingly, the 5-HT7R is also involved in bidirectional modulation of cerebellar synaptic plasticity, since its activation induces LTD at the parallel fiber-Purkinje cell synapse, whereas it blocks LTP induced by parallel fiber stimulation . These results suggest that the receptor might be involved in motor learning, a cognitive function depending on the activity of cerebellar circuits .
Altogether, these findings strongly suggest that the 5-HT7R plays a role in modulating synaptic plasticity and neuronal connectivity in both developing and mature brain circuits, although the molecular and cellular mechanisms underlying this modulation are only partially understood (Figure 1).
reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7013427/
More information: Lei Xing et al, Serotonin Receptor 2A Activation Promotes Evolutionarily Relevant Basal Progenitor Proliferation in the Developing Neocortex, Neuron (2020). DOI: 10.1016/j.neuron.2020.09.034