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Neurotransmitters and neuromodulators during early human development
Early Human Development 65 (2001) 21 – 37 www.elsevier.com/locate/earlhumdev
Review article
Neurotransmitters and neuromodulators during early human development Eric Herlenius, Hugo Lagercrantz* Neonatal Unit, Department of Women and Child Health, Astrid Lindgren Children’s Hospital, Karolinska Institutet, S-171 76 Stockholm, Sweden Received 22 March 2001; received in revised form 4 June 2001; accepted 5 June 2001
Abstract Background: Neurotransmitters such as monoamines appear in the embryo before the neurones are differentiated. They may have other functions than neurotransmission during embryogenesis such as differentiation and neuronal growth. For example, serotonin may act as a morphogen. A number of neuropeptides are expressed during ontogenesis, but their function has been difficult to establish. Maybe some of them remain as evolutionary residues. Fast-switching neurotransmitters like the excitatory amino acids and the more ionotropic receptors dominate in the human brain, but appear probably later during evolution as well as during ontogeny. Methods: The distribution of catecholamines during development has been analysed with a fluorescense method, while most of the other neuortransmitters have been mapped with immunohistochemical methods. The classical method to determine the physiological role of a neurotransmitter or modulator is to study the physiological effect of its antagonist, blocking the endogenous activity. By transgenic technique, the genes encoding for enzymes involved in the synthesis of neurotransmitters can be knocked-out. Major findings: Pharmacological blocking of endogenous activity has, for example, demonstrated that adenosine suppresses fetal respiration. Knocking out the dopamine beta-hydroxylase gene results in fetal death, suggesting that noradrenaline is essential for survival. Some neurotransmitters change their effect during embryogenesis, e.g. GABA which is excitatory in the embryo, but inhibitory after birth due to a switch from a high to low chloride content in the nerve cells. It is possible that this is of importance for the wiring of neuronal network in early life. NMDA receptors dominate in the foetus, while kainate and AMPA receptors appear later. At birth, there is a surge of neurotransmitters such as catecholamines, which may be of importance for the neonatal adaptation. Conclusions: Neurotransmitters and modulators are not only important for the neural trafficking in the embryo, but also for the development of the neuronal circuits. Prenatal or
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Corresponding author. Tel.: +46-8-5177-4700; fax: +46-8-5177-5121. E-mail address: [email protected] (H. Lagercrantz).
0378-3782/01/$ – see front matter D 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 3 7 8 2 ( 0 1 ) 0 0 1 8 9 - X
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neonatal stress (hypoxia), as well as various drugs, may disturb the wiring and cause long-term behavioural effects (fetal and neonatal programming). D 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neurotransmitters; Neuromodulators; Early human development
1. Introduction Although genes mainly determine the development of the scaffold of the CNS, the detailed wiring of the neuronal circuits is to a large degree self-generated dependent on the action of neurotransmitters and neuromodulators. They can promote, amplify, block, inhibit or attenuate the micro-electric signals which are passed on to neurones. Thereby, they give rise to the signalling patterns between myriads of neuronal networks providing the physical networks of cerebral neurones. Neurotransmitters such as the catecholamines appear in the embryos of vertebrate and invertebrate animals even before neurones are differentiated [1]. Some of the cells in the neuronal crest contain noradrenaline from the outset, but become cholinergic due to environmental influences [2]. Many neuroactive molecules change their functional role in the CNS during development. The same molecule may be crucial for differentiation, neuronal growth and establishment of neuronal networks in the immature CNS while switching to a more modulatory role of the ongoing traffic in the mature CNS. Receptor subunits may exchange during development, i.e. the NMDA receptors, whose subunits allow longer open channel time during early development then switch to a shorter, more stable adult subunit composition. This is of importance for the plasticity of the immature brain and subsequently, for memory storage and preservation in the adult brain [3]. Noradrenaline and acetylcholine are regarded as classical neurotransmitters and dominate in the peripheral nervous system (Fig. 1). They appear at an early stage during both phylogenesis and ontogenesis. Many of the neuropeptides were first identified in the gastrointestinal tract and probably appear early during CNS development. They act slowly since they have to be synthesised and packaged in the cell soma and carried to the terminals before they can be released. The recently evolved and more sophisticated mammalian brain requires more fast-switching neurotransmitters acting directly on ion-channels. Therefore, excitatory and inhibitory amino acids seem to dominate in the mature CNS, where the monoamines and neuropeptides may act more as neuromodulators (see Ref. [4]). The distinction between a transmitter and a modulator is far from clear, since several of the neuroactive agents described to date change their role during brain development or have different actions depending on brain region or innervated neurones. Furthermore, a given transmitter may have different effects depending on brain region, postsynaptic receptor configuration, G-protein coupling and second messenger system. A neuroactive agent can be expressed in high amounts during certain stages of development, but then persists in only a few synapses [5]. It is possible that this agent
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Table 1 Major neurotransmitters and neuromodulators presented in presumed order of appearance during ontogeny Purines Adenosine, ATP Monoamines Serotonin (indoleamines) Dopamine, noradrenaline, adrenaline Neuropeptides Opioids: enkephalins, endorphin, dynorphin Tachykinins: substance P, neurokinin Glucagon-related: vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase activating peptide (PACAP) Neuropeptide Y (NPY) Somatostatin Neurotensin, calcitonin gene-related peptide (CGRP) Acetylcholine Amino acids Glycine, GABA, glutamate, aspartate
either has only a transitory role in a critical window in development or that it remains mainly as an evolutionary residue, with minor functions in, e.g. mammals. If the synthesis of some of these neurotransmitters/modulators is blocked pharmacologically or knocked-out by transgenic techniques, it does not seem to affect survival or even important physiological functions. This illustrates the plasticity of the brain during early development. Other neuroactive agents seem to be able to take over. Markers for neurotransmitters and neuromodulators during CNS development generally appear first in the caudal and phylogenetically older part of the brain probably due to earlier neurogenesis (see Ref. [6]). Classification of the main neurotransmitters and modulators according to principal biochemical differences and tentative ontogenetic appearance is depicted in Table 1.
2. Receptors The neurotransmitters or modulators can act on either metabotropic or ionotropic receptors (see reviews by, e.g. Ref. [7]). The action of the metabotropic receptors is based on their effects on G- or N-proteins in the lipid bilayer of the membrane to affect their enzymes and channels. This effect is slower (tens of milliseconds) than for the ionotropic receptors. Metabotropic receptors are probably expressed at an earlier stage during ontogeny and play a more modulatory role in the mature CNS. The ionotropic receptors respond rapidly and are also termed class I receptors. They act on ion gates, which they can open or close in less than a millisecond. The ion channels consist of transmembrane proteins, which can be selective for cations (activatory receptors) or anions (inhibitory). The ionotropic nicotinic acetylcholine receptor (nAcR), the GABAA-R and glycine receptor GlyR are members of the same evolutionary super
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family and have a similar structure. A fetal subunit of the acetylcholine receptor (gammaAchR) is replaced by an adult type (epsilon-AchR) in the muscle end-plate to increase the conductance [8].
3. Ontogeny of neurotransmitter systems The choice of neurotransmitter of a precursor neuron depends on the environment. In a series of remarkable experiments, Le Douarin [9] demonstrated that when the sympathetic trunk crest from a quail was transplanted into the vagal region of a chick host, the nerves became cholinergic. Conversely, when vagal neurones were transplanted into the sympathetic trunk, the nerves became adrenergic. The expression of neurotransmitter type seemed to be dependent on a tissue factor. When sympathetic ganglia cells were cultivated in a medium from a heart cell culture, the adrenergic neurones became cholinergic [2]. The choice of transmitter could also be affected by corticosteroids. Thus, environmental factors are important for the differentiation and which neuroactive agent will be used in communication with the surrounding cells.
4. Catecholamines Catecholamines can be found in protozoa as well as in the very early embryo. The synthesising enzyme tyrosine hydroxylase has been detected the first day of/after incubation of the chicken; dopamine the second day, and noradrenaline and adrenaline the third day. High concentrations of catecholamines have been found in Hensen’s node, corresponding to the notochord of the mammalian embryo (see Ref. [1]). Noradrenaline is essential for normal brain development. The noradrenergic system regulates the development of the Cajal-Retzius cells, which are the first neurones to be born in the cortex and proposed to be instrumental in neuronal migration and laminar formation [10]. Administration of 6-OH-dopamine prevents the natural-programmed cell death of these neurones and delays the formation of cortical layers. Lesioning the noradrenergic projections or blocking neurotransmission with receptor antagonist prevents astrogliosis and glial cell proliferation. Depleting noradrenaline during the perinatal period results in subtle dendritic changes and possibly also alterations in cortical differentiation (see Ref. [11]). The role of noradrenaline has been investigated by targeted disruption of the dopamine b-hydroxylase (DBH) gene. The DBH locus of the DBH proximal promoter and the first exon were replaced with a neomycin-resistance cassette [12]. This resulted in fetal death, probably due to cardiovascular failure. Only about 5% of the homozygotic mice survived until adulthood, presumably due to some placental transfer of noradrenaline. Most of the mice could be rescued to birth by providing them with dihydroxyphenylserine (DDPS), a precursor that can be converted to noradrenaline in the absence of DBH. These mice had a reduced ability of acquisition and retention for some tasks. Interestingly, female mice seemed to have deficient ability to take care of
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their offspring. Thus, there seems to be a critical window during early development when noradrenaline is involved in forming the pathways responsible for maternal behaviour [13].
Fig. 1. Shows schematic sagittal illustrations of cell bodies and projections of monoamine neurotransmitter systems. Acetylcholinergic pathways, dopaminergic pathways, serotoninergic pathways and noradrenergic pathways in the human brain. Modified from L. Heimer: The human brain and the spinal cord. Springer, NY, 1995.
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Fig. 2. Arbitrary levels of monoamines and acetylcholine in man versus age (10-logarithmic scale). Data from Refs. [14,58,59].
Dopamine plays a very important role in motor and cognitive programmes. Dopaminergic neurones appear early during development 6 – 8 weeks in human [14] (Fig. 2), earlier in females than in males. The dopamine turnover is relatively high during the perinatal period compared to adults. Extremely high levels of D1 receptors have been reported in the pallidum during the perinatal period [15]. D1 receptor stimulation regulates transcription of other genes, and it is possible that abnormal perinatal stimulation can result in long-term consequences (see below). Disturbances of the development of the dopaminergic system may lead to dyskinesia, dystonia, tics, obsessive-compulsive disorders and abnormal eye movements. This has been observed in DA-depleted rats after 6-hydroxydopamine treatments but with preserved noradrenaline effect. Tyrosine– hydroxylase gene-deleted mice were hypoactive and suffered from adipsia and aphagia, which could be treated with L-dopa [16].
5. Serotonin Serotonin can already be detected in the fertilised egg and is involved in early morphogenesis of the heart, the craniofacial epithelia and other structures. If embryos are cultured in the presence of serotonin uptake inhibitors or receptor ligands, specific craniofacial malformations occur. Serotoninergic cells in the raphe are among the earliest to be generated in the brain (about E11 to E15 in the rat and between weeks 5 and 12 in the human foetus) (Fig. 2). These cells send axons to the forebrain and may be of importance in the differentiation of neuronal progenitors [17]. Excess of serotonin prevents the normal development of the somatosensory cortex, which has been demonstrated in monoamine oxidase knockout mice [18]. At birth, serotonergic neurones penetrate all cortical layers, but then decline markedly after about 3 weeks. Depletion of serotonin after birth seems to have little effect on cortical development. There are some indirect evidences that reduced serotonergic innervation of the cortex results in impaired behaviour in juvenile rats. Autism has been suggested to be related to hyperserotonism.
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6. Drugs affecting monoaminergic activity Cocaine is probably the most well-known drug interacting with the catecholaminergic systems in the brain during development [19]. It inhibits the presynaptic transport mechanisms, removing and terminating the action of dopamine and noradrenaline. While cocaine potentiates the catecholamine effects in the adult, it inhibits the activity during the immediate postnatal period in most brain regions. Prenatal cocaine exposure results in disturbance of neuronal migration and consequently leads to severe neurobehavioural disturbances. Prenatal cocaine exposure in humans causes abnormal motor behaviour immediately after birth and abnormal behaviour is apparent at 2 and 3 years follow-up, probably mainly due to disturbance of the dopaminergic system. Neuroleptic drugs administrated during pregnancy can block dopamine receptors and cause long-lasting effects (see Ref. [15]).
7. Acetylcholine Acetylcholine is one of the major neurotransmitters in the brain of importance for cortical activation, memory and learning. It has a major role in the control motor tone and movement and probably counterbalances the effect of dopamine (see Ref. [20]). The cholinergic innervation of the cortex occurs later than the monoaminergic around week 20 in the human foetus. Mature levels in rodents are not reached until after 8 weeks postnatally (see Ref. [11]). The concentrations of ACh reach about 20% of the adult levels at E15 in the whole brain of the rat and about 40% at day P7 (Fig. 2). The levels of choline acetyltrasferase (ChAT) are much lower (1% and 8%) at the corresponding ages, indicating low firing rates of the cholinergic neurones. Conversely, the receptors reach adult levels earlier. The cholinergic markers appear sooner in the pons-medulla, probably due to earlier neurogenesis in the caudal and phylogenetically older part of the brain (see Ref. [6]). The cholinergic afferents seem to have an important role in the differentiation of the cortex. After a considerable reduction of the cholinergic innervation, a delay of cortical cytodifferentiation was revealed (see Ref. [11]). The cholinergic innervation has been found to be disturbed in Down’s syndrome, lead and ethanol toxicity and asphyxia. Perinatal manipulations of the cholinergic system result in major changes of cortical structure. These changes can be correlated to cognitive deficits but do not affect motor behaviour.
8. Amino acid transmitters The amino acids are involved in the wiring of neuronal networks and building CNS cytoarchitecture [21] (Fig. 3). Amino acid transmitters are the most abundant transmitters in the central nervous system. However, they were recognised as neurotransmitters in the mammalian brain much later than the monoamines and acetylcholine. This was probably due to the fact that they are involved in intermediate metabolism and constitute important building blocks in the proteins.
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Fig. 3. Expression and arbitrary levels of receptors of amino acid transmitter versus age in man (10-logarithmic scale). Data from Refs. [58 – 62]. NMDA receptors are expressed relatively earlier than the kainate and AMPA receptors. GABA operates mainly as an excitatory transmitter on immature neurones. GABA receptors can facilitate the activation of NMDA receptors, playing the role conferred to AMPA receptors later on in development. It is assumed that the NMDA receptors are more involved in the wiring of the brain while the kainate and AMPA receptors are responsible for the fast traffic in the more mature brain. GABA operates mainly as an excitatory transmitter on immature neurones. GABA receptors can facilitate the activation of NMDA receptors, playing the role conferred to AMPA receptors later on in development. Embryonic age (E) expressed in weeks, postnatal age in years.
Glutamate and aspartate are the dominating excitatory amino acids (EAA) and the primary neurotransmitter in about half of all the synapses in the mammalian forebrain. They constitute the major transmitters of the pyramidal cells, the dominating neurones in the cortex. This has been demonstrated by injection of radioactively labelled D-(3H) glutamate into the appropriate projection areas (see Ref. [5]). EAA pathways undergo striking developmental changes, involving transient overshoots, especially during critical periods as evident in visual cortex and hippocampus. EAA terminals are overproduced during the early postnatal period, for example after 1– 2 years in the human cortex (see Fig. 3), which may be related to the high generation of synapses during those periods. Glutamate acts on at least five types of receptors. The slower acting metabotropic receptors, eight subclasses are hitherto known, are expressed at a relatively early stage. Of the ionotropic receptors, the NMDA receptors dominate in the immature brain when synaptic transmission is weak and extremely plastic (Fig. 3). The NMDA receptors permit entry of Na + and Ca2 + when opened and may mediate neurotoxic effects during perinatal asphyxia. Furthermore, Ca2 + entry through NMDA channels seems to be crucially involved in the appearance of long-term potentiation (LTP) and memory storage. During maturation, the AMPA and kainic ionotropic receptors predominate and carry most of the fast neuronal traffic in the brain [4].
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Dark rearing or blocking the activity with tetrodotoxin results in preservation of the NMDA receptors in the visual cortex. Dark rearing also preserves the immature form of the NMDA receptors containing NR2B subunit and the expression of NR2A is delayed. This subunit switch is essential for development of rapid synaptic transmission [3]. Fetal rats exposed to NMDA antagonists were found to have excessive apoptosis in the same way as the asphyxiated perinatal brain. NMDA receptor stimulation by excessive glutamate release leads to Ca+ + influx, which may induce subsequent neuronal apoptosis. ‘‘Thus, either too much or too little NMDA receptor activity can be life-threatening to the developing neurones’’ [22]. Gamma-aminobutyric acid (GABA) is the dominating neurotransmitter in the nonpyramidal cells, as demonstrated by uptake of (3H)-GABA and immunochemical labelling of the GABA-synthesising enzyme glutamic acid (GAD). Perhaps 25 –40% of all nerve terminals contain GABA. GABA is regarded as the main inhibitory transmitter in the mature animal, but has a different role during early development. There are two types of GABA receptors: GABAA and GABAB. The GABAA-receptor (GABAA-R) is an ionotropic receptor that gates a chloride channel. It is a transmembrane protein built of several subunits where, for example benzodiazepines, barbiturates and ethanol, can bind to specific sites and modulate the opening properties of the chloride channel. The GABAB-R is coupled to a G-protein, is present in lower levels in the CNS than the GABAA-receptor and starts to function late in CNS development (postnatal life in rodents). During early development, the Cl concentration is high in the nerve cells. When GABA opens the Cl channels, a depolarisation (i.e. excitation) occurs. During maturation, the Cl concentration decreases which results in an opposite effect of GABA, i.e. Cl ions are pumped out and the cell becomes hyperpolarised. In this way, GABA switches from an excitatory to an inhibitory neurotransmitter [23]. This switch occurs around birth in the rat [23] but it is not known when it occurs in the human. Thus, GABA operates mainly as an excitatory transmitter on immature neurones. As described above, glutaminergic synapses initially lack functional AMPA receptors and the NMDA channels are blocked by Mg2 + at resting membrane potentials. GABA depolarises immature neurones, which may result in Ca2 + influx by removing the Mg2 + blockage of NMDA channels. Thus, GABAA-receptors play the role conferred to AMPA receptors in the more matured CNS [21,24]. An increase in the intracellular Ca2 + concentration activates a wide range of intracellular cascades and is involved in neuronal growth and differentiation. Furthermore, GABA excitation and Ca2 + influx may act as triggers for plasticity of synaptic connections and for establishing and patterning of neural networks. The GABAA-receptors have a strong affinity for benzodiazepines. Several anxiolytic and anticonvulsant drugs increase the ability for GABA to open chloride channels. In neonatal neurones, GABA currents are potentiated by barbiturates but are insensitive to benzodiazepines [25]. As GABA has a trophic role during early brain development, interference with the function of GABAergic transmission during this period may affect the development of neuronal wiring, plasticity of neuronal network, and also have a profound influence on neural organisation.
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In fact, ethanol, abused by some mothers during pregnancy, interacts with the GABAA-receptor. The sensitive time window in rat cerebral cortex for ethanol exposure is situated between P3 and P10. It is worth noting that GABA, during this same period, seem to have mainly depolarising and trophic effects on developing cortical neurones through effects on cell proliferation and migration [26]. In humans, the intellectual deficits produced by abnormalities of brain growth are the most important components of fetal alcohol syndrome [27]. Craniofacial abnormalities in human foetuses related to first trimester alcohol exposure are similar to the facial defects seen in GABAA subunit receptor knockout mice [28]. Thus, ethanol interaction with the GABAA-receptor could be implicated in the pathogenesis of fetal alcohol syndrome. Glycine has both excitatory and inhibitory actions and can be regarded as the phylogenetically older inhibitory transmitter restricted to the brain stem and spinal cord in the adult. A similar switch as regarding the GABAA-receptors from excitatory to inhibitory effects seems to occur with maturation [23]. The NMDA receptor has a modulatory site where glycine in submicromolar concentrations increases the frequency of NMDA-receptor channel opening. Conditions that alter the extracellular concentration of glycine can dramatically alter NMDA-receptormediated responses.
9. Neuropeptides More than 50 neuropeptides have been identified. In contrast to most of the other neurotransmitters/modulators, the neuropeptides are synthesised and packaged in large dense-cored vesicles in the cell soma and are carried to the nerve terminals by axonal transport at a rate of 1.5 mm/h. It is obvious that by this relatively slow process, the neuropeptides cannot act as fast-switching neurotransmitters. Rather, they have a neuromodulatory role. They are often stored together with other neurotransmitters, i.e. monoamines or EAA, and it is possible that they play a role in setting of the sensitivity [29]. Some of them are probably of less physiological importance and occur in the body mainly as evolutionary residues [30]. Still they are of great neuropharmacological interest and their analogues or antagonists can be used as drugs. The most well-known examples are the opioids and naloxone. b-Endorphin is expressed early during fetal brain development and seems to be involved in fetal growth and postnatal development at least in the rat [31]. mReceptor binding sites are present during mid-fetal time and have a high density in cardiorespiratory-related brainstem nuclei, whereas the d-opioid receptors primarily appear during the postnatal period in rats [32]. Although, opioid binding sites progressively increase in the developing brain (Fig. 4), the effect of opioids appears to be dependent on the status of neuronal maturation. In addition, many neuronal populations exhibit transient expression of one or the other opioid genes but the physiological role of this is not clear. Opioid agonists inhibit mitosis and DNA synthesis in the developing brain and endogenous opioids exert potent regulatory effects on brain development and morphogenesis, as demonstrated by the admin-
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Fig. 4. Expression and arbitrary levels of some major neuropeptides versus age in man (10-logarithmic scale). Data from Refs. [34,38,63,64].
istration of exogenous opioid agonist and antagonist during the fetal period [33]. Human neonates who have been exposed in utero to opioids, such as heroine, have a smaller head circumference and reduced body weight due to a decrease in cell number [27]. Substance P is a primary sensory transmitter mediating pain sensations via the thin C-fibres. Substance P is also involved in the transmission of chemoreceptor and barometric input from the carotid and aortic chemo- and baro-receptors. In humans, there is an increase towards birth and then a levelling off during the first 6 months [34] (Fig. 4). Substance P may play a role in neurogenesis. It seems to counteract damage induced by neurotoxins and accelerates regeneration of cortical catecholamine fibres [35]. Increased expression of mRNA coding for pre-protachykinin A, the substance P precursor, has been recorded in respiratory-related nuclei in both the rabbit (see Ref. [36]) and the rat (see Ref. [37]). Increased expression of the substance P precursor has also been detected in patches in the nucleus caudatus and putamen of the human newborn brain [38]. Thus, there are suggestions that substance P is involved in the resetting and adaption of the organism to extrauterine life. Vasoactive intestitinal polypeptide (VIP) appears to have important trophic effects on the development of the cerebral cortex. Radio-labelled VIP has been demonstrated to pass the placental barrier and stimulate growth of the brain in the mouse. VIP promotes astrocytogenesis in the germinative zone [39]. Calcitonin gene-related peptide (CGRP) is an example of a neuropeptide with trophic effects, which might be involved in synaptogenesis. It has been demonstrated to stimulate the synthesis of ACh receptors in cultured muscle cells from the chick. The expression of CGRP is most prominent from E11 to E19, when the motor end-plates are formed [40] (see also Ref. [29]). Somatostatin is mainly an inhibitory neuromodulator co-existing with GABA. Somatostatin-related immunoflourescence (SRIF) neurones are transiently expressed to a high degree between E14 and E21 in the fetal rat. The peak is at E17 [5]. Whether this high expression is crucial for the formation of the cortex or just reflects an evolutionary residue is not really known [41].
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10. Purines Purines are fundamental components in the energy turnover of all cells but also modulate neuronal activity through synaptic or non-synaptic release and interaction with specific receptors. The purinergic receptors are divided into type-1 receptors (P1) sensitive to adenosine and AMP, and type-2 (P2) sensitive for ATP and ADP. The action of purines is related as a rapid breakdown of ATP increases the levels of adenosine. The purine nucleotide ATP is the main energy source of cells, but is also stored in synaptic vesicles and released together with classical transmitters such as noradrenaline and acetylcholine. The ratio between ATP and catecholamines in chromaffin granules has been found to be higher during early life than later, suggesting that it is a phylogenetic and ontogenetic old signalling substance [42]. Adenosine is a constituent of all body fluids, including the extracellular space of the central nervous system. It has multiple effects on organs and cells of the body. Thus, its levels are tightly regulated by a series of enzymatic steps [43]. Adenosine can be regarded more as a neuromodulator such that it does not seem to be stored in vesicles with a regulated release from nerve terminals. During basal conditions, adenosine levels are 30 – 300 nM and can rise following stimuli that cause an imbalance between ATP synthesis and ATP breakdown. Thus, the levels during ischemia or hypoxia can rise 100-fold [43,44]. The extracellular concentrations of adenosine might be higher in the fetal brain than postnatally, since fetal PaO2 can decrease below the level (30 mm Hg) when a significant increase in extracellular adenosine can be expected [44]. Overall, adenosine decreases oxygen consumption and has neuroprotective effects [43]. However, hypoxia also induces a decrease in neonatal respiration. Theophylline and caffeine are adenosine antagonists that cause ventilation to increase and decrease the incidence of neonatal apnoeas when given systemically, mainly due to the antagonistic effect of theophylline on adenosine A1-receptors in the medulla oblongata (see Ref. [45]).
11. Transition at birth The levels of most neurotransmitters and neuromodulators increase concomitantly with synapse formation. Some of them surge during the perinatal period (such as glutamate, catecholamines and some neuropeptides) and then level off. The interesting question is to what extent the expression of neuroactive agents is related to the functional state of the foetus and the newborn. On one hand, there is an intense firing and wiring in the fetal brain, particularly during active sleep. Therefore, an inhibitory neurotransmitter, such as GABA, seems to be mainly excitatory in the fetal period (see above). Amino acid transmitters also act via NMDA receptors, which are important for the wiring and plasticity of the immature brain, while the main excitatory fast-switching receptors (AMPA) are expressed later.
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Activities such as respiratory movements are suppressed. The foetus seldom or never becomes aroused or wakes up. The sympathetic tonus is low. Furthermore, the foetus is adapted to the low oxygen level in the womb. If it is challenged by asphyxia, it is not excited as an adult responding with a flight or fight reaction, but rather becomes immobilised, stops breathing and becomes bradycardic (see Ref. [36]). This paralytic state of the foetus can be caused by inhibition of the chemical neurotransmission or lack of expression of excitatory neuroactive agents. Adenosine is such a neuromodulator, which might be involved in this suppression of the fetal brain. It has a general sedatory effect. Its concentration increases during energy failure and hypoxia and it has been suggested that it can act as a modulator to cope with the hypoxic situation [46]. Adenosine A1-R activation depresses breathing substantially in the foetus and the neonate by inhibiting synaptic transmission and hyperpolarising certain neurones [47]. Neuropeptides which might be involved in the suppression of fetal activity are NPY, somatostatin and endogenous opioids. The levels of NPY are relatively high in the fetal brain and decline after birth. Plasma levels of endorphins and enkephalins are increased in the umbilical cord at birth. Blocking endogenous opioids with naloxone increases breathing in the newborn rabbit. The healthy newborn baby is aroused and awake the first 2 h after birth and starts continuous breathing movements. Factors like squeezing and squashing of the foetus, increased sensory input and cooling are probably of importance. We can hypothesise that there is a surge of excitatory neurotransmitters and down regulation of inhibitory ones in the brain. The increased neuronal activity is indicated by the increased expression of immediate early genes [48]. The arousal and vigilance of the newborn can probably partially be due to activation of the noradrenergic system in the brain, particularly locus coeruleus from where noradrenergic neurones are distributed in the whole brain (see above). The noradrenaline turnover as indicated by the ratio of the metabolite MHPG and NA was increased 2– 3-fold in the newborn rat (see Ref. [36]). There are indirect indications that there is also a noradrenaline surge in the human brain, by the finding of high level of plasma catecholamines after birth. The most important driving mechanism of respiration, coupling the ventilation to metabolic demand is the CO2-drive. This sensitivity seems to be mediated by a cholinergic mechanism [49]. The CO2-drive seems to be strongly upregulated at birth. An increased expression of immediate early genes (c-fos) has been recorded at the ventral surface of the newborn rat [37]. Whether this could trigger an upregulation of the cholinergic neurotransmission is not yet known. A neuropeptide involved in respiratory control at birth has been found to be substantially upregulated at birth. mRNA encoding for the substance P precursor preprotachykinin A is increased about 4-fold in respiratory nuclei but not in others during the first day after birth in rabbit pups (see Ref. [36]). A rapid decrease of the inhibitory neuromodulator adenosine in the brain occurs as partial pressure of oxygen in arterial blood rapidly increases after birth. In addition, a decreased sensitivity during the first postnatal days for adenosine seems to contribute to the maintenance of continuous breathing [45,47].
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12. Pre and perinatal programming The concept of fetal and neonatal programming first described by David Barker (see Ref. [50]) also applies to the ontogeny of neurotransmitters and neuromodulators, i.e. an early stimulus or insult at a critical period can result in long-term changes in the structure and the function of the organism. For example, it can be postulated that prenatal or perinatal stress can disturb the timetable of the expression of neurotransmitters and neuromodulators and their receptors. Hydrocortisone given to neonatal rats has been found to enhance the maturation of the monoaminergic systems in the brain [51]. Administration of extra glucocorticosteroids to the rat foetus induces alterations of dopamine receptor responses, which affects the spontanous motor-control both in shortand long-term perspectives [52]. Chronic prenatal hypoxia alters the monoamine turnover in the locus coeruleus and nucleus tractus solitarius in the adolescent rat [53]. This was related to disturbed control of respiratory behaviour. Human handling of newborn rats for 15 min during the first weeks of life appeared to affect ascending serotonergic projections into the hipoocampus and long-lasting increase in glucocorticoid receptors (see Ref. [54]). There are also clinical studies indicating that prenatal stress is associated with attention deficit disorders in children [55], possibly due to disturbance of monoaminergic turnover and subsequent wrong programming. Schizophrenic patients seem to have experienced more pregnancy and birth complications than their healthy siblings [56]. For example, mothers of schizophrenic patients suffered more often from severe infections during pregnancy, possibly affecting cytokines and indirectly, the development of monoaminergic circuits in the fetal brain [57].
13. Conclusions Monoamines are expressed in the very early embryo, at which stage the notochord already contains high noradrenaline levels. They may have an important role for neurotransmission in the foetus. Purines and neuropeptides are probably also expressed at an early stage, in a similar way as they occur early during phylogenesis. In the adult mammal, the fast-switching excitatory amino acids dominate. However, they also seem to be important for the wiring of the brain and the plasticity before birth. NMDA receptors that might mediate these effects dominate and are then substituted by AMPA receptors. The main inhibitory amino acids GABA and glycine seem to be excitatory before birth, which could be of major importance for the wiring of neuronal circuits during development. Prenatal or neonatal stress, for example hypoxia, can affect the programming of neurotransmitter and receptor expression, which can lead to long-term behavioural effects.
Acknowledgements This article is a shorter version of a chapter entitled ‘Neurotransmitters and and neuromodulators’ published in: The Newborn Brain. Neuroscience and Clinical
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applications (Eds. H. Lagercrantz, M. Hanson, P. Evrard, C. Rodeck), Cambridge Univ. Press, 2001. Supported by the Swedish Medical Research Council (5234), The Axelson-Johnson Foundation, Ma¨rta and Gunnar Philipson Foundation.
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Review article
Neurotransmitters and neuromodulators during early human development Eric Herlenius, Hugo Lagercrantz* Neonatal Unit, Department of Women and Child Health, Astrid Lindgren Children’s Hospital, Karolinska Institutet, S-171 76 Stockholm, Sweden Received 22 March 2001; received in revised form 4 June 2001; accepted 5 June 2001
Abstract Background: Neurotransmitters such as monoamines appear in the embryo before the neurones are differentiated. They may have other functions than neurotransmission during embryogenesis such as differentiation and neuronal growth. For example, serotonin may act as a morphogen. A number of neuropeptides are expressed during ontogenesis, but their function has been difficult to establish. Maybe some of them remain as evolutionary residues. Fast-switching neurotransmitters like the excitatory amino acids and the more ionotropic receptors dominate in the human brain, but appear probably later during evolution as well as during ontogeny. Methods: The distribution of catecholamines during development has been analysed with a fluorescense method, while most of the other neuortransmitters have been mapped with immunohistochemical methods. The classical method to determine the physiological role of a neurotransmitter or modulator is to study the physiological effect of its antagonist, blocking the endogenous activity. By transgenic technique, the genes encoding for enzymes involved in the synthesis of neurotransmitters can be knocked-out. Major findings: Pharmacological blocking of endogenous activity has, for example, demonstrated that adenosine suppresses fetal respiration. Knocking out the dopamine beta-hydroxylase gene results in fetal death, suggesting that noradrenaline is essential for survival. Some neurotransmitters change their effect during embryogenesis, e.g. GABA which is excitatory in the embryo, but inhibitory after birth due to a switch from a high to low chloride content in the nerve cells. It is possible that this is of importance for the wiring of neuronal network in early life. NMDA receptors dominate in the foetus, while kainate and AMPA receptors appear later. At birth, there is a surge of neurotransmitters such as catecholamines, which may be of importance for the neonatal adaptation. Conclusions: Neurotransmitters and modulators are not only important for the neural trafficking in the embryo, but also for the development of the neuronal circuits. Prenatal or
*
Corresponding author. Tel.: +46-8-5177-4700; fax: +46-8-5177-5121. E-mail address: [email protected] (H. Lagercrantz).
0378-3782/01/$ – see front matter D 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 3 7 8 2 ( 0 1 ) 0 0 1 8 9 - X
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neonatal stress (hypoxia), as well as various drugs, may disturb the wiring and cause long-term behavioural effects (fetal and neonatal programming). D 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neurotransmitters; Neuromodulators; Early human development
1. Introduction Although genes mainly determine the development of the scaffold of the CNS, the detailed wiring of the neuronal circuits is to a large degree self-generated dependent on the action of neurotransmitters and neuromodulators. They can promote, amplify, block, inhibit or attenuate the micro-electric signals which are passed on to neurones. Thereby, they give rise to the signalling patterns between myriads of neuronal networks providing the physical networks of cerebral neurones. Neurotransmitters such as the catecholamines appear in the embryos of vertebrate and invertebrate animals even before neurones are differentiated [1]. Some of the cells in the neuronal crest contain noradrenaline from the outset, but become cholinergic due to environmental influences [2]. Many neuroactive molecules change their functional role in the CNS during development. The same molecule may be crucial for differentiation, neuronal growth and establishment of neuronal networks in the immature CNS while switching to a more modulatory role of the ongoing traffic in the mature CNS. Receptor subunits may exchange during development, i.e. the NMDA receptors, whose subunits allow longer open channel time during early development then switch to a shorter, more stable adult subunit composition. This is of importance for the plasticity of the immature brain and subsequently, for memory storage and preservation in the adult brain [3]. Noradrenaline and acetylcholine are regarded as classical neurotransmitters and dominate in the peripheral nervous system (Fig. 1). They appear at an early stage during both phylogenesis and ontogenesis. Many of the neuropeptides were first identified in the gastrointestinal tract and probably appear early during CNS development. They act slowly since they have to be synthesised and packaged in the cell soma and carried to the terminals before they can be released. The recently evolved and more sophisticated mammalian brain requires more fast-switching neurotransmitters acting directly on ion-channels. Therefore, excitatory and inhibitory amino acids seem to dominate in the mature CNS, where the monoamines and neuropeptides may act more as neuromodulators (see Ref. [4]). The distinction between a transmitter and a modulator is far from clear, since several of the neuroactive agents described to date change their role during brain development or have different actions depending on brain region or innervated neurones. Furthermore, a given transmitter may have different effects depending on brain region, postsynaptic receptor configuration, G-protein coupling and second messenger system. A neuroactive agent can be expressed in high amounts during certain stages of development, but then persists in only a few synapses [5]. It is possible that this agent
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Table 1 Major neurotransmitters and neuromodulators presented in presumed order of appearance during ontogeny Purines Adenosine, ATP Monoamines Serotonin (indoleamines) Dopamine, noradrenaline, adrenaline Neuropeptides Opioids: enkephalins, endorphin, dynorphin Tachykinins: substance P, neurokinin Glucagon-related: vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase activating peptide (PACAP) Neuropeptide Y (NPY) Somatostatin Neurotensin, calcitonin gene-related peptide (CGRP) Acetylcholine Amino acids Glycine, GABA, glutamate, aspartate
either has only a transitory role in a critical window in development or that it remains mainly as an evolutionary residue, with minor functions in, e.g. mammals. If the synthesis of some of these neurotransmitters/modulators is blocked pharmacologically or knocked-out by transgenic techniques, it does not seem to affect survival or even important physiological functions. This illustrates the plasticity of the brain during early development. Other neuroactive agents seem to be able to take over. Markers for neurotransmitters and neuromodulators during CNS development generally appear first in the caudal and phylogenetically older part of the brain probably due to earlier neurogenesis (see Ref. [6]). Classification of the main neurotransmitters and modulators according to principal biochemical differences and tentative ontogenetic appearance is depicted in Table 1.
2. Receptors The neurotransmitters or modulators can act on either metabotropic or ionotropic receptors (see reviews by, e.g. Ref. [7]). The action of the metabotropic receptors is based on their effects on G- or N-proteins in the lipid bilayer of the membrane to affect their enzymes and channels. This effect is slower (tens of milliseconds) than for the ionotropic receptors. Metabotropic receptors are probably expressed at an earlier stage during ontogeny and play a more modulatory role in the mature CNS. The ionotropic receptors respond rapidly and are also termed class I receptors. They act on ion gates, which they can open or close in less than a millisecond. The ion channels consist of transmembrane proteins, which can be selective for cations (activatory receptors) or anions (inhibitory). The ionotropic nicotinic acetylcholine receptor (nAcR), the GABAA-R and glycine receptor GlyR are members of the same evolutionary super
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family and have a similar structure. A fetal subunit of the acetylcholine receptor (gammaAchR) is replaced by an adult type (epsilon-AchR) in the muscle end-plate to increase the conductance [8].
3. Ontogeny of neurotransmitter systems The choice of neurotransmitter of a precursor neuron depends on the environment. In a series of remarkable experiments, Le Douarin [9] demonstrated that when the sympathetic trunk crest from a quail was transplanted into the vagal region of a chick host, the nerves became cholinergic. Conversely, when vagal neurones were transplanted into the sympathetic trunk, the nerves became adrenergic. The expression of neurotransmitter type seemed to be dependent on a tissue factor. When sympathetic ganglia cells were cultivated in a medium from a heart cell culture, the adrenergic neurones became cholinergic [2]. The choice of transmitter could also be affected by corticosteroids. Thus, environmental factors are important for the differentiation and which neuroactive agent will be used in communication with the surrounding cells.
4. Catecholamines Catecholamines can be found in protozoa as well as in the very early embryo. The synthesising enzyme tyrosine hydroxylase has been detected the first day of/after incubation of the chicken; dopamine the second day, and noradrenaline and adrenaline the third day. High concentrations of catecholamines have been found in Hensen’s node, corresponding to the notochord of the mammalian embryo (see Ref. [1]). Noradrenaline is essential for normal brain development. The noradrenergic system regulates the development of the Cajal-Retzius cells, which are the first neurones to be born in the cortex and proposed to be instrumental in neuronal migration and laminar formation [10]. Administration of 6-OH-dopamine prevents the natural-programmed cell death of these neurones and delays the formation of cortical layers. Lesioning the noradrenergic projections or blocking neurotransmission with receptor antagonist prevents astrogliosis and glial cell proliferation. Depleting noradrenaline during the perinatal period results in subtle dendritic changes and possibly also alterations in cortical differentiation (see Ref. [11]). The role of noradrenaline has been investigated by targeted disruption of the dopamine b-hydroxylase (DBH) gene. The DBH locus of the DBH proximal promoter and the first exon were replaced with a neomycin-resistance cassette [12]. This resulted in fetal death, probably due to cardiovascular failure. Only about 5% of the homozygotic mice survived until adulthood, presumably due to some placental transfer of noradrenaline. Most of the mice could be rescued to birth by providing them with dihydroxyphenylserine (DDPS), a precursor that can be converted to noradrenaline in the absence of DBH. These mice had a reduced ability of acquisition and retention for some tasks. Interestingly, female mice seemed to have deficient ability to take care of
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their offspring. Thus, there seems to be a critical window during early development when noradrenaline is involved in forming the pathways responsible for maternal behaviour [13].
Fig. 1. Shows schematic sagittal illustrations of cell bodies and projections of monoamine neurotransmitter systems. Acetylcholinergic pathways, dopaminergic pathways, serotoninergic pathways and noradrenergic pathways in the human brain. Modified from L. Heimer: The human brain and the spinal cord. Springer, NY, 1995.
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Fig. 2. Arbitrary levels of monoamines and acetylcholine in man versus age (10-logarithmic scale). Data from Refs. [14,58,59].
Dopamine plays a very important role in motor and cognitive programmes. Dopaminergic neurones appear early during development 6 – 8 weeks in human [14] (Fig. 2), earlier in females than in males. The dopamine turnover is relatively high during the perinatal period compared to adults. Extremely high levels of D1 receptors have been reported in the pallidum during the perinatal period [15]. D1 receptor stimulation regulates transcription of other genes, and it is possible that abnormal perinatal stimulation can result in long-term consequences (see below). Disturbances of the development of the dopaminergic system may lead to dyskinesia, dystonia, tics, obsessive-compulsive disorders and abnormal eye movements. This has been observed in DA-depleted rats after 6-hydroxydopamine treatments but with preserved noradrenaline effect. Tyrosine– hydroxylase gene-deleted mice were hypoactive and suffered from adipsia and aphagia, which could be treated with L-dopa [16].
5. Serotonin Serotonin can already be detected in the fertilised egg and is involved in early morphogenesis of the heart, the craniofacial epithelia and other structures. If embryos are cultured in the presence of serotonin uptake inhibitors or receptor ligands, specific craniofacial malformations occur. Serotoninergic cells in the raphe are among the earliest to be generated in the brain (about E11 to E15 in the rat and between weeks 5 and 12 in the human foetus) (Fig. 2). These cells send axons to the forebrain and may be of importance in the differentiation of neuronal progenitors [17]. Excess of serotonin prevents the normal development of the somatosensory cortex, which has been demonstrated in monoamine oxidase knockout mice [18]. At birth, serotonergic neurones penetrate all cortical layers, but then decline markedly after about 3 weeks. Depletion of serotonin after birth seems to have little effect on cortical development. There are some indirect evidences that reduced serotonergic innervation of the cortex results in impaired behaviour in juvenile rats. Autism has been suggested to be related to hyperserotonism.
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6. Drugs affecting monoaminergic activity Cocaine is probably the most well-known drug interacting with the catecholaminergic systems in the brain during development [19]. It inhibits the presynaptic transport mechanisms, removing and terminating the action of dopamine and noradrenaline. While cocaine potentiates the catecholamine effects in the adult, it inhibits the activity during the immediate postnatal period in most brain regions. Prenatal cocaine exposure results in disturbance of neuronal migration and consequently leads to severe neurobehavioural disturbances. Prenatal cocaine exposure in humans causes abnormal motor behaviour immediately after birth and abnormal behaviour is apparent at 2 and 3 years follow-up, probably mainly due to disturbance of the dopaminergic system. Neuroleptic drugs administrated during pregnancy can block dopamine receptors and cause long-lasting effects (see Ref. [15]).
7. Acetylcholine Acetylcholine is one of the major neurotransmitters in the brain of importance for cortical activation, memory and learning. It has a major role in the control motor tone and movement and probably counterbalances the effect of dopamine (see Ref. [20]). The cholinergic innervation of the cortex occurs later than the monoaminergic around week 20 in the human foetus. Mature levels in rodents are not reached until after 8 weeks postnatally (see Ref. [11]). The concentrations of ACh reach about 20% of the adult levels at E15 in the whole brain of the rat and about 40% at day P7 (Fig. 2). The levels of choline acetyltrasferase (ChAT) are much lower (1% and 8%) at the corresponding ages, indicating low firing rates of the cholinergic neurones. Conversely, the receptors reach adult levels earlier. The cholinergic markers appear sooner in the pons-medulla, probably due to earlier neurogenesis in the caudal and phylogenetically older part of the brain (see Ref. [6]). The cholinergic afferents seem to have an important role in the differentiation of the cortex. After a considerable reduction of the cholinergic innervation, a delay of cortical cytodifferentiation was revealed (see Ref. [11]). The cholinergic innervation has been found to be disturbed in Down’s syndrome, lead and ethanol toxicity and asphyxia. Perinatal manipulations of the cholinergic system result in major changes of cortical structure. These changes can be correlated to cognitive deficits but do not affect motor behaviour.
8. Amino acid transmitters The amino acids are involved in the wiring of neuronal networks and building CNS cytoarchitecture [21] (Fig. 3). Amino acid transmitters are the most abundant transmitters in the central nervous system. However, they were recognised as neurotransmitters in the mammalian brain much later than the monoamines and acetylcholine. This was probably due to the fact that they are involved in intermediate metabolism and constitute important building blocks in the proteins.
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Fig. 3. Expression and arbitrary levels of receptors of amino acid transmitter versus age in man (10-logarithmic scale). Data from Refs. [58 – 62]. NMDA receptors are expressed relatively earlier than the kainate and AMPA receptors. GABA operates mainly as an excitatory transmitter on immature neurones. GABA receptors can facilitate the activation of NMDA receptors, playing the role conferred to AMPA receptors later on in development. It is assumed that the NMDA receptors are more involved in the wiring of the brain while the kainate and AMPA receptors are responsible for the fast traffic in the more mature brain. GABA operates mainly as an excitatory transmitter on immature neurones. GABA receptors can facilitate the activation of NMDA receptors, playing the role conferred to AMPA receptors later on in development. Embryonic age (E) expressed in weeks, postnatal age in years.
Glutamate and aspartate are the dominating excitatory amino acids (EAA) and the primary neurotransmitter in about half of all the synapses in the mammalian forebrain. They constitute the major transmitters of the pyramidal cells, the dominating neurones in the cortex. This has been demonstrated by injection of radioactively labelled D-(3H) glutamate into the appropriate projection areas (see Ref. [5]). EAA pathways undergo striking developmental changes, involving transient overshoots, especially during critical periods as evident in visual cortex and hippocampus. EAA terminals are overproduced during the early postnatal period, for example after 1– 2 years in the human cortex (see Fig. 3), which may be related to the high generation of synapses during those periods. Glutamate acts on at least five types of receptors. The slower acting metabotropic receptors, eight subclasses are hitherto known, are expressed at a relatively early stage. Of the ionotropic receptors, the NMDA receptors dominate in the immature brain when synaptic transmission is weak and extremely plastic (Fig. 3). The NMDA receptors permit entry of Na + and Ca2 + when opened and may mediate neurotoxic effects during perinatal asphyxia. Furthermore, Ca2 + entry through NMDA channels seems to be crucially involved in the appearance of long-term potentiation (LTP) and memory storage. During maturation, the AMPA and kainic ionotropic receptors predominate and carry most of the fast neuronal traffic in the brain [4].
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Dark rearing or blocking the activity with tetrodotoxin results in preservation of the NMDA receptors in the visual cortex. Dark rearing also preserves the immature form of the NMDA receptors containing NR2B subunit and the expression of NR2A is delayed. This subunit switch is essential for development of rapid synaptic transmission [3]. Fetal rats exposed to NMDA antagonists were found to have excessive apoptosis in the same way as the asphyxiated perinatal brain. NMDA receptor stimulation by excessive glutamate release leads to Ca+ + influx, which may induce subsequent neuronal apoptosis. ‘‘Thus, either too much or too little NMDA receptor activity can be life-threatening to the developing neurones’’ [22]. Gamma-aminobutyric acid (GABA) is the dominating neurotransmitter in the nonpyramidal cells, as demonstrated by uptake of (3H)-GABA and immunochemical labelling of the GABA-synthesising enzyme glutamic acid (GAD). Perhaps 25 –40% of all nerve terminals contain GABA. GABA is regarded as the main inhibitory transmitter in the mature animal, but has a different role during early development. There are two types of GABA receptors: GABAA and GABAB. The GABAA-receptor (GABAA-R) is an ionotropic receptor that gates a chloride channel. It is a transmembrane protein built of several subunits where, for example benzodiazepines, barbiturates and ethanol, can bind to specific sites and modulate the opening properties of the chloride channel. The GABAB-R is coupled to a G-protein, is present in lower levels in the CNS than the GABAA-receptor and starts to function late in CNS development (postnatal life in rodents). During early development, the Cl concentration is high in the nerve cells. When GABA opens the Cl channels, a depolarisation (i.e. excitation) occurs. During maturation, the Cl concentration decreases which results in an opposite effect of GABA, i.e. Cl ions are pumped out and the cell becomes hyperpolarised. In this way, GABA switches from an excitatory to an inhibitory neurotransmitter [23]. This switch occurs around birth in the rat [23] but it is not known when it occurs in the human. Thus, GABA operates mainly as an excitatory transmitter on immature neurones. As described above, glutaminergic synapses initially lack functional AMPA receptors and the NMDA channels are blocked by Mg2 + at resting membrane potentials. GABA depolarises immature neurones, which may result in Ca2 + influx by removing the Mg2 + blockage of NMDA channels. Thus, GABAA-receptors play the role conferred to AMPA receptors in the more matured CNS [21,24]. An increase in the intracellular Ca2 + concentration activates a wide range of intracellular cascades and is involved in neuronal growth and differentiation. Furthermore, GABA excitation and Ca2 + influx may act as triggers for plasticity of synaptic connections and for establishing and patterning of neural networks. The GABAA-receptors have a strong affinity for benzodiazepines. Several anxiolytic and anticonvulsant drugs increase the ability for GABA to open chloride channels. In neonatal neurones, GABA currents are potentiated by barbiturates but are insensitive to benzodiazepines [25]. As GABA has a trophic role during early brain development, interference with the function of GABAergic transmission during this period may affect the development of neuronal wiring, plasticity of neuronal network, and also have a profound influence on neural organisation.
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In fact, ethanol, abused by some mothers during pregnancy, interacts with the GABAA-receptor. The sensitive time window in rat cerebral cortex for ethanol exposure is situated between P3 and P10. It is worth noting that GABA, during this same period, seem to have mainly depolarising and trophic effects on developing cortical neurones through effects on cell proliferation and migration [26]. In humans, the intellectual deficits produced by abnormalities of brain growth are the most important components of fetal alcohol syndrome [27]. Craniofacial abnormalities in human foetuses related to first trimester alcohol exposure are similar to the facial defects seen in GABAA subunit receptor knockout mice [28]. Thus, ethanol interaction with the GABAA-receptor could be implicated in the pathogenesis of fetal alcohol syndrome. Glycine has both excitatory and inhibitory actions and can be regarded as the phylogenetically older inhibitory transmitter restricted to the brain stem and spinal cord in the adult. A similar switch as regarding the GABAA-receptors from excitatory to inhibitory effects seems to occur with maturation [23]. The NMDA receptor has a modulatory site where glycine in submicromolar concentrations increases the frequency of NMDA-receptor channel opening. Conditions that alter the extracellular concentration of glycine can dramatically alter NMDA-receptormediated responses.
9. Neuropeptides More than 50 neuropeptides have been identified. In contrast to most of the other neurotransmitters/modulators, the neuropeptides are synthesised and packaged in large dense-cored vesicles in the cell soma and are carried to the nerve terminals by axonal transport at a rate of 1.5 mm/h. It is obvious that by this relatively slow process, the neuropeptides cannot act as fast-switching neurotransmitters. Rather, they have a neuromodulatory role. They are often stored together with other neurotransmitters, i.e. monoamines or EAA, and it is possible that they play a role in setting of the sensitivity [29]. Some of them are probably of less physiological importance and occur in the body mainly as evolutionary residues [30]. Still they are of great neuropharmacological interest and their analogues or antagonists can be used as drugs. The most well-known examples are the opioids and naloxone. b-Endorphin is expressed early during fetal brain development and seems to be involved in fetal growth and postnatal development at least in the rat [31]. mReceptor binding sites are present during mid-fetal time and have a high density in cardiorespiratory-related brainstem nuclei, whereas the d-opioid receptors primarily appear during the postnatal period in rats [32]. Although, opioid binding sites progressively increase in the developing brain (Fig. 4), the effect of opioids appears to be dependent on the status of neuronal maturation. In addition, many neuronal populations exhibit transient expression of one or the other opioid genes but the physiological role of this is not clear. Opioid agonists inhibit mitosis and DNA synthesis in the developing brain and endogenous opioids exert potent regulatory effects on brain development and morphogenesis, as demonstrated by the admin-
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Fig. 4. Expression and arbitrary levels of some major neuropeptides versus age in man (10-logarithmic scale). Data from Refs. [34,38,63,64].
istration of exogenous opioid agonist and antagonist during the fetal period [33]. Human neonates who have been exposed in utero to opioids, such as heroine, have a smaller head circumference and reduced body weight due to a decrease in cell number [27]. Substance P is a primary sensory transmitter mediating pain sensations via the thin C-fibres. Substance P is also involved in the transmission of chemoreceptor and barometric input from the carotid and aortic chemo- and baro-receptors. In humans, there is an increase towards birth and then a levelling off during the first 6 months [34] (Fig. 4). Substance P may play a role in neurogenesis. It seems to counteract damage induced by neurotoxins and accelerates regeneration of cortical catecholamine fibres [35]. Increased expression of mRNA coding for pre-protachykinin A, the substance P precursor, has been recorded in respiratory-related nuclei in both the rabbit (see Ref. [36]) and the rat (see Ref. [37]). Increased expression of the substance P precursor has also been detected in patches in the nucleus caudatus and putamen of the human newborn brain [38]. Thus, there are suggestions that substance P is involved in the resetting and adaption of the organism to extrauterine life. Vasoactive intestitinal polypeptide (VIP) appears to have important trophic effects on the development of the cerebral cortex. Radio-labelled VIP has been demonstrated to pass the placental barrier and stimulate growth of the brain in the mouse. VIP promotes astrocytogenesis in the germinative zone [39]. Calcitonin gene-related peptide (CGRP) is an example of a neuropeptide with trophic effects, which might be involved in synaptogenesis. It has been demonstrated to stimulate the synthesis of ACh receptors in cultured muscle cells from the chick. The expression of CGRP is most prominent from E11 to E19, when the motor end-plates are formed [40] (see also Ref. [29]). Somatostatin is mainly an inhibitory neuromodulator co-existing with GABA. Somatostatin-related immunoflourescence (SRIF) neurones are transiently expressed to a high degree between E14 and E21 in the fetal rat. The peak is at E17 [5]. Whether this high expression is crucial for the formation of the cortex or just reflects an evolutionary residue is not really known [41].
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10. Purines Purines are fundamental components in the energy turnover of all cells but also modulate neuronal activity through synaptic or non-synaptic release and interaction with specific receptors. The purinergic receptors are divided into type-1 receptors (P1) sensitive to adenosine and AMP, and type-2 (P2) sensitive for ATP and ADP. The action of purines is related as a rapid breakdown of ATP increases the levels of adenosine. The purine nucleotide ATP is the main energy source of cells, but is also stored in synaptic vesicles and released together with classical transmitters such as noradrenaline and acetylcholine. The ratio between ATP and catecholamines in chromaffin granules has been found to be higher during early life than later, suggesting that it is a phylogenetic and ontogenetic old signalling substance [42]. Adenosine is a constituent of all body fluids, including the extracellular space of the central nervous system. It has multiple effects on organs and cells of the body. Thus, its levels are tightly regulated by a series of enzymatic steps [43]. Adenosine can be regarded more as a neuromodulator such that it does not seem to be stored in vesicles with a regulated release from nerve terminals. During basal conditions, adenosine levels are 30 – 300 nM and can rise following stimuli that cause an imbalance between ATP synthesis and ATP breakdown. Thus, the levels during ischemia or hypoxia can rise 100-fold [43,44]. The extracellular concentrations of adenosine might be higher in the fetal brain than postnatally, since fetal PaO2 can decrease below the level (30 mm Hg) when a significant increase in extracellular adenosine can be expected [44]. Overall, adenosine decreases oxygen consumption and has neuroprotective effects [43]. However, hypoxia also induces a decrease in neonatal respiration. Theophylline and caffeine are adenosine antagonists that cause ventilation to increase and decrease the incidence of neonatal apnoeas when given systemically, mainly due to the antagonistic effect of theophylline on adenosine A1-receptors in the medulla oblongata (see Ref. [45]).
11. Transition at birth The levels of most neurotransmitters and neuromodulators increase concomitantly with synapse formation. Some of them surge during the perinatal period (such as glutamate, catecholamines and some neuropeptides) and then level off. The interesting question is to what extent the expression of neuroactive agents is related to the functional state of the foetus and the newborn. On one hand, there is an intense firing and wiring in the fetal brain, particularly during active sleep. Therefore, an inhibitory neurotransmitter, such as GABA, seems to be mainly excitatory in the fetal period (see above). Amino acid transmitters also act via NMDA receptors, which are important for the wiring and plasticity of the immature brain, while the main excitatory fast-switching receptors (AMPA) are expressed later.
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Activities such as respiratory movements are suppressed. The foetus seldom or never becomes aroused or wakes up. The sympathetic tonus is low. Furthermore, the foetus is adapted to the low oxygen level in the womb. If it is challenged by asphyxia, it is not excited as an adult responding with a flight or fight reaction, but rather becomes immobilised, stops breathing and becomes bradycardic (see Ref. [36]). This paralytic state of the foetus can be caused by inhibition of the chemical neurotransmission or lack of expression of excitatory neuroactive agents. Adenosine is such a neuromodulator, which might be involved in this suppression of the fetal brain. It has a general sedatory effect. Its concentration increases during energy failure and hypoxia and it has been suggested that it can act as a modulator to cope with the hypoxic situation [46]. Adenosine A1-R activation depresses breathing substantially in the foetus and the neonate by inhibiting synaptic transmission and hyperpolarising certain neurones [47]. Neuropeptides which might be involved in the suppression of fetal activity are NPY, somatostatin and endogenous opioids. The levels of NPY are relatively high in the fetal brain and decline after birth. Plasma levels of endorphins and enkephalins are increased in the umbilical cord at birth. Blocking endogenous opioids with naloxone increases breathing in the newborn rabbit. The healthy newborn baby is aroused and awake the first 2 h after birth and starts continuous breathing movements. Factors like squeezing and squashing of the foetus, increased sensory input and cooling are probably of importance. We can hypothesise that there is a surge of excitatory neurotransmitters and down regulation of inhibitory ones in the brain. The increased neuronal activity is indicated by the increased expression of immediate early genes [48]. The arousal and vigilance of the newborn can probably partially be due to activation of the noradrenergic system in the brain, particularly locus coeruleus from where noradrenergic neurones are distributed in the whole brain (see above). The noradrenaline turnover as indicated by the ratio of the metabolite MHPG and NA was increased 2– 3-fold in the newborn rat (see Ref. [36]). There are indirect indications that there is also a noradrenaline surge in the human brain, by the finding of high level of plasma catecholamines after birth. The most important driving mechanism of respiration, coupling the ventilation to metabolic demand is the CO2-drive. This sensitivity seems to be mediated by a cholinergic mechanism [49]. The CO2-drive seems to be strongly upregulated at birth. An increased expression of immediate early genes (c-fos) has been recorded at the ventral surface of the newborn rat [37]. Whether this could trigger an upregulation of the cholinergic neurotransmission is not yet known. A neuropeptide involved in respiratory control at birth has been found to be substantially upregulated at birth. mRNA encoding for the substance P precursor preprotachykinin A is increased about 4-fold in respiratory nuclei but not in others during the first day after birth in rabbit pups (see Ref. [36]). A rapid decrease of the inhibitory neuromodulator adenosine in the brain occurs as partial pressure of oxygen in arterial blood rapidly increases after birth. In addition, a decreased sensitivity during the first postnatal days for adenosine seems to contribute to the maintenance of continuous breathing [45,47].
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12. Pre and perinatal programming The concept of fetal and neonatal programming first described by David Barker (see Ref. [50]) also applies to the ontogeny of neurotransmitters and neuromodulators, i.e. an early stimulus or insult at a critical period can result in long-term changes in the structure and the function of the organism. For example, it can be postulated that prenatal or perinatal stress can disturb the timetable of the expression of neurotransmitters and neuromodulators and their receptors. Hydrocortisone given to neonatal rats has been found to enhance the maturation of the monoaminergic systems in the brain [51]. Administration of extra glucocorticosteroids to the rat foetus induces alterations of dopamine receptor responses, which affects the spontanous motor-control both in shortand long-term perspectives [52]. Chronic prenatal hypoxia alters the monoamine turnover in the locus coeruleus and nucleus tractus solitarius in the adolescent rat [53]. This was related to disturbed control of respiratory behaviour. Human handling of newborn rats for 15 min during the first weeks of life appeared to affect ascending serotonergic projections into the hipoocampus and long-lasting increase in glucocorticoid receptors (see Ref. [54]). There are also clinical studies indicating that prenatal stress is associated with attention deficit disorders in children [55], possibly due to disturbance of monoaminergic turnover and subsequent wrong programming. Schizophrenic patients seem to have experienced more pregnancy and birth complications than their healthy siblings [56]. For example, mothers of schizophrenic patients suffered more often from severe infections during pregnancy, possibly affecting cytokines and indirectly, the development of monoaminergic circuits in the fetal brain [57].
13. Conclusions Monoamines are expressed in the very early embryo, at which stage the notochord already contains high noradrenaline levels. They may have an important role for neurotransmission in the foetus. Purines and neuropeptides are probably also expressed at an early stage, in a similar way as they occur early during phylogenesis. In the adult mammal, the fast-switching excitatory amino acids dominate. However, they also seem to be important for the wiring of the brain and the plasticity before birth. NMDA receptors that might mediate these effects dominate and are then substituted by AMPA receptors. The main inhibitory amino acids GABA and glycine seem to be excitatory before birth, which could be of major importance for the wiring of neuronal circuits during development. Prenatal or neonatal stress, for example hypoxia, can affect the programming of neurotransmitter and receptor expression, which can lead to long-term behavioural effects.
Acknowledgements This article is a shorter version of a chapter entitled ‘Neurotransmitters and and neuromodulators’ published in: The Newborn Brain. Neuroscience and Clinical
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applications (Eds. H. Lagercrantz, M. Hanson, P. Evrard, C. Rodeck), Cambridge Univ. Press, 2001. Supported by the Swedish Medical Research Council (5234), The Axelson-Johnson Foundation, Ma¨rta and Gunnar Philipson Foundation.
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