Brain mapping across 16 autism mouse models reveals a spectrum of functional connectivity subtypes.

Autism Spectrum Disorder (ASD) is characterized by substantial, yet highly heterogeneous abnormalities in functional brain connectivity. However, the origin and significance of this phenomenon remain unclear. To unravel ASD connectopathy and relate it to underlying etiological heterogeneity, we carried out a bi-center cross-etiological investigation of fMRI-based connectivity in the mouse, in which specific ASD-relevant mutations can be isolated and modeled minimizing environmental contributions. By performing brain-wide connectivity mapping across 16 mouse mutants, we show that different ASD-associated etiologies cause a broad spectrum of connectional abnormalities in which diverse, often diverging, connectivity signatures are recognizable. Despite this heterogeneity, the identified connectivity alterations could be classified into four subtypes characterized by discrete signatures of network dysfunction. Our findings show that etiological variability is a key determinant of connectivity heterogeneity in ASD, hence reconciling conflicting findings in clinical populations. The identification of etiologically-relevant connectivity subtypes could improve diagnostic label accuracy in the non-syndromic ASD population and paves the way for personalized treatment approaches.

Increased susceptibility to kainic acid-induced seizures in Engrailed-2 knockout mice.

The En2 gene, coding for the homeobox-containing transcription factor Engrailed-2 (EN2), has been associated to autism spectrum disorder (ASD). Due to neuroanatomical and behavioral abnormalities, which partly resemble those observed in ASD patients, En2 knockout (En2(-/-)) mice have been proposed as a model for ASD. In the mouse embryo, En2 is involved in the specification of midbrain/hindbrain regions, being predominantly expressed in the developing cerebellum and ventral midbrain, and its expression is maintained in these structures until adulthood. Here we show that in the adult mouse brain, En2 mRNA is expressed also in the hippocampus and cerebral cortex. Hippocampal En2 mRNA content decreased after seizures induced by kainic acid (KA). This suggests that En2 might also influence the functioning of forebrain areas during adulthood and in response to seizures. Indeed, a reduced expression of parvalbumin and somatostatin was detected in the hippocampus of En2(-/-) mice as compared to wild-type (WT) mice, indicating an altered GABAergic innervation of limbic circuits in En2(-/-) mice. In keeping with these results, En2(-/-) mice displayed an increased susceptibility to KA-induced seizures. KA (20 mg/kg) determined more severe and prolonged generalized seizures in En2(-/-) mice, when compared to WT animals. Seizures were accompanied by a widespread c-fos and c-jun mRNA induction in the brain of En2(-/-) but not WT mice. Long-term histopathological changes (CA1 cell loss, upregulation of neuropeptide Y) also occurred in the hippocampus of KA-treated En2(-/-) but not WT mice. These findings suggest that En2(-/-) mice might be used as a novel tool to study the link between epilepsy and ASD.

Antiproliferative role of dopamine: loss of D2 receptors causes hormonal dysfunction and pituitary hyperplasia.

The function of dopamine (DA) in the nervous system is paralleled by its neuroendocrine control of pituitary gland functions. Here, we document the neuroendocrine function of dopamine by studying the pituitary gland of mice lacking DA D2 receptors (D2R). These mice present a striking, progressive increase in lactotroph number, which ultimately leads to tumors in aged animals. Females develop tumors much earlier than males. An estrogen-mediated lactotroph proliferation cannot account for this sexual dimorphism, since D2R-null females are hypoestrogenic and, thus, have estrogen levels similar to males. In contrast, prolactin levels are six times higher in females than in males. We show that active prolactin receptors are present in the pituitary and their expression increases in concomitance with tumor expansion. These results point to prolactin as an autocrine proliferative factor in the pituitary gland. Additionally, they demonstrate an antiproliferative function for DA regulated through D2 receptor activation.

Action of botulinum neurotoxins in the central nervous system: antiepileptic effects.

Botulinum neurotoxins (BoNTs) are metalloproteases which act on nerve terminals and cause a long-lasting inhibition of neurotransmitter release. BoNTs act by cleaving core proteins of the neurotransmitter release machinery, namely the SNARE (soluble NSF-attachment receptors) proteins. The action of BoNTs in the peripheral nervous system (PNS) has been extensively documented, and knowledge gained in this field laid the foundations for the use of BoNTs in human disorders characterized by hyperfunction of peripheral nerve terminals. Much less is known about the action of BoNTs on the central nervous system (CNS). In vitro studies have demonstrated that BoNTs can affect the release of several neurotransmitters from central neurons. Recent studies have provided the first characterization of the effects of BoNT/E on CNS neurons in vivo. It has been shown that BoNT/E injected into the rat hippocampus inhibits glutamate release and blocks spike activity of pyramidal neurons. Intrahippocampal injection of BoNT/E resulted in significant inhibition of seizure activity in experimental models of epilepsy, suggesting a potential therapeutic use of BoNTs in the CNS.

Brain-derived neurotrophic factor signaling is altered in the forebrain of Engrailed-2 knockout mice.

Engrailed-2 (En2), a homeodomain transcription factor involved in regionalization and patterning of the midbrain and hindbrain regions has been associated to autism spectrum disorders (ASDs). En2 knockout (En2(-/-)) mice show ASD-like features accompanied by a significant loss of GABAergic subpopulations in the hippocampus and neocortex. Brain-derived neurotrophic factor (BDNF) is a crucial factor for the postnatal development of forebrain GABAergic neurons, and altered GABA signaling has been hypothesized to underlie the symptoms of ASD. Here we sought to determine whether interneuron loss in the En2(-/-) forebrain might be related to altered expression of BDNF and its signaling receptors. We first evaluated the expression of different BDNF mRNA isoforms in the neocortex and hippocampus of wild-type (WT) and En2(-/-) mice. Quantitative RT-PCR showed a marked down-regulation of several splicing variants of BDNF mRNA in the neocortex but not hippocampus of adult En2(-/-) mice, as compared to WT controls. Accordingly, levels of mature BDNF protein were lower in the neocortex but not hippocampus of En2(-/-) mice, as compared to WT. Increased levels of phosphorylated TrkB and decreased levels of p75 receptor were also detected in the neocortex of mutant mice. Accordingly, the expression of low density lipoprotein receptor (LDLR) and RhoA, two genes regulated via p75 was significantly altered in forebrain areas of mutant mice. These data indicate that BDNF signaling alterations might be involved in the anatomical changes observed in the En2(-/-) forebrain and suggest a pathogenic role of altered BDNF signaling in this mouse model of ASD.

Neuroprotective role of dopamine against hippocampal cell death.

Glutamate excitotoxicity plays a key role in the induction of neuronal cell death occurring in many neuropathologies, including epilepsy. Systemic administration of the glutamatergic agonist kainic acid (KA) is a well characterized model to study epilepsy-induced brain damage. KA-evoked seizures in mice result in hippocampal cell death, with the exception of some strains that are resistant to KA excitotoxicity. Little is known about the factors that prevent epilepsy-related neurodegeneration. Here we show that dopamine has such a function through the activation of the D2 receptor (D2R). D2R gene inactivation confers susceptibility to KA excitotoxicity in two mouse strains known to be resistant to KA-induced neurodegeneration. D2R-/- mice develop seizures when administered KA doses that are not epileptogenic for wild-type (WT) littermates. The spatiotemporal pattern of c-fos and c-jun mRNA induction well correlates with the occurrence of seizures in D2R-/- mice. Moreover, KA-induced seizures result in extensive hippocampal cell death in D2R-/- but not WT mice. In KA-treated D2R-/- mice, hippocampal neurons die by apoptosis, as indicated by the presence of fragmented DNA and the induction of the proapoptotic protein BAX. These results reveal a central role of D2Rs in the inhibitory control of glutamate neurotransmission and excitotoxicity.

Reduced phosphorylation of synapsin I in the hippocampus of Engrailed-2 knockout mice, a model for autism spectrum disorders.

Mice lacking the homeodomain transcription factor Engrailed-2 (En2(-/-) mice) are a well-characterized model for autism spectrum disorders (ASD). En2(-/-) mice present molecular, neuropathological and behavioral deficits related to ASD, including down-regulation of ASD-associated genes, cerebellar hypoplasia, interneuron loss, enhanced seizure susceptibility, decreased sociability and impaired cognition. Specifically, impaired spatial learning in the Morris water maze (MWM) is associated with reduced expression of neurofibromin and increased phosphorylation of extracellular-regulated kinase (ERK) in the hippocampus of En2(-/-) adult mice. In the attempt to better understand the molecular cascades underlying neurofibromin-dependent cognitive deficits in En2 mutant mice, we investigated the expression and phosphorylation of synapsin I (SynI; a major target of neurofibromin-dependent signaling) in the hippocampus of wild-type (WT) and En2(-/-) mice before and after MWM. Here we show that SynI mRNA and protein levels are down-regulated in the hippocampus of naïve and MWM-treated En2(-/-) mice, as compared to WT controls. This down-regulation is paralleled by reduced levels of SynI phosphorylation at Ser549 and Ser553 residues in the hilus of mutant mice, before and after MWM. These data indicate that in En2(-/-) hippocampus, neurofibromin-dependent pathways converging on SynI phosphorylation might underlie hippocampal-dependent learning deficits observed in En2(-/-) mice.

Monocular deprivation decreases brain-derived neurotrophic factor immunoreactivity in the rat visual cortex.

Neurotrophins play a crucial role in the development and activity-dependent plasticity of the visual cortex [Berardi N. et al. (1994) Proc. natn. Acad. Sci. U.S.A. 91, 684-688; Bonhoeffer T. (1996) Curr. Opin. Neurobiol. 6, 119-126; Cellerino A. and Maffei L. (1996) Prog. Neurobiol. 49, 53-71; Domenici L. et al. (1994) NeuroReport 5, 2041-2044; Galuske R. A. W. et al (1996) Eur. J. Neurosci. 8, 1554-1559; Katz L. C. and Shatz C. J. (1996) Science 274, 1133-1138; Maffei L. et al. (1992) J. Neurosci. 12, 4651-4662; Pizzorusso T. and Maffei L. (1996) Curr. Opin. Neurol. 9, 122-125; Thoenen H. (1995) Science 270, 593-598]. As a possible mechanism of action, it has been postulated that the activity-dependent expression of neurotrophins by cortical cells could regulate synapse stabilization during the first period of postnatal life (critical period). Indeed, brain-derived neurotrophic factor messenger RNA expression in the visual cortex is regulated by neuronal activity as well as during development [Castrén E. et al. (1992) Proc. natn. Acad. Sci. U.S.A. 89, 9444-9448]. Moreover, we showed that monocular deprivation decreases brain-derived neurotrophic factor messenger RNA levels in the visual cortex receiving input from the deprived eye [Bozzi Y. et al. (1995) Neuroscience 69, 1133-1144]. What is missing, however, is the demonstration that brain-derived neurotrophic factor protein expression follows that of brain-derived neurotrophic factor messenger RNA. The aim of the present study is to fill this important gap in order to support the hypothesis that brain-derived neurotrophic factor is fundamental in the plasticity of the visual cortex. We found that brain-derived neurotrophic factor immunoreactivity peaks during the critical period and that it is preferentially localized in layers II-III and V-VI. We also demonstrated that monocular deprivation determines a decrease of brain-derived neurotrophic factor immunoreactivity exclusively in the visual cortex contralateral to the deprived eye. Our results support the proposed role for brain-derived neurotrophic factor in the development and activity-dependent plasticity of the visual cortex [Cabelli R. J. et al. (1995) Science 267, 1662-1666].

Monocular deprivation decreases the expression of messenger RNA for brain-derived neurotrophic factor in the rat visual cortex.

We found that deprivation of pattern vision in one eye, that leaves luminance detection performance unaffected, is sufficient to reduce brain-derived neurotrophic factor (but not trkB) messenger RNA in the visual cortex of young and adult rats. Monocular deprivation by means of eyelids' suture was performed during or after the critical period and the cortical amount of brain-derived neurotrophic factor messenger RNA was analysed by in situ hybridization and RNAase protection after 15-30 days of deprivation. A reduction of brain-derived neurotrophic factor messenger RNA was observed in the visual cortex contralateral to the deprived eye in rats monocularly deprived during the critical period. The same reduction was also found in rats monocularly deprived after the end of the critical period, when anatomical or physiological signs of monocular deprivation are absent. The pharmacological blockade of retinal activity equally affected the expression of brain-derived neurotrophic factor messenger RNA in young and adults. Quantitative RNAase protection assays revealed that the cortical level of brain-derived neurotrophic factor messenger RNA was reduced to the same extent when intraocular injections of tetrodotoxin were performed within or after the critical period. A developmental study of brain-derived neurotrophic factor messenger RNA expression in rat visual cortex showed a marked increase around the time of natural eye-opening followed by a plateau from postnatal day 20 until adult age. Messenger RNA for the kinasic domain of brain-derived neurotrophic factor receptor (trkB) was found in the dorsal lateral geniculate nucleus and the visual cortex during development and in adults. Our results suggest that the reduction of brain-derived neurotrophic factor messenger RNA induced by monocular deprivation is related to the absence of pattern vision rather than to the competitive interactions that underlie the effects of monocular deprivation during the critical period.

Dopamine D2 receptors in signal transduction and behavior.

The dopamine D2 receptor belongs to the family of seven transmembrane domain G-protein-coupled receptors and is highly expressed in the central nervous system and the pituitary gland. The binding of dopamine to the D2 receptor is crucial for the regulation of diverse physiological functions, such as the control of locomotor activity and the synthesis of peptide hormones. Two alternatively spliced transcripts are generated from the D2 receptor gene and code for the D2L and D2S isoforms, which are 444 and 415 amino acids in length, respectively. These isoforms exhibit similar pharmacological characteristics and are expressed in the same cell types, with a ratio that normally favors expression of the longer isoform. The D2L isoform differs from D2S by the insertion of 29 amino acids in the putative third intracellular loop of the receptor. This loop is involved in the coupling of the receptor to different G proteins. Experiments have shown that the D2 isoforms have different G-protein-coupling affinities, suggesting that these receptors might serve different functions in vivo. Additionally, this difference in coupling affinity could be a mechanism to amplify the signal transduced by the binding of dopamine to D2 receptors. Important insights into D2 receptor function in vivo have been obtained by knocking out the D2 gene in mice. The Parkinsonian-like phenotype of D2-null mice demonstrates the importance of the D2 receptor for locomotor function.

Activity, non-selective attention and emotionality in dopamine D2/D3 receptor knock-out mice.

In order to assess the role of dopamine (DA) D2 and D3 receptors in the modulation of behaviour, we analysed exploration in a spatial novelty in mouse model systems. Genetically engineered mice mutants have been used that carry normal, partial or no expression of D2R, D3R, or both D2R/D3R (double mutants) DA receptor subtypes. Adult male mice were exposed for 30 min to a Làte-maze. The behaviour was analysed for indices of activity, orienting (rearing frequency), scanning times (rearing duration) and defecation score (emotionality). D2R - / - and + / - as well as the D2R/D3R double homozygous mutants were less active than wild-type (WT) controls in travelled distance. In contrast D3R + / - were more active than WT mice in the first part of the test. As to orienting frequency, the D2R - / - were less active than WT during the entire test-period, whereas the D2 + / - mutants were less active than WT only in the second part of the test. Moreover, the D3R - / - and + / - mutants showed less and more rearing frequency than WT, respectively, during the entire test. Finally, the D2/D3R - / - double mutants were also less active than WT during the entire test period. As to scanning times, D2R + / - and - / - mutants were higher than WT during the entire test or only in the second part, respectively. The D3R + / - and - / - were not different from WT, whereas the D2/D3R - / - double mutants showed shorter scanning times only in the first part of the test. As to emotionality index, the defecation score, was lower only in D3R + / - mutants. Thus, the dopamine D2 and D3 receptor subtypes appear to be differentially involved in the modulation of activity, orienting and scanning phases of attention. Lastly double mutation experiments reveal an interaction between D2R and D3R with the former prevailing on the latter.

Increased dopaminergic innervation in the brain of conditional mutant mice overexpressing Otx2: effects on locomotor behavior and seizure susceptibility.

The homeobox-containing transcription factor Otx2 controls the identity, fate and proliferation of mesencephalic dopaminergic (mesDA) neurons. Transgenic mice, in which Otx2 was conditionally overexpressed by a Cre recombinase expressed under the transcriptional control of the Engrailed1 gene (En1(Cre/+); tOtx2(ov/+)), show an increased number of mesDA neurons during development. In adult mice, Otx2 is expressed in a subset of neurons in the ventral tegmental area (VTA) and its overexpression renders mesDA more resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-HCl (MPTP) neurotoxin. Here we further investigated the neurological consequences of the increased number of mesDA neurons in En1(Cre/+); tOtx2(ov/+) adult mice. Immunohistochemistry for the active, glycosylated form of the dopamine transporter (glyco-Dat) showed that En1(Cre/+); tOtx2(ov/+) adult mice display an increased density of mesocortical DAergic fibers, as compared to control animals. Increased glyco-Dat staining was accompanied by a marked hypolocomotion in En1(Cre/+); tOtx2(ov/+) mice, as detected in the open field test. Since conditional knockout mice lacking Otx2 in mesDA precursors (En1(Cre/+); Otx2(floxv/flox) mice) show a marked resistance to kainic acid (KA)-induced seizures, we investigated the behavioral response to KA in En1(Cre/+); tOtx2(ov/+) and control mice. No difference was observed between mutant and control mice, but En1(Cre/+); tOtx2(ov/+) mice showed a markedly different c-fos mRNA induction profile in the cerebral cortex and hippocampus after KA seizures, as compared to controls. Accordingly, an increased density of parvalbumin (PV)-positive inhibitory interneurons was detected in the deep layers of the frontal cortex of naïve En1(Cre/+); tOtx2(ov/+) mice, as compared to controls. These data indicate that Otx2 overexpression results in increased DAergic innervation and PV cell density in the fronto-parietal cortex, with important consequences on spontaneous locomotor activity and seizure-induced gene expression. Our results strengthen the notion that Otx2 mutant mouse models are a powerful genetic tool to unravel the molecular and behavioral consequences of altered development of the DAergic system.

Loss of survivin in neural precursor cells results in impaired long-term potentiation in the dentate gyrus and CA1-region.

In adult mammals, newborn neural precursor cells (NPCs) derived from either the subventricular zone (SVZ) or the subgranular zone (SGZ) migrate into the olfactory bulb and the dentate gyrus (DG), respectively, where some of them mature into excitatory and inhibitory neurons. There is increasing evidence that this neurogenesis process is important for some types of learning and synaptic plasticity and vice versa. Survivin, a member of the inhibitor-of-apoptosis protein (IAP) family, has been suggested to have a central role in the regulation of neurogenesis. The protein is abundantly expressed in nervous tissue during embryonic development while being restricted postnatally to proliferating and migrating NPCs in SVZ and SGZ. Here we examined adult Survivin(Camcre) mice with a conditional deletion of the survivin gene in embryonic neurogenic regions. Although the deletion of survivin had no effect on basic excitability in DG and CA1-region, there was a marked impairment of long-term potentiation (LTP) in these areas. Our data support a function of survivin in hippocampal synaptic plasticity and learning and underline the importance of adult brain neurogenesis for proper operation of the hippocampal tri-synaptic circuit and the physiological functions that depend on it.

Absence of the dopamine D2 receptor leads to a decreased expression of GDNF and NT-4 mRNAs in restricted brain areas.

Neurotrophic factors (NTFs) control the metabolic and electrophysiological properties of dopaminergic neurons in the brain. At the level of the substantia nigra, NTFs have been proposed to control dopamine release by regulating the firing rate of dopaminergic cells. This function is normally controlled by presynaptic dopaminergic autoreceptors. Dopamine has also been proposed to regulate the expression of NTFs and their receptors in the nigrostriatal pathway. Thus, an interaction between the signalling cascades activated by NTFs and dopamine receptors might possibly influence the physiology of dopaminergic neurons. Among dopamine receptors, D2 receptors (D2R) are the most abundant on dopaminergic neurons, where they exert autoreceptor functions. To test for an interaction between the NTF and dopaminergic pathways we have analysed the expression of NTFs and their receptors in D2R-deficient (D2R -/-) mice. Our study shows that the mRNA levels of brain-derived neurotrophic factor (BDNF), neurotrophin-3 and their corresponding receptors are not modified in the dopaminergic system of D2R -/- adult mice compared with wild-type littermates. However, a marked reduction of glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-4 (NT-4) mRNAs is observed in the striatum and parietal cortex of D2R -/- mice, respectively. These results implicate dopamine, acting through D2 receptors, in the local control of specific NTF expression. The down-regulation of GDNF and NT-4 expression might also contribute to the locomotor phenotype of D2R -/- mice.

Regulation of constitutive exocytic transport by membrane receptors. A biochemical and morphometric study.

Biochemical and morphometric approaches were combined to examine whether constitutive secretory transport might be controlled by plasma membrane receptors, as this possibility would have significant physiological implications. Indeed, IgE receptor stimulation in rat basophilic leukemia cells potently increased the rate of transport of soluble pulse-labeled 35S-sulfated glycosaminoglycans from distal Golgi compartments to the cell surface. This effect was largely protein kinase C (PKC)-dependent. Direct activation of PKC also stimulated constitutive transport of glycosaminoglycans, as indicated by the use of agonistic and antagonistic PKC ligands. PKC ligands also had potent, but different, effects on the exocytic transport from distal Golgi compartments to the plasma membrane of a membrane-bound protein (vesicular stomatitis virus glycoprotein), which was slightly stimulated by activators and profoundly suppressed by inhibitors of PKC. Morphological analysis showed impressive changes of the organelles of the secretory pathway in response to IgE receptor stimulation and to direct PKC activation (enhanced number of buds and vesicles originating from the endoplasmic reticulum and Golgi and increase in surface and volume of Golgi compartments), suggestive of an overall activation of exocytic movements. These results show that rapid and large changes in constitutive transport fluxes and in the morphology of the exocytic apparatus can be induced by membrane receptors (as well as by direct PKC stimulation).