Persistent Angiogenesis in the Autism Brain: An Immunocytochemical Study of Postmortem Cortex, Brainstem and Cerebellum.

In the current work, we conducted an immunocytochemical search for markers of ongoing neurogenesis (e.g. nestin) in auditory cortex from postmortem sections of autism spectrum disorder (ASD) and age-matched control donors. We found nestin labeling in cells of the vascular system, indicating blood vessels plasticity. Evidence of angiogenesis was seen throughout superior temporal cortex (primary auditory cortex), fusiform cortex (face recognition center), pons/midbrain and cerebellum in postmortem brains from ASD patients but not control brains. We found significant increases in both nestin and CD34, which are markers of angiogenesis localized to pericyte cells and endothelial cells, respectively. This labeling profile is indicative of splitting (intussusceptive), rather than sprouting, angiogenesis indicating the blood vessels are in constant flux rather than continually expanding.

Time-dependent modulation of serotonin and its receptors 1A and 2A expression in allergic contact dermatitis.

BACKGROUND: Serotonin implication in allergic contact dermatitis was earlier suggested. OBJECTIVE: To study the expression of serotonin (5-HT) and its receptors 1A and 2A during the kinetics of allergic contact dermatitis (ACD). METHOD: Biopsies from 10 female nickel-allergic patients were obtained at 0, 6, 24, 48 and 72 h after nickel application using a patch testing procedure and then analysed by immunohistochemistry. RESULTS: Higher 5-HT epidermal immunoreactivity was seen at 0 and 6 h compared with 24, 48 and 72 h. The number of 5-HT labelled platelets was higher at 48 and 72 h compared with 0, 6 and 24 h. There was a decrease in the number of 5-HT1AR positive cells at 24, 48 and 72 h compared with 0 h. Furthermore, epidermal 5-HT1AR immunoreactivity was stronger at 48 and 72 h compared with 0 h. Total (dermis plus epidermis) number of 5-HT2AR positive cells was gradually increased in a time-dependent manner at 6, 24, 48 and 72 h compared with 0 h. CONCLUSIONS: Our results suggest the implication of 5-HT, 5-HT1AR and 5-HT2AR in the development of human ACD and the possibility to target those receptors at an early stage of this inflammatory condition.

S100beta protein expression: gender- and age-related daily changes.

S100beta is a soluble protein released by glial cells mainly under the activation of the 5-HT1A receptor. It has been reported as a neuro-trophic and -tropic factor that promotes neurite maturation and outgrowth during development. This protein also plays a role in axonal stability and the plasticity underlying long-term potentiation in adult brains. The ability of S100beta to rapidly regulate neuronal morphology raises the interesting point of whether there are daily rhythm or gender differences in S100beta level in the brain. To answer this question, the S100beta expression in adult female and male rats, as well as in adult female CD-21 and S100beta -/- female mice, were investigated. Scintillation counting and morphometric analysis of the immunoreactivity of S100beta, showed rhythmic daily expression. The female and male rats showed opposite cycles. Females presented the highest value at the beginning of the rest phase (5:00 h), while in males the maximum value appeared in the beginning of the motor activity period (21:00 h). These results confirm previous S100beta evaluations in human serum and cerebrospinal fluid reporting the protein's function as a biomarker for brain damage (Gazzolo et al. in Clin Chem 49:967-970, 2003; Clin Chim Acta 330:131-133, 2003; Pediatr Res 58:1170-1174, 2005), similar behavior was also observed for GFAP in relation to Alzheimer Disease (Fukuyama et al. in Eur Neurol 46:35-38, 2001). The data should be taken into account when considering S100beta as a biomarker of health condition. In addition, the results raise questions on which structure or condition imposes these rhythms as well as on the physiological meaning of the observed gender differences.

Neuroimmune mechanisms in patients with atopic dermatitis during chronic stress.

OBJECTIVE: To identify pathoaetiological neuroimmune mechanisms in patients with atopic dermatitis (AD) and chronic stress, focusing at nerve density, sensory neuropeptides, and the serotonergic system. METHODS: Eleven patients with AD with histories of stress worsening were included. Biopsies from involved and non-involved skin were processed for immunohistochemistry. Salivary cortisol test was done as a marker for chronic stress. RESULTS: There were more acanthosis and fewer nerve fibres in epidermis and papillary dermis of involved compared with non-involved skin. Whereas there was no significant change in the number of substance P and calcitonin gene-related peptide-positive nerve fibres between the involved and non-involved skin, there was an increase in the epidermal fraction of 5-hydroxtrytamine 1A (5-HT1A) receptor and serotonin transporter protein (SERT) immunoreactivity in the involved skin. The number of 5-HT2AR, CD3-positive cells, and SERT-positive cells, most of them being CD3 positive, was increased in involved skin. There was an increase in mast cells in the involved skin, and these cells were often located close to the basement membrane. There was a strong tendency to a correlation between 5-HT2AR positive cells in the papillary dermis of involved skin and low cortisol ratios, being an indicator of chronic stress. CONCLUSION: A changed innervation and modulation of the serotonergic system are indicated in chronic atopic eczema also during chronic stress.

Neuronal instability: implications for Rett's syndrome.

The maturational changes in the brain and spinal cord do not linearly proceed from immature in infants to mature in adults. Dendrites dynamically extend or retract as neurotrophic factors fluctuate. In certain cases mature neurons can be seen soon after birth, and in other cases immature neurons can be identified in the aged brain. Monoamine 'neurotransmitter'; such as serotonin (5-HT), dopamine and norepinephrine appear to function as Maintenance Growth Factors since they must be present in order to produce their maturational actions. Serotonin neurons contain TRK-B receptors and are sensitive to availability of the trophic factor, BDNF. 5-HT also functions by promoting the release of the glial extension factor, S-100beta. 5-HT and S-100beta can provide maturational signals to a variety of neurons, in both cortical and subcortical areas, and appear to be involved in regulating the maturation and release of acetylcholine and dopamine. We have shown that activation of the 5-HT1A receptor is particularly effective in inducing growth of stunted neurons. The mechanism of action of the 5-HT1A receptor involves both a direct inhibition on c-AMP and pCREB formation in postsynaptic neurons and a release of S-100beta from glial cells. Both these events are capable of stabilization and elaboration of the cytoskeleton of the neuron and inhibition of apoptosis. 5-HT1A receptors have been shown to effectively reverse stunted neurons and microencephaly produced in animal models of fetal alcohol syndrome and prenatal cocaine administration. I discuss the implications for regressive disorders such as Rett's syndrome and autism, and the feasibility of treatments with 5-HT1A agonists in children with developmental disorders.

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.

Colchicine-induced cytoskeletal collapse and apoptosis in N-18 neuroblastoma cultures is rapidly reversed by applied S-100beta.

Brain connections depend on a stable association between dendrites and axons whose cytoskeleton is stabilized by the proteins MAP-2 and tau, respectively. The glial protein S-100beta inhibits the phosphorylation by PKC of these two microtubule-associated proteins. In order to determine if exogenous S-100beta can directly influence the cytoskeleton of living cells, cultures of N-18 cells (neuroblastoma clonal cell line) are treated for 30 min in serum-free medium with 10(-6) M colchicine. In normal media, colchicine induces a rapid retraction of processes, membrane blebbing, nuclear collapse, and cell death. The observed cellular changes, due to cytoskeletal collapse after exposure to colchicine, are similar and consistent with the loss of processes and cytoplasmic blebbing seen in cells undergoing apoptosis. The addition of 20 ng/ml of S-100beta after the initial 30-min exposure to colchicine prevents apoptosis, nuclear collapse and induces the regrowth of retracted processes. Cells were treated with the Hoechst Stain, a fluorescent marker that binds to nuclear material, to determine the occurrence of apoptosis in our cultures. In our control cultures, receiving no drugs, we found that 15.1% of the cells were apoptotic. When colchicine was added to the culture medium we found that 31.6% of the cells became apoptotic. However, when colchicine was followed by exposure to S-100beta we found that only 5.4% of the cells were apoptotic. Our results suggest that extracellular application of the glial protein S-100beta is sufficient to reverse colchicine-induced cytoskeletal collapse and prevent the resultant apoptosis of the cells. The increased levels of S-100beta seen after brain injury and in certain neurological and psychiatric disorders may be considered as beneficial for brain recovery.

Pyramidal cell axons show a local specialization for GABA and 5-HT inputs in monkey and human cerebral cortex.

Various mechanisms are thought to control excitation of pyramidal cells of the cerebral cortex. With immunocytochemical methods, we found that the proximal portions of numerous pyramidal cell axons (Pyr-axons) in the human and monkey neocortex are immunoreactive for the serotonin (5-HT) receptor 5-HT-(1A). With double-labeling experiments and confocal laser microscopy, we found that most (93.4%) of the 5-HT(1A)-immunoreactive Pyr-axons present in layers II and III were innervated by parvalbumin-immunoreactive chandelier cell axon terminals. In addition, Pyr-axons were compartmentalized: 5-HT-(1A) receptors were found proximal to inputs from chandelier cells. Although we found close appositions between GABAergic chandelier cell axon terminals and Pyr-axons, suggesting synaptic connections, we did not observe 5-HT-immunoreactive fibers in close proximity to the Pyr-axons. These results suggested that Pyr-axons are under the influence of 5-HT in a paracrine manner (via 5-HT-(1A) receptors) and, more distally, are under the influence of gamma-aminobutyric acid (GABA) in a synaptic manner (through the axons of chandelier cells). The local axonal specialization might represent a powerful inhibitory mechanism by which the responses of large populations of pyramidal cells can be globally controlled by subcortical serotonin afferents, in addition to local inputs from GABAergic interneurons.

Trophic interactions between brain-derived neurotrophic factor and s100beta on cultured serotonergic neurons.

Brain-derived neurotrophic factor (BDNF) and S100beta stimulate serotonergic neurons in fetal rat raphe primary cultures grown under serum-free conditions. BDNF (50 ng/ml) treatment for 3 h enhanced S100beta immunoreactivity in both raphe and hippocampal glial cells. Combined treatment with BDNF and S100beta for 3 days increased the soma area of 5-HT neurons, but not the neurite length. Our results suggest that BDNF and S100beta, which regulate different signal transduction cascades, interact to exert complimentary effects on neuronal maturation by acting sequentially, not concurrently.

Long term depletion of serotonin leads to selective changes in glutamate receptor subunits.

The present study was carried out to clarify possible modulation mechanism of serotonin (5-HT) on glutamatergic neurotransmission in the rat cerebral cortex. 5-HT was depleted by a 5-HT metabolite blocker (para-chlorophenylalanine; pCPA) for a week. Receptor binding experiments using (S)-[(3)H]alpha-amino-3-hydroxy-5-methylisoxazol-4-propionic acid (AMPA) showed a considerable increase in B(max) value of the membrane samples prepared from the cerebral cortex of rats compared with that of control animals received saline. In contrast, B(max) value of the [(3)H]MK-801 binding experiments for NMDA receptor was not changed by pCPA-treatment. Changes in the density of each AMPA receptor subtype were examined in the cerebral cortex by immunoblot analyses using antibodies against AMPA receptor subunits. The density of immunoreactive bands with receptor subtype specific antibodies against GluR2/3 and GluR2 receptors was increased, whereas that of GluR1 receptors was decreased. Considering GluR2 receptor subtype inhibits Ca(2+) influx into neurons, the present study suggests that 5-HT appears to modulate synaptic plasticity by regulating the density of each AMPA receptor subtype.

Homologous regulation of 5-HT1A receptor mRNA in adult rat hippocampal dentate gyrus.

Short-term adrenalectomy induces a loss of mature granular neuronal phenotypes in the hippocampal dentate gyrus; injection of 5-HT1A receptor agonist reverses this effect. Adrenalectomy also induces an increase of expression of 5-HT1A receptor mRNA in the dentate gyrus. This study tested the effect of 5-HT1A agonist on this adrenalectomy-induced increase of 5-HT1A mRNA. Five, 9 and 18 days after adrenalectomy, 5-HT1A receptor mRNA is increased in the granular layer of the dentate gyrus. The increase is nearly 100% at day 18 after adrenalectomy. 5-HT1A agonist treatment decreased 5-HT1A mRNA both at 9 (20%) and 18 days (34%) after adrenalectomy. Our results indicated that a 5-HT1A agonist can partly reverse the adrenalectomy-induced increase of 5-HT1A mRNA and loss of mature granular neuronal phenotypes in hippocampal dentate gyrus.

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.

Growth inhibitory effects of a mu opioid on cultured cholinergic neurons from fetal rat ventral forebrain, brainstem, and spinal cord.

Cholinergic pathways play a role in respiration in the mammalian brain, and agents that affect respiratory function such as opioid peptides might have positive or negative neurotrophic effects during the development of these cholinergic connections. Rat fetal nerve cell cultures from developmental stages E14-E18 were established in 96-well plates from ventral forebrain (VFB), an area rich in cholinergic neurons, and from brainstem and rostral spinal cord, areas where respiratory control systems and cholinergic neurons co-exist. High affinity 3H-choline uptake was highest in E14 VFB cultures and decreased to 20% of this value by E16 and E18. Choline uptakes in E14 brainstem and spinal cord were only 20% and 13%, respectively, of E14 VFB uptake. A mu opioid receptor agonist, d-ala2-mePhe4-gly(ol)5]-enkephalin (DAMGO), was tested for its effect on somal area and neurite outgrowth in E16 cultures. Cholinergic neurons were identified by immunostaining with choline acetyltransferase antibody. DAMGO (10(-8) M) significantly decreased somal area in VFB cultures and spinal cord, but had no effect on somal area in brainstem. Naltrexone (10(-6) M) reversed this inhibition. Spinal cord cell neurite outgrowth was inhibited by DAMGO, and this inhibition was reversed by naltrexone. DAMGO had no significant effect on neurite length in VFB. Brainstem neurite length was paradoxically increased by both DAMGO and naltrexone. It was concluded that mu-selective opioid peptides inhibit growth of cultured cholinergic neurons in VFB and spinal cord, but not in the brainstem. There was no evidence for endogenous opioid activity in either VFB or spinal cord cultures.

Agonist- and antagonist-induced plasticity of rat 5-HT1A receptor in hippocampal cell culture.

We examined the response and regulation of 5-HT1A receptor on hippocampal cultured fetal neurons grown in the absence of serotonin and steroids using three experimental designs: 1) functional response using an antibody against phosphorylated cyclic adenosine monophosphate response element binding protein (pCREB); 2) transcriptional regulation using in situ hybridization; and 3) translational expression using antipeptide 5-HT1A receptor antibody. Pretreatment of cultured hippocampal cells with the agonist 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH-DPAT) (10(-8) M) or ipsapirone (IPS) (10(-9) M) for 10 min blocked the forskolin-stimulated increase in pCREB immunoreactivity. In situ hybridization radioautography revealed that IPS (10(-9) M) decreased the 5-HT1A receptor mRNA expression (-33%) after a 24-h treatment. The decrease in 5-HT1A receptor mRNAwas accompanied by a change in protein immunoreactivity using a 5-HT1A receptor antipeptide antibody. Computer-assisted morphometric analyses showed a reduction in the 5-HT1A receptor immunoreactive (IR) intensity as compared to control 24 h after treatment with 8-OH-DPAT (10(-7)-10(-12) M) and IPS (10(-9) M). Thus, fetal hippocampal neurons have a functional 5-HT1A receptor that is downregulated at both the transcription and translation levels. In addition, we found increased 5-HT1A receptor-IR intensity (+17% approximately +39%) 24 h after treatment with the antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635) (10(-7)-10(-12) M). Our results indicate that the 5-HT1A receptor is sensitive to both agonists (downregulation) and antagonists (upregulation) in hippocampal fetal neurons grown in the absence of serotonin and steroids.

Characterization of the translocation of protein kinase C (PKC) by 3,4-methylenedioxymethamphetamine (MDMA/ecstasy) in synaptosomes: evidence for a presynaptic localization involving the serotonin transporter (SERT).

3, 4-methylenedioxymethamphetamine (MDMA or Ecstasy) is a substituted amphetamine whose acute and long-term effects on the serotonin system are dependent on an interaction with the 5-HT uptake transporter (SERT). Although much of the work dedicated to the study of this compound has focused on its ability to release monoamines, this drug has many important metabolic consequences on neurons and glial cells. The identification of these physiological responses will help to bridge the gap that exists in the information between the acute and neurotoxic effects of amphetamines. Substituted amphetamines have the ability to produce a long-term translocation of protein kinase C (PKC) in vivo, and this action may be crucial to the development of serotonergic neurotoxicity. Our earlier results suggested that PKC activation occurred through pre- and postsynaptic mechanisms. Because the primary site of action of these drugs is the 5-HT transporter, we now expand on our previous results and attempt to characterize MDMA's ability to translocate PKC within cortical 5-HT nerve terminals. In synaptosomes, MDMA produced a concentration-dependent increase in membrane-bound PKC (as measured by 3H-phorbol 12, 13 dibutyrate, 3H-PDBu) bindings sites. This response was abolished by cotreatment with the specific serotonin reuptake inhibitor (SSRI), fluoxetine, but not by the 5-HT2A/2C antagonist, ketanserin. In contrast, full agonists to 5-HT1A and 5-HT2 receptors did not produce significant PKC translocation. MDMA-mediated PKC translocation also requires the presence of extracellular calcium ions. Using assay conditions where extracellular calcium was absent prevented in vitro activation of PKC by MDMA. Prolonged PKC translocation has been hypothesized to contribute to the calcium-dependent neurotoxicity produced by substituted amphetamines. In addition, many physiological processes within 5-HT nerve terminals, including 5-HT reuptake and vesicular serotonin release, are susceptible to modification by PKC-dependent protein phosphorylation. Our results suggest that prolonged activation of PKC within the 5-HT nerve terminal may contribute to lasting changes in the homeostatic function of 5-HT neurons, leading to the degeneration of specific cellular elements after repeated MDMA exposure.

Molecular characterization of antipeptide antibodies against the 5-HT1A receptor: evidence for state-dependent antibody binding.

Differential immunohistochemical labeling is often observed using different antibodies against the same protein. Two polyclonal antipeptide antibodies against the 5-HT1A receptor have been generated by our group. The S1A-170 (aa 170-186) and 258 (aa 258-274) are specific for sites in the second extracellular loop and third intracellular loop, respectively [E.C. Azmtia, I. Yu, H.M. Akbari, N. Kheck, P.M. Whitaker-Azmitia and D.R. Marshak, Antipeptide antibodies against the 5-HT1A receptor, J. Chem. Neuroanat., 5 (1992) 289-298]. Comparison of the labeling patterns of these two antibodies and other antipeptide antibodies against the 5-HT1A receptor revealed that although similar populations of cells were labeled, individual antibodies favor certain staining patterns. Immunocytochemistry and western blotting results of transfected cell lines and brain tissue revealed the following: (1) both the S1A-170 and S1A-258 are specific for the 5-HT1A receptor when used for immunocytochemistry in transfected HEK-293 and COS-1 cells; (2) when expressed in cultured cell lines, the 5-HT1A receptor is differentially glycosylated dependent on cell type, and the S1A-258 is specific for only certain species on immunoblots; and (3) the S1A-258 and L5B7 [M. Riad, S. El Mestikawy, D. Derge, H. Gozlan, and M. Hamon, Visualization and quantification of central 5-HT1A receptors with specific antibodies, Neurochem. Int., 4 (1991) 413-423] label common bands at 40 and 70 kDa on immunoblots of hippocampal proteins, but show opposite staining intensities. These results provide evidence for the immunocytochemical specificity of both the S1A-170 and S1A-258 and suggest that the discrepancies noted in immunohistochemistry may be due in part to different molecular conformations.