Prenatal Cocaine Disrupts Serotonin Signaling-Dependent Behaviors: Implications for Sex Differences, Early Stress and Prenatal SSRI Exposure

Prenatal cocaine (PC) exposure negatively impacts the developing nervous system, including numerous changes in serotonergic signaling. Cocaine, a competitive antagonist of the serotonin transporter, similar to selective serotonin reuptake inhibitors (SSRIs), also blocks dopamine and norepinephrine transporters, leaving the direct mechanism through which cocaine disrupts the developing serotonin system unclear. In order to understand the role of the serotonin transporter in cocaine’s effect on the serotonergic system, we compare reports concerning PC and prenatal antidepressant exposure and conclude that PC exposure affects many facets of serotonergic signaling (serotonin levels, receptors, transporters) and that these effects differ significantly from what is observed following prenatal SSRI exposure. Alterations in serotonergic signaling are dependent on timing of exposure, test regimens, and sex. Following PC exposure, behavioral disturbances are observed in attention, emotional behavior and stress response, aggression, social behavior, communication, and like changes in serotonergic signaling, these effects depend on sex, age and developmental exposure. Vulnerability to the effects of PC exposure can be mediated by several factors, including allelic variance in serotonergic signaling genes, being male (although fewer studies have investigated female offspring), and experiencing the adverse early environments that are commonly coincident with maternal drug use. Early environmental stress results in disruptions in serotonergic signaling analogous to those observed with PC exposure and these may interact to produce greater behavioral effects observed in children of drug-abusing mothers. We conclude that based on past evidence, future studies should put a greater emphasis on including females and monitoring environmental factors when studying the impact of PC exposure

The sea urchin embryo as a model for mammalian developmental neurotoxicity: ontogenesis of the high-affinity choline transporter and its role in cholinergic trophic activity.

Embryonic development in the sea urchin requires trophic actions of the same neurotransmitters that participate in mammalian brain assembly. We evaluated the development of the high-affinity choline transporter, which controls acetylcholine synthesis. A variety of developmental neurotoxicants affect this transporter in mammalian brain. [3H]Hemicholinium-3 binding to the transporter was found in the cell membrane fraction at stages from the unfertilized egg to pluteus, with a binding affinity comparable with that seen in mammalian brain. Over the course of development, the concentration of transporter sites rose more than 3-fold, achieving concentrations comparable with those of cholinergically enriched mammalian brain regions. Dimethylaminoethanol (DMAE), a competitive inhibitor of choline transport, elicited dysmorphology beginning at the mid-blastula stage, with anomalies beginning progressively later as the concentration of DMAE was lowered. Pretreatment, cotreatment, or delayed treatment with acetylcholine or choline prevented the adverse effects of DMAE. Because acetylcholine was protective at a lower threshold, the DMAE-induced defects were most likely mediated by its effects on acetylcholine synthesis. Transient removal of the hyaline layer enabled a charged transport inhibitor, hemicholinium-3, to penetrate sufficiently to elicit similar anomalies, which were again prevented by acetylcholine or choline. These results indicate that the developing sea urchin possesses a high-affinity choline transporter analogous to that found in the mammalian brain, and, as in mammals, the functioning of this transporter plays a key role in the developmental, trophic activity of acetylcholine. The sea urchin model may thus be useful in high-throughput screening of suspected developmental neurotoxicants

An anxiety-like phenotype in mice selectively bred for aggression

Using selective bi-directional breeding procedures, two different lines of mice were developed. The NC900 line is highly reactive and attacks their social partners without provocation, whereas aggression in NC100 animals is uncommon in social environments. The enhanced reactivity of NC900 mice suggests that emotionality may have been selected with aggression. As certain forms of anxiety promote exaggerated defensive responses, we tested NC900 mice for the presence of an anxiety-like phenotype. In the open field, light-dark exploration, and zero maze tests, NC900 mice displayed anxiety-like responses. These animals were less responsive to the anxiolytic actions of diazepam in the zero maze than NC100 animals; diazepam also reduced the reactivity and attack behaviors of NC900 mice. The NC900 mice had reduced diazepam-sensitive GABAA receptor binding in brain regions associated with aggression and anxiety. Importantly, there was a selective reduction in levels of the GABAA receptor α2 subunit protein in NC900 frontal cortex and amygdala; no changes in α1 or γ2 subunit proteins were observed. These findings suggest that reductions in the α2 subunit protein in selected brain regions may underlie the anxiety and aggressive phenotype of NC900 mice. Since anxiety and aggression are comorbid in certain psychiatric conditions, such as borderline personality and posttraumatic stress disorder, investigations with NC900 mice may provide new insights into basic mechanisms that underlie these and related psychiatric conditions

GABA A Receptors Mediate Trophic Effects of GABA on Embryonic Brainstem Monoamine Neurons In Vitro

The inhibitory neurotransmitter GABA may act as a trophic signal for developing monoamine neurons in embryonic rat brain, because GABA neurons and their receptors appear in brainstem during generation of monoamine neurons. To test this hypothesis, we used dissociated cell cultures from embryonic day 14 rat brainstem, which contains developing serotonin (5-HT), noradrenaline (tyrosine hydroxylase; TH), and GABA neurons. Immunocytochemistry and reverse transcription-PCR (RT-PCR) revealed the presence of multiple alpha, beta, gamma, and delta subunits in these cultures. Competitive RT-PCR demonstrated high levels of beta3 subunit transcripts. Expression of functional GABAA receptors was demonstrated using 36Cl- flux assays. To investigate GABAergic regulation of neuronal survival and growth, cultures were treated for 1-3 d in vitro with 10 microM GABA and/or GABAA antagonist (bicuculline or the pesticide dieldrin). The effects of treatments were quantified by analysis of immunoreactive 5-HT, TH, and GABA neurons. GABAA receptor ligands differentially regulated neuronal survival and growth depending on neurotransmitter phenotype. GABA exerted positive effects on monoamine neurons, which were countered by bicuculline (and dieldrin, 5-HT neurons only). By itself, bicuculline produced inhibitory effects on both 5-HT and TH neurons, whereas dieldrin potently inhibited 5-HT neurons only. GABA neurons responded positively to both antagonists, but more strongly to bicuculline. Taken together, these results demonstrate that the activation/inhibition of GABAA receptors produces opposite effects on the development of embryonic monoamine and GABA neurons. This suggests that these neurotransmitter phenotypes may express GABAA receptors that differ in fundamental ways, and these differences determine the developmental responses of these cells to GABAergic stimuli

Early postnatal administration of 5,7-dihydroxytryptamine: Effects on substance P and thyrotropin-releasing hormone neurons and terminals in rat brain

Substance P, thyrotropin-releasing hormone (TRH) and serotonin are putative neurotransmitters which have been proposed to coexist in some brain neurons Our previous immunocytochemical and biochemical studies have demonstrated that 85–100% of all serotonin neurons are destroyed following neonatal 5,7-dihydroxtryptamine (5,7-DHT) treatment In this study, we have determined the effect of neonatal 5,7-DHT and desipramine (DMI) treatment on the biochemical content and immunocytochemical localization of substance P and TRH throughout the brain Interestingly, we have observed that virtually all substance P- and TRH-immunoreactive cells in the ventral pons-medulla are destroyed by the neurotoxin. However, peptide-containing neurons in other regions were not affected Additionally, we measured the peptide content and found that TRH is significantly decreased in the spinal cord (−50%) and pons-medulla (−20%), but not in other brain regions Substance P content was not significantly altered in any region, even after a greater than 90% reduction of serotonin These data indicate that the co-localized substance P and TRH forms a small proportion of the total peptide in brai

An Invertebrate Model of the Developmental Neurotoxicity of Insecticides: Effects of Chlorpyrifos and Dieldrin in Sea Urchin Embryos and Larvae

Chlorpyrifos targets mammalian brain development through a combination of effects directed at cholinergic receptors and intracellular signaling cascades that are involved in cell differentiation. We used sea urchin embryos as an invertebrate model system to explore the cellular mechanisms underlying the actions of chlorpyrifos and to delineate the critical period of developmental vulnerability. Sea urchin embryos and larvae were exposed to chlorpyrifos at different stages of development ranging from early cell cleavages through the prism stage. Although early cleavages were unaffected even at high chlorpyrifos concentrations, micromolar concentrations added at the mid-blastula stage evoked a prominent change in cell phenotype and overall larval structure, with appearance of pigmented cells followed by their accumulation in an extralarval cap that was extruded from the animal pole. At higher concentrations (20-40 microM), these abnormal cells constituted over 90% of the total cell number. Studies with cholinergic receptor blocking agents and protein kinase C inhibitors indicated two distinct types of effects, one mediated through stimulation of nicotinic cholinergic receptors and the other targeting intracellular signaling. The effects of chlorpyrifos were not mimicked by chlorpyrifos oxon, the active metabolite that inhibits cholinesterase, nor by nonorganophosphate cholinesterase inhibitors. Dieldrin, an organochlorine that targets GABA(A )receptors, was similarly ineffective. The effects of chlorpyrifos and its underlying cholinergic and signaling-related mechanisms parallel prior findings in mammalian embryonic central nervous system. Invertebrate test systems may thus provide both a screening procedure for potential neuroteratogenesis by organophosphate-related compounds, as well as a system with which to uncover novel mechanisms underlying developmental vulnerability

The sea urchin embryo, an invertebrate model for mammalian developmental neurotoxicity, reveals multiple neurotransmitter mechanisms for effects of chlorpyrifos: Therapeutic interventions and a comparison with the monoamine depleter, reserpine

Lower organisms show promise for the screening of neurotoxicants that might target mammalian brain development. Sea urchins use neurotransmitters as embryonic growth regulatory signals, so that adverse effects on neural substrates for mammalian brain development can be studied in this simple organism. We compared the effects of the organophosphate insecticide, chlorpyrifos in sea urchin embryos with those of the monoamine depleter, reserpine, so as to investigate multiple neurotransmitter mechanisms involved in developmental toxicity and to evaluate different therapeutic interventions corresponding to each neurotransmitter system. Whereas reserpine interfered with all stages of embryonic development, the effects of chlorpyrifos did not emerge until the mid-blastula stage. After that point, the effects of the two agents were similar. Treatment with membrane permeable analogs of the monoamine neurotransmitters, serotonin and dopamine, prevented the adverse effects of either chlorpyrifos or reserpine, despite the fact that chlorpyrifos works simultaneously through actions on acetylcholine, monoamines and other neurotransmitter pathways. This suggests that different neurotransmitters, converging on the same downstream signaling events, could work together or in parallel to offset the developmental disruption caused by exposure to disparate agents. We tested this hypothesis by evaluating membrane permeable analogs of acetylcholine and cannabinoids, both of which proved effective against chlorpyrifos- or reserpine-induced teratogenesis. Invertebrate test systems can provide both a screening procedure for mammalian neuroteratogenesis and may uncover novel mechanisms underlying developmental vulnerability as well as possible therapeutic approaches to prevent teratogenesis

Ontogeny of serotonin and serotonin2A receptors in rat auditory cortex

Maturation of the mammalian cerebral cortex is, in part, dependent upon multiple coordinated afferent neurotransmitter systems and receptor-mediated cellular linkages during early postnatal development. Given that serotonin (5-HT) is one such system, the present study was designed to specifically evaluate 5-HT tissue content as well as 5-HT2A receptor protein levels within the developing auditory cortex (AC). Using high performance liquid chromatography (HPLC), 5-HT and the metabolite, 5-hydroxyindoleacetic acid (5-HIAA), was measured in isolated AC, which demonstrated a developmental dynamic, reaching young adult levels early during the second week of postnatal development. Radioligand binding of 5-HT2A receptors with the 5-HT2A/2C receptor agonist, 125I-DOI ((+/−)-1-(2,5- dimethoxy-4-iodophenyl)-2-aminopropane HCl; in the presence of SB206553, a selective 5-HT2C receptor antagonist, also demonstrated a developmental trend, whereby receptor protein levels reached young adult levels at the end of the first postnatal week (P8), significantly increased at P10 and at P17, and decreased back to levels not significantly different from P8 thereafter. Immunocytochemical labeling of 5-HT2A receptors and confocal microscopy revealed that 5-HT2A receptors are largely localized on layer II/III pyramidal cell bodies and apical dendrites within AC. When considered together, the results of the present study suggest that 5-HT, likely through 5-HT2A receptors, may play an important role in early postnatal AC development

Allometry and Ecology of the Bilaterian Gut Microbiome.

Classical ecology provides principles for construction and function of biological communities, but to what extent these apply to the animal-associated microbiota is just beginning to be assessed. Here, we investigated the influence of several well-known ecological principles on animal-associated microbiota by characterizing gut microbial specimens from bilaterally symmetrical animals (Bilateria) ranging from flies to whales. A rigorously vetted sample set containing 265 specimens from 64 species was assembled. Bacterial lineages were characterized by 16S rRNA gene sequencing. Previously published samples were also compared, allowing analysis of over 1,098 samples in total. A restricted number of bacterial phyla was found to account for the great majority of gut colonists. Gut microbial composition was associated with host phylogeny and diet. We identified numerous gut bacterial 16S rRNA gene sequences that diverged deeply from previously studied taxa, identifying opportunities to discover new bacterial types. The number of bacterial lineages per gut sample was positively associated with animal mass, paralleling known species-area relationships from island biogeography and implicating body size as a determinant of community stability and niche complexity. Samples from larger animals harbored greater numbers of anaerobic communities, specifying a mechanism for generating more-complex microbial environments. Predictions for species/abundance relationships from models of neutral colonization did not match the data set, pointing to alternative mechanisms such as selection of specific colonists by environmental niche. Taken together, the data suggest that niche complexity increases with gut size and that niche selection forces dominate gut community construction.IMPORTANCEThe intestinal microbiome of animals is essential for health, contributing to digestion of foods, proper immune development, inhibition of pathogen colonization, and catabolism of xenobiotic compounds. How these communities assemble and persist is just beginning to be investigated. Here we interrogated a set of gut samples from a wide range of animals to investigate the roles of selection and random processes in microbial community construction. We show that the numbers of bacterial species increased with the weight of host organisms, paralleling findings from studies of island biogeography. Communities in larger organisms tended to be more anaerobic, suggesting one mechanism for niche diversification. Nonselective processes enable specific predictions for community structure, but our samples did not match the predictions of the neutral model. Thus, these findings highlight the importance of niche selection in community construction and suggest mechanisms of niche diversification

Acquisition of suppressive function by conventional T cells limits antitumor immunity upon Treg depletion

Regulatory T (Treg) cells contribute to immune homeostasis but suppress immune responses to cancer. Strategies to disrupt Treg cell–mediated cancer immunosuppression have been met with limited clinical success, but the underlying mechanisms for treatment failure are poorly understood. By modeling Treg cell–targeted immunotherapy in mice, we find that CD4+ Foxp3− conventional T (Tconv) cells acquire suppressive function upon depletion of Foxp3+ Treg cells, limiting therapeutic efficacy. Foxp3− Tconv cells within tumors adopt a Treg cell–like transcriptional profile upon ablation of Treg cells and acquire the ability to suppress T cell activation and proliferation ex vivo. Suppressive activity is enriched among CD4+ Tconv cells marked by expression of C-C motif receptor 8 (CCR8), which are found in mouse and human tumors. Upon Treg cell depletion, CCR8+ Tconv cells undergo systemic and intratumoral activation and expansion, and mediate IL-10–dependent suppression of antitumor immunity. Consequently, conditional deletion of Il10 within T cells augments antitumor immunity upon Treg cell depletion in mice, and antibody blockade of IL-10 signaling synergizes with Treg cell depletion to overcome treatment resistance. These findings reveal a secondary layer of immunosuppression by Tconv cells released upon therapeutic Treg cell depletion and suggest that broader consideration of suppressive function within the T cell lineage is required for development of effective Treg cell–targeted therapies