Jean M. Lauder

Mouse behavioral tasks relevant to autism: Phenotypes of 10 inbred strains

Three defining clinical symptoms of autism are aberrant reciprocal social interactions, deficits in social communication, and repetitive behaviors, including motor stereotypies and insistence on sameness. We developed a set of behavioral tasks designed to model components of these core symptoms in mice. Male mice from 10 inbred strains were characterized in assays for sociability, preference for social novelty, and reversal of the spatial location of the reinforcer in T-maze and Morris water maze tasks. Six strains, C57BL/6J, C57L/J, DBA/2J, FVB/NJ, C3H/HeJ, and AKR/J, showed significant levels of sociability, while A/J, BALB/cByJ, BTBR T(+)tf/J, and 129S1/SvImJ mice did not. C57BL/6J, C57L/J, DBA/2J, FVB/NJ, BALB/cByJ, and BTBR T(+)tf/J showed significant preference for social novelty, while C3H/HeJ, AKR/J, A/J, and 129S1/SvImJ did not. Normal scores on relevant control measures confirmed general health and physical abilities in all strains, ruling out artifactual explanations for social deficits. Elevated plus maze scores confirmed high anxiety-like behaviors in A/J, BALB/cByJ, and 129S1/SvImJ, which could underlie components of their low social approach. Strains that showed high levels of performance on acquisition of a T-maze task were also able to reach criterion for reversal learning. On the Morris water maze task, DBA/2J, AKR/J, BTBR T(+)tf/J, and 129S1/SvImJ failed to show significant quadrant preference during the reversal probe trial. These results highlight a dissociation between social task performance and reversal learning. BTBR T(+)tf/J is a particularly interesting strain, displaying both low social approach and resistance to change in routine on the water maze, consistent with an autism-like phenotype. Our multitask strategy for modeling symptoms of autism will be useful for investigating targeted and random gene mutations, QTLs, and microarray analyses.

Ontogeny of the Serotonergic System in the Rat: Serotonin as a Developmental Signala

The serotonergic system is an early forming component of the CNS circuitry, beginning its development on gestational days 11-12 in the rat. Owing to its early presence in the embryonic nervous system, 5-HT has been proposed to act as a developmental signal for receptive cells. In vivo and in vitro evidence that 5-HT can influence both biochemical and morphological differentiation of raphe neurons and receptive target cells suggests that this neurotransmitter may have an organizing function in the developing nervous system which involves effects on neurite outgrowth and other aspects of neuronal differentiation, including synaptogenesis. Such functions may be mediated by a variety of 5-HT receptors located on both neuronal and non-neuronal cells. The apparent function of 5-HT as a differentiation signal in the developing nervous system raises important issues regarding the use of psychoactive serotonergic drugs by pregnant women, since these drugs may act as neural teratogens in the unborn child.

Development of the serotonergic system in the rat embryo: An immunocytochemical study ☆

The development of central serotonergic neurons has been examined immunocytochemically utilizing an antiserum to serotonin (5-HT). Cells of the B4-B9 complex are first detected early on embryonic day 13 (E13; 7 mm crown rump length, CRL) and increase rapidly in number through E15 when they appear as bilateral columns situated from just caudal to the mesencephalic flexure to the pontine flexure. Aggregation of cells into subgroups is apparent soon after 5-HT neurons leave the ventricular zone, allowing the identification of certain subdivisions of the B4-B9 complex long before they assume their adult locations. The initial detection of 5-HT immunoreactive cells in the medulla occurs 1-2 days after the appearance of cells in the B4-B9 complex, although it has been reported that the time of origin of medullary raphe neurons (B1-B3) occurs before that of raphe neurons in the midbrain and pons (B4-B9). The first medullary 5-HT neurons, comprising the B3 subdivision occur ventro-laterally on E14 (10-11 mm CRL) at least 1-2 days before midline 5-HT neurons are visualized in the B1 and B2 groups. Thus, in contrast to cells in the B4-B9 complex, medullary 5-HT neurons complete much of their migration before they can be detected immunocytochemically, indicating that the time of onset of transmitter synthesis and storage may differ during differentiation of cells sharing a common neurotransmitter phenotype. The formation of ascending 5-HT fiber projections occurs rapidly from cells of the B4-B9 complex. Within 24 hours after the initial detection of 5-HT fiber immunoreactivity in such cells at E13, their axons are seen entering the caudal diencephalon (E14). These fibers have traversed the diencephalon and floor of the telencephalon by E15-E16 and reach the frontal neocortical pole by E17. The main ascending bundle of 5-HT axons courses through the diencephalon in the vicinity of the medial forebrain bundle, although some fibers also diverge and travel along certain pre-existing non-5HT pathways. However, examples are also found of acute directional changes in 5-HT fiber growth which do not appear to be associated with pre-formed non-5HT pathways. The pattern of ascending fiber outgrowth suggests a priority routing system which provides certain regions with 5-HT axons in a preferential sequence irrespective of the distance of these areas from 5-HT cell groups or from major bundles of ascending 5-HT fibers.

Prenatal ontogeny of the gabaergic system in the rat brain: An immunocytochemical study

Prenatal development of the GABAergic system in the rat brain has been studied using an antiserum to GABA-glutaraldehyde-hemocyanin conjugates, specific for GABAergic neurons. The gamma-aminobutyric acid (GABA) system has been found to differentiate very early relative to other transmitter-identified neurons, such that by embryonic day 13 a well developed fiber network exists in the brainstem, mesencephalon and diencephalon, including a large projection in the posterior commissure and adjacent areas on the surface of the mesencephalon and tectum. Although no cell bodies are visible at this time, it appears that these fibers originate from the caudal brainstem and spinal cord. GABAergic cell bodies begin to appear on embryonic day 14 in the lateral cortical anlage. By embryonic day 16, they are also visible in the basal forebrain and in all regions of cortex where they are located in three zones: in layer I, below the cortical plate, and in the intermediate zone. Also contained in the outer part of layer I is a dense fiber plexus which stains intensely for GABA. These fibers may be part of the first contingent of cortical afferents to invade the telencephalic vesicle, an event which is thought to be a stimulus for the beginning of neuronal differentiation in this region. By E18, two bands of immunoreactivity are visible in layer I, which probably contain both cell bodies and fibers. The trajectories taken by growing GABAergic fibers in the brainstem, mesencephalon and diencephalon at embryonic day 13 and at subsequent stages of development are coincident with regions of both monoaminergic and peptidergic differentiation and appear to correspond to recently reported patterns of benzodiazepine receptors which appear slightly later. The early differentiation of the GABAergic system could indicate a trophic role for GABA in early brain development, possibly involving receptors for this neurotransmitter or related substances.

Neurotransmitters as morphogens

Publisher Summary This chapter presents considerable evidence for morphogenetic functions of neurotransmitters during embryonic development, a role that may derive from their appearance early in evolution, still evidenced today by their presence in primitive organisms. Use of the term “morphogen” in the context of neurotransmitters acting as regulators of morphogenetic processes gives it a more general meaning. Turing has originally defined it in a rather broad sense, even including hormones as possible morphogens. Neurotransmitter substances, including the catecholamines, serotonin acetylcholine and γ-aminobutyric acid (GABA), are found in a variety of primitive organisms and early invertebrate embryos in which no nervous system is present. In such cases, these substances appear to act as both intra- and intercellular signals involved in the control of cell proliferation, ciliary activity, motility, cell shape changes and morphogenetic cell movements. In the adult nervous system, neurotransmitters are discrete intercellular signalling devices that mediate communication between cells in complex neural circuitries. However, they may have evolved to this specialized role from more general humoral functions in primitive organisms where they were used as both intra- and intercellular signalling devices.

Serotonin and serotonin-like substances as regulators of early embryogenesis and morphogenesis

Abstract. The problem of pre-nervous neurotransmitter systems arose from studies carried out on different groups of invertebrates and vertebrates in the late 1950s to early 1960s. These investigations were motivated by an hypothesis formulated by K. S. Koshtoyants concerning the similarity between pre-nervous control processes and neuronal functions. Here, we review new data related to the embryogenetic and morphogenetic functions of serotonin (5-HT) and 5-HT-like substances in early embryos of sea urchins, mouse, and other species. Accumulating evidence across animal phyla indicates that 5-HT, together with other classical neurotransmitters, regulates basic developmental processes, including cell proliferation, migration, differentiation, and morphogenesis. Future investigations of cellular and molecular mechanisms underlying phylogenetically old functions of neurotransmitters could provide new insights into the evolutionary emergence of the vertebrate nervous system.

Social Approach in Genetically-Engineered Mouse Lines Relevant to Autism

Profound impairment in social interaction is a core symptom of autism, a severe neurodevelopmental disorder. Deficits can include a lack of interest in social contact and low levels of approach and proximity to other children. In this study, a three‐chambered choice task was used to evaluate sociability and social novelty preference in five lines of mice with mutations in genes implicated in autism spectrum disorders. Fmr1tm1Cgr/Y(Fmr1−/y) mice represent a model for fragile X, a mental retardation syndrome that is partially comorbid with autism. We tested Fmr1−/ymice on two genetic backgrounds, C57BL/6J and FVB/N‐129/OlaHsd (FVB/129). Targeted disruption of Fmr1 resulted in low sociability on one measure, but only when the mutation was expressed on FVB/129. Autism has been associated with altered serotonin levels and polymorphisms in SLC6A4 (SERT), the serotonin transporter gene. Male mice with targeted disruption of Slc6a4 displayed significantly less sociability than wild‐type controls. Mice with conditional overexpression of Igf‐1 (insulin‐like growth factor‐1) offered a model for brain overgrowth associated with autism. Igf‐1 transgenic mice engaged in levels of social approach similar to wild‐type controls. Targeted disruption in other genes of interest, En2 (engrailed‐2) and Dhcr7, was carried on genetic backgrounds that showed low levels of exploration in the choice task, precluding meaningful interpretations of social behavior scores. Overall, results show that loss of Fmr1 or Slc6a4 gene function can lead to deficits in sociability. Findings from the fragile X model suggest that the FVB/129 background confers enhanced susceptibility to consequences of Fmr1 mutation on social approach.

From oocyte to neuron: Do neurotransmitters function in the same way throughout development?

Summary1.Classical neurotransmitters (such as acetylcholine, biogenic amines, and GABA) are functionally active througout ontogenesis.2.Based on accumulated evidence, reviewed herein, we present an hypothetical scheme describing developmental changes in this functional activity, from the stage of maturing oocytes through neuronal differentiation. This scheme reflects not only the spatio-temporal sequence of these changes, but also the genesis of neurotransmitter functions, from “protosynapses” in oocytes and cleaving embryos to the development of functional neuronal synapses.3.Thus, it appears that neurotransmitters participate in various forms of intra- and intercellular signalling throughout all stages of ontogenesis.

Prenatal infection and risk for schizophrenia: IL-1β, IL-6, and TNFα inhibit cortical neuron dendrite development

Prenatal exposure to infection increases risk for schizophrenia, and we have hypothesized that inflammatory cytokines, generated in response to maternal infection, alter neuron development and increase risk for schizophrenia. We sought to study the effect of cytokines generated in response to infection—interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), and interleukin-6 (IL-6)—on the dendritic development of cortical neurons. Primary mixed neuronal cultures were obtained from E18 rats and exposed to 0, 100, or 1000 units (U)/ml of IL-1β, TNFα, IL-6, or IL-1β+TNFα for 44 h. MAP-2-positive neurons were randomly identified for each condition and the number of primary dendrites, nodes, and total dendrite length was determined. We found that 100 U of TNFα significantly reduced the number of nodes (27%, p=0.02) and total dendritic length (14%, p=0.04), but did not affect overall neuron survival. A measure of 100 U IL-1β+TNFα significantly reduced the number of primary dendrites (17%, p=0.006), nodes (32%, p=0.001), and total dendritic length (30%, p<0.0001), although it did not affect overall neuron survival. At 1000 U, each cytokine significantly reduced the number of primary dendrites (14–24%), nodes (28–37%), as well as total dendritic length (25–30%); neuron survival was reduced by 14–21%. These results indicate that inflammatory cytokines can significantly reduce dendrite development and complexity of developing cortical neurons, consistent with the neuropathology of schizophrenia. These findings also support the hypothesis that cytokines play a key mechanistic role in the link between prenatal exposure to infection and risk for schizophrenia.

Identification of somatomedin/insulin-like growth factor immunoreactive cells in the human fetus.

ABSTRACT: Somatomedins/insulin-like growth factors (Sm/IGFs) are present in blood and in extracts from multiple tissues of the human fetus and induce the proliferation of cultured human fetal cells. To identify the cellular location of immunoreactive Sm/IGF in human fetal tissues, we have performed immunocytochemistry in tissues from prostaglandin-induced human fetal abortuses of 12 to 20 wk in gestation. Every tissue studied except the cerebral cortex contains Sm/IGF immunoreactive cells. Cells staining positively include hepatocytes, hepatic hemopoietic cells, columnar epithelia of the pulmonary airways, intestine and kidney tubules, adrenal cortical cells, dermal cells, skeletal and cardiac muscle fibers, and pancreatic islet and acinar cells. Immunostaining was specific for Sm/IGFs, but because of the cross-reactivity of the antibodies it was not possible to determine whether the immunoreactivity represented Sm-C/IGF I, IGF II, or both. Liver contained the greatest proportion of immunoreactive cells, while the thymus and spleen had only a few immunostained cells. With the possible exception of dermal and some adrenal cortical cells, the immunoreactive cells do not appear to be the primary sites of Sm/IGF synthesis, because parallel in situ hybridization histochemical studies using Sm/IGF oligodeoxyribonucleotide probes show that Sm/IGF mRNAs are localized predominantly to fibroblasts and mesenchymal cells. Therefore the immunoreactive cells identified in this study may define sites of action of Sm/IGFs.