Efrain C. Azmitia

Modern views on an ancient chemical: serotonin effects on cell proliferation, maturation, and apoptosis.

Evolutionarily, serotonin existed in plants even before the appearance of animals. Indeed, serotonin may be tied to the evolution of life itself, particularly through the role of tryptophan, its precursor molecule. Tryptophan is an indole-based, essential amino acid which is unique in its light-absorbing properties. In plants, tryptophan-based compounds capture light energy for use in metabolism of glucose and the generation of oxygen and reduced cofactors. Tryptophan, oxygen, and reduced cofactors combine to form serotonin. Serotonin-like molecules direct the growth of light-capturing structures towards the source of light. This morphogenic property also occurs in animal cells, in which serotonin alters the cytoskeleton of cells and thus influences the formation of contacts. In addition, serotonin regulates cell proliferation, migration and maturation in a variety of cell types, including lung, kidney, endothelial cells, mast cells, neurons and astrocytes). In brain, serotonin has interactions with seven families of receptors, numbering at least 14 distinct proteins. Of these, two receptors are important for the purposes of this review. These are the 5-HT1A and 5-HT2A receptors, which in fact have opposing functions in a variety of cellular and behavioral processes. The 5-HT1A receptor develops early in the CNS and is associated with secretion of S-100beta from astrocytes and reduction of c-AMP levels in neurons. These actions provide intracellular stability for the cytoskeleton and result in cell differentiation and cessation of proliferation. Clinically, 5-HT1A receptor drugs decrease brain activity and act as anxiolytics. The 5-HT2A receptor develops more slowly and is associated with glycogenolysis in astrocytes and increased Ca(++) availability in neurons. These actions destabilize the internal cytoskeleton and result in cell proliferation, synaptogenesis, and apoptosis. In humans, 5-HT2A receptor drugs produce hallucinations. The dynamic interactions between the 5-HT1A and 5-HT2A receptors and the cytoskeleton may provide important insights into the etiology of brain disorders and provide novel strategies for their treatment.

Stimulation of astroglial 5-HT1A receptors releases the serotonergic growth factor, protein S-100, and alters astroglial morphology

Stimulation of astroglial 5-HT1A receptors causes astroglial cells to acquire a more mature morphology and to release a factor (or factors) which promotes growth of serotonergic neurons. By using an antibody-blocking approach, we have shown that at least one of the growth-promoting factors thus released is the astroglial-specific protein S-100. This may be a particularly important observation, in view of studies implicating S-100 in both Downs syndrome and Alzheimers disease.

Cellular localization of the 5-HT1A receptor in primate brain neurons and glial cells

Activation of 5-HT1A receptors produces many different physiologic responses, which may be due to their localization on divers cells in the brain. A 5-HT1A receptor antipeptide (aa170-186) antibody was produced that showed both high titer for peptide binding and immunocytochemical staining. Studies performed in perfusion-fixed brain tissue showed immunoreactive neurons, glial, and ependymal cells in the rat, mouse, cat, and monkey. Results from our studies of Macaca fascicularis brains are presented. We observed two main neuronal labeling patterns in the primate brain: (1) A general, diffuse somatodendritic distribution of 5-HT1A receptor immunoreactivity is seen in the raphe nuclei where the dendritic shaft, its branches and spines, and the entire perikaryon are immunolabeled. This pattern is also observed in the nucleus locus coeruleus, in scattered large brainstem reticular neurons, and in dentate gyrus hilar interneurons. (2) A discrete localization of 5-HT1A receptor immunoreactivity on the initial axon segment (axon hillock) is noted in pyramidal neurons of layer III and V of cerebral cortex, Cornu Ammonus (1-4) of the hippocampus, and in most brainstem and cervical spinal cord motorneurons. In addition to neuronal labeling, 5-HT1A receptor immunoreactivity is seen in the cell body and processes of astrocytes, and other nonneuronal cells. This pattern is particularly evident in the white matter of cerebral cortex and spinal cord, the pontine nulcei, the brainstem tectum, and the hilus of the dentate gyrus. The clinical implications of 5-HT1A cellular localization are briefly discussed.

S-100B but not NGF, EGF, insulin or calmodulin is a CNS serotonergic growth factor

The effects of S-100B, nerve growth factor (NGF), epidermal growth factor (EGF) and insulin were tested in cultured mesencephalic neurons. Only chronic S-100B showed enhancement (maximal at 3.2 ng/ml is 171%) after 3 days of incubation of the [3H]5-HT uptake capacity by serotonergic neurons. A single application at initial plating of S-100B (maximal at 5 ng/ml is 185%), but not calmodulin, increased the development of the [3H]5-HT uptake capacity by the cultured serotonergic neurons. Morphometric analysis of cultured 5-HT immunoreactive (IR) neurons showed an increase (135 and 147%) in neurite length 30 h after S-100B application of 16 and 3.2 ng/ml (respectively). These results suggest that S-100B is a serotonergic growth factor in the mammalian brain.

Corticosterone Regulation of Tryptophan Hydroxylase in Midbrain of the Rat

The tryptophan hydroxylase activity in the rat midbrain decreases after adrenalectomy and is restored by treatment with corticosterone. Cycloheximide, adminiistered intracisternally, prevents the restoration of the enzyme activity by corticosterone. Cycloheximide administration to adrenalectomized rats resutlts in a further decrease in the enzyme activity. an indication that the enzyme has a rapid turnover even in the absence of corticosterone.

The substituted amphetamines 3,4-methylenedioxymethamphetamine, methamphetamine, p-chloroamphetamine and fenfluramine induce 5-hydroxytryptamine release via a common mechanism blocked by fluoxetine and cocaine.

The abilities of the substituted amphetamines 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine, p-chloroamphetamine (PCA) and fenfluramine to induce synaptosomal [3H]serotonin (5-HT) release were compared using a novel microassay system. The rank order of release potencies was found to be (+/-)PCA congruent to (+)-fenfluramine greater than (+)-MDMA much greater than (+)-methamphetamine. Combination of two drugs at their EC50 did not cause more release than either drug alone at an equivalent concentration. In addition, the 5-HT uptake blockers fluoxetine and cocaine inhibited the release induced by MDMA, methamphetamine, PCA and fenfluramine to the same percentage. However, threshold concentrations of the substituted amphetamines known to inhibit uptake did not attenuate the release caused by higher concentrations of these compounds. These results suggests that MDMA, methamphetamine, PCA and fenfluramine cause 5-HT release via a common mechanism. Furthermore, these results indicate that the 5-HT uptake blockade induced by these substituted amphetamines in vitro is different from that induced by either fluoxetine or cocaine.

Autoregulation of fetal serotonergic neuronal development: Role of high affinity serotonin receptors

A microculture technique was used to study the factors regulating the development of fetal rat serotonergic neurons. Mesencephalic raphe cells from E14 rats co-cultured with hippocampal cells from E18 were grown for up to 4 days in the presence of various agents known to alter serotonergic function in the mature brain. Pargyline (a non-specific monoamine oxidase inhibitor) alone and with serotonin (10(-8) to 10(-6) M) both inhibited growth of serotonergic neurons as assessed by uptake of [3H]serotonin. 5-methoxytryptamine (5-MT), a serotonin agonist with selectivity for serotonin autoreceptors, inhibited growth at low concentrations, but this inhibition was overcome at higher concentrations. Using immunocytochemistry with a primary anti-serotonin antibody, 5-MT was observed to produce stunted processes, increase autoinnervation and lead to neuronal death. A model is proposed whereby high affinity serotonin receptors in fetal brainstem tissue and in fetal forebrain tissue regulate direction and extent of growth. We have confirmed the presence of these receptors using a direct binding assay.

Serotonin Neurons, Neuroplasticity, and Homeostasis of Neural Tissue

Homeostasis is the process by which the internal milieu of the body is able to maintain equilibrium in the face of constant insults from the external world. Endocrine, immune, and vascular systems play pivotal roles in adjusting internal biochemical reactions to counteract assaults from the outside. Despite the vast accumulation of data over the last 50 years, a role for serotonin in brain homeostasis has not been proposed. In this chapter I will review the plasticity and anatomy of serotonergic neurons in integrating external sensory and motor systems as well as internal endocrine, glial and vascular signals with the various cellular elements comprising neural tissue. Steroids and neuropeptides have both been shown to alter the morphology of serotonergic neurons. In turn, alterations in serotonin levels in the adult brain can change the morphology of its target cells. A pivotal role for serotonin in the homeostasis of neural tissue is consistent with the function of serotonin throughout evolution and explains the large number of biological systems, behavioral activities, and clinical diseases associated with serotonergic neurons.

Prenatal cocaine exposure disrupts the development of the serotonergic system

Prenatal cocaine exposure has been found to result in a number of neurobehavioral abnormalities in both clinical and laboratory studies. We have previously shown that cocaine inhibits the growth of developing serotonin neurons in culture. This study examines the effects of cocaine on the developing serotonin system in vivo. Pregnant rats were injected with cocaine (40 mg/kg s.c.) from gestational day 13 to parturition. One group of rats was additionally injected on postnatal days 1-5 with cocaine (10 mg/kg s.c.). [3H]Paroxetine, a selective ligand for the serotonin uptake carrier, was used to quantify serotonin terminal fiber density at one day, one week, and four weeks postnatal. Cocaine exposure was found to significantly decrease [3H]paroxetine-labelled sites and thus the density of serotonin fibers in the cortex and hippocampus at one day and one week postnatal. By four weeks postnatal, no significant effect was observed, indicating that a recovery had occurred. Serotonin immunocytochemistry performed at one month revealed normal fiber distribution in the cortex but a loss of fibers in the CA1 and CA2 hippocampal fields. Postnatal treatment alleviated the effects of prenatal cocaine exposure, resulting in [3H]paroxetine binding levels at one week which were comparable to and, in the cortex, even higher than those of saline controls. We conclude that cocaine delays the maturation of the serotonin system when administered prenatally but may accelerate maturation when administered both pre- and postnatally.

Stimulation of astroglial serotonin receptors produces culture media which regulates growth of serotonergic neurons.

Our work has been concerned with the role of high affinity serotonin receptors in regulating the development of the serotonergic system. In previous studies, we have found evidence that these receptors occur on astroglial cells and that their number is developmentally linked. The current work is aimed at investigating the mechanism by which these receptors may regulate serotonin neuronal growth. Primary cultures of astroglial cells were exposed to serotonin (5-HT) or the selective receptor agonists 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-D-PAT, for 5-HT1a receptors) or trifluoro-methyl-phenyl-piperazine (TFMPP) and m-chlorophenylpiperazine (mCPP) (for 5-HT1b receptors). Media was collected after 4 or 24 h, and added to primary cultures of serotonergic neurons. Growth was determined by specific uptake of radiolabeled serotonin into the cultures. Our results show the presence of a factor(s) in the glial-conditioned media which can be stimulatory or toxic to serotonin neurons, depending on the neuronal plating density. This factor is significantly present after 24 h, is found in both brainstem and cortical astroglial-conditioned media and appears to be linked to the 5-HT1a receptor. Thus, it appears possible that the serotonergic neuronal system can regulate its own development through an action on astroglial cells.