Cadherin‐8 expression,synaptic localization,and molecular control of neuronal form in prefrontal corticostriatal circuits

Neocortical interactions with the dorsal striatum support many motor and executive functions, and such underlying functional networks are particularly vulnerable to a variety of developmental, neurological, and psychiatric brain disorders, including autism spectrum disorders, Parkinson's disease, and Huntington's disease. Relatively little is known about the development of functional corticostriatal interactions, and in particular, virtually nothing is known of the molecular mechanisms that control generation of prefrontal cortex–striatal circuits. Here, we used regional and cellular in situ hybridization techniques coupled with neuronal tract tracing to show that Cadherin‐8 (Cdh8), a homophilic adhesion protein encoded by a gene associated with autism spectrum disorders and learning disability susceptibility, is enriched within striatal projection neurons in the medial prefrontal cortex and in striatal medium spiny neurons forming the direct or indirect pathways. Developmental analysis of quantitative real‐time polymerase chain reaction and western blot data show that Cdh8 expression peaks in the prefrontal cortex and striatum at P10, when cortical projections start to form synapses in the striatum. High‐resolution immunoelectron microscopy shows that Cdh8 is concentrated at excitatory synapses in the dorsal striatum, and Cdh8 knockdown in cortical neurons impairs dendritic arborization and dendrite self‐avoidance. Taken together, our findings indicate that Cdh8 delineates developing corticostriatal circuits where it is a strong candidate for regulating the generation of normal cortical projections, neuronal morphology, and corticostriatal synapses. J. Comp. Neurol. 523:75–92, 2015. © 2014 Wiley Periodicals, Inc.     

Impaired dopaminergic neuron development and locomotor function in zebrafish with loss of pink1 function

Mutations in the human PTEN‐induced kinase 1 (PINK1) gene are linked to recessive familial Parkinson’s disease. Animal models of altered PINK1 function vary greatly in their phenotypic characteristics. Drosophila pink1 mutants exhibit mild dopaminergic neuron degeneration and locomotion defects. Such defects are not observed in mice with targeted null mutations in pink1, although these mice exhibit impaired dopamine release and synaptic plasticity. Here, we report that in zebrafish, morpholino‐mediated knockdown of pink1 function did not cause large alterations in the number of dopaminergic neurons in the ventral diencephalon. However, the patterning of these neurons and their projections are perturbed. This is accompanied by locomotor dysfunction, notably impaired response to tactile stimuli and reduced swimming behaviour. All these defects can be rescued by expression of an exogenous pink1 that is not a target of the morpholinos used. These results indicate that normal PINK1 function during development is necessary for the proper positioning of populations of dopaminergic neurons and for the establishment of neuronal circuits in which they are implicated.     

A large Italian family with Gilles de la Tourette syndrome: clinical study and analysis of the SLITRK1 gene.

Our objective was to report the clinical characteristics and to investigate the role of SLITRK1 gene in a large Italian family with Tourette syndrome (TS). The diagnosis of TS and chronic motor tics (CMT) was made according to "The Tourette Syndrome Classification Study Group" (1993). Psychiatric diagnoses were made by administering the Structured Clinical Interview for DSM and the Yale-Brown Obsessive Compulsive Scale. Genetic study included direct sequencing and copy number analysis of the SLITRK1 gene, and haplotype analysis. We found tics or other behavioral manifestations in 15 subjects. Of these, 5 received a diagnosis of definite TS, 5 were classified as having definite CMT, 2 had definite nonspecific tic disorder, and 3 patients had obsessive-compulsive disorder without motor or phonic tics. Tics mainly involved the craniocervical district. Many patients with tics had coexisting psychiatric disorders, especially obsessive-compulsive disorder, performed poorly at school and had social problems. Direct sequencing and copy number analysis of the SLITRK1 gene, and haplotype analysis suggested that the SLITRK1 locus was not involved in this family. In conclusion, the distinctive clinical features in this family are the motor tics mainly involving the face and the neck and the severe coexisting psychiatric disorders. The negative results of the SLITRK1 analysis point to genetic heterogeneity in TS.     

Abnormal water diffusivity in corticostriatal projections in children with Tourette syndrome

The fronto‐striato‐thalamic circuit has been implicated in the pathomechanism of Tourette Syndrome (TS). To study white and gray matter comprehensively, we used a novel technique called Tract‐Based Spatial Statistics (TBSS) combined with voxel‐based analysis (VBA) of diffusion tensor MR images in children with TS as compared to typically developing controls. These automated and unbiased methods allow analysis of cerebral white matter and gray matter regions. We compared 15 right‐handed children with TS (mean age: 11.6 ± 2.5 years, 12 males) to 14 age‐matched right‐handed healthy controls (NC; mean age: 12.29 ± 3.2 years, 6 males). Tic severity and neurobehavioral scores were correlated with FA and ADC values in regions found abnormal by these methods. For white matter, TBSS analysis showed regions of increased ADC in the corticostriatal projection pathways including left external capsule and left and right subcallosal fasciculus pathway in TS group compared to NC group. Within the TS group, ADC for the left external capsule was negatively associated with tic severity (r= −0.586, P = 0.02). For gray matter, VBA revealed increased ADC for bilateral orbitofrontal cortex, left putamen, and left insular cortex. ADC for the right and left orbitofrontal cortex was highly correlated with internalizing problems (r = 0.665; P = 0.009, r = 0.545; P = 0.04, respectively). Altogether, this analysis revealed focal diffusion abnormalities in the corticostriatal pathway and in gray matter structures involved in the fronto‐striatal circuit in TS. These diffusion abnormalities could serve as a neuroimaging marker related to tic severity and neurobehavioral abnormalities in TS subjects. Hum Brain Mapp, 2010. © 2010 Wiley‐Liss, Inc.     

Activation of α2A‐containing nicotinic acetylcholine receptors mediates nicotine‐induced motor output in embryonic zebrafish

It is well established that cholinergic signaling has critical roles during central nervous system development. In physiological and behavioral studies, activation of nicotinic acetylcholine receptors (nAChRs) has been implicated in mediating cholinergic signaling. In developing spinal cord, cholinergic transmission is associated with neural circuits responsible for producing locomotor behaviors. In this study, we investigated the expression pattern of the α2A nAChR subunit as previous evidence suggested it could be expressed by spinal neurons. In situ hybridization and immunohistochemistry revealed that the α2A nAChR subunits are expressed in spinal Rohon–Beard (RB) neurons and olfactory sensory neurons in young embryos. To examine the functional role of the α2A nAChR subunit during embryogenesis, we blocked its expression using antisense modified oligonucleotides. Blocking the expression of α2A nAChR subunits had no effect on spontaneous motor activity. However, it did alter the embryonic nicotine‐induced motor output. This reduction in motor activity was not accompanied by defects in neuronal and muscle elements associated with the motor output. Moreover, the anatomy and functionality of RB neurons was normal even in the absence of the α2A nAChR subunit. Thus, we propose that α2A‐containing nAChRs are dispensable for normal RB development. However, in the context of nicotine‐induced motor output, α2A‐containing nAChRs on RB neurons provide the substrate that nicotine acts upon to induce the motor output. These findings also indicate that functional neuronal nAChRs are present within spinal cord at the time when locomotor output in zebrafish first begins to manifest itself.     

Alpha‐synuclein overexpression in mice alters synaptic communication in the corticostriatal pathway

α‐Synuclein (α‐Syn) is a presynaptic protein implicated in Parkinson's disease (PD). Mice overexpressing human wildtype (WT) α‐Syn under the Thy1 promoter show high levels of α‐Syn in cortical and subcortical regions, exhibit progressive sensorimotor anomalies, as well as non‐motor abnormalities and are considered models of pre‐manifest PD as there is little evidence of early loss of dopaminergic (DA) neurons. We used whole‐cell patch clamp recordings from visually identified striatal medium‐sized spiny neurons (MSSNs) in slices from α‐Syn and WT littermate control mice at 35, 90 and 300 days of age to examine corticostriatal synaptic function. MSSNs displayed significant decreases in the frequency of spontaneous excitatory postsynaptic currents (EPSCs) in α‐Syn mice at all ages. This difference persisted in the presence of tetrodotoxin, indicating it was independent of action potentials. Stimulation thresholds for evoking EPSCs were significantly higher and responses were smaller in α‐Syn mice. These data suggest a decrease in neurotransmitter release at the corticostriatal synapse. At 90 days the frequency of spontaneous GABA_A receptor‐mediated synaptic currents was decreased in MSSNs but increased in cortical pyramidal neurons. These observations indicate that high levels of expression of α‐Syn alter corticostriatal synaptic function early and they provide evidence for early synaptic dysfunction in a pre‐manifest model of PD. Of importance, these changes are opposite to those found in DA‐depletion models, suggesting that before degeneration of DA neurons in the substantia nigra synaptic adaptations occur at the corticostriatal synapse that may initiate subtle preclinical manifestations. © 2009 Wiley‐Liss, Inc.     

Differential developmental refinement of the intrinsic electrophysiological properties of CA1 pyramidal neurons from the rat dorsal and ventral hippocampus

The dorsal and ventral regions of the rat longitudinal hippocampal axis are functionally distinct. That is, each region is associated with different behavioral tasks and disease susceptibilities due to underlying anatomical, and physiological differences. These differences are especially pronounced in area CA1, where significant differences in morphology, synaptic physiology, intrinsic excitability, and gene expression have been reported between CA1 pyramidal neurons from the dorsal (DHC) and ventral hippocampus (VHC). However, despite a significant amount of recent attention, a cogent picture of the intrinsic electrophysiological profile of DHC and VHC neurons has remained elusive, due, in part, to experiments performed on rats at different developmental time points. Moreover, the resulting intrinsic electrophysiological profiles are sufficiently different as to warrant a thorough investigation of the spatial and temporal changes in the intrinsic excitability of CA1 pyramidal neurons across developmental time. Accordingly, in this study, I have characterized the intrinsic electrophysiological properties of CA1 pyramidal neurons from acute hippocampal slices prepared from the DHC and VHC throughout an approximately 3‐week developmental period (P14–P37). DHC and VHC neurons exhibited distinct intra‐region changes (DHC or VHC) and inter‐region differences (DHC versus VHC) in their intrinsic electrophysiological properties, which yielded two developmental timelines: (a) a common developmental timeline describing changes observed in both DHC and VHC neurons, and (b) a differential developmental timeline highlighting unique features observed in DHC neurons. Specifically, DHC neurons exhibited significant inter‐region differences in RMP, input resistance, threshold, and spike frequency adaptation relative to VHC neurons, as well as an intra‐region change in the rebound slope (a proxy for I_h). These observations both integrate and reconcile previous work performed with rats at different developmental stages and suggest a distinct developmental trajectory for DHC neurons that might shed light on the normal physiological functions and disease susceptibility of the DHC.     

NMDA Receptor Enhances Correlation of Spontaneous Activity in Neonatal Barrel Cortex

Correlated spontaneous activity plays critical role in the organization of neocortical circuits during development. However, cortical mechanisms regulating activity correlation are still elusive. In this study, using two-photon calcium imaging of the barrel cortex layer 4 (L4) in living neonatal mice, we found that NMDA receptors (NMDARs) in L4 neurons are important for enhancement of spontaneous activity correlation. Disruption of GluN1 (Grin1), an obligatory NMDAR subunit, in a sparse population of L4 neurons reduced activity correlation between GluN1 knock-out (GluN1KO) neuron pairs within a barrel. This reduction in activity correlation was even detected in L4 neuron pairs in neighboring barrels and most evident when either or both of neurons are located on the barrel edge. Our results provide evidence for the involvement of L4 neuron NMDARs in spatial organization of the spontaneous firing activity of L4 neurons in the neonatal barrel cortex.SIGNIFICANCE STATEMENT Precise wiring of the thalamocortical circuits is necessary for proper sensory information processing, and thalamus-derived correlated spontaneous activity is important for thalamocortical circuit formation. The molecular mechanisms involved in the correlated activity transfer from the thalamus to the neocortex are largely unknown. In vivo two-photon calcium imaging of the neonatal barrel cortex revealed that correlated spontaneous activity between layer four neurons is reduced by mosaic knock-out (KO) of the NMDA receptor (NMDAR) obligatory subunit GluN1. Our results suggest that the function of NMDARs in layer four neurons is necessary for the communication between presynaptic and postsynaptic partners during thalamocortical circuit formation.     

Visualizing corticotropin-releasing hormone receptor type 1 expression and neuronal connectivities in the mouse using a novel multifunctional allele

The corticotropin‐releasing hormone (CRH) and its type 1 receptor (CRHR1) play a central role in coordinating the endocrine, autonomic, and behavioral responses to stress. A prerequisite to functionally dissect the complexity of the CRH/CRHR1 system is to unravel the identity of CRHR1‐expressing neurons and their connectivities. Therefore, we used a knockin approach to genetically label CRHR1‐expressing cells with a tau‐lacZ (tZ) reporter gene. The distribution of neurons expressing β‐galactosidase in the brain and the relative intensity of labeling is in full accordance with previously described Crhr1 mRNA expression. Combining the microtubule‐binding properties of TAU with the Cre‐loxP system allowed to direct the β‐galactosidase to proximal dendrites, and in particular to axons. Thereby, we were able to visualize projections of CRHR1 neurons such as glutamatergic and dopaminergic afferent connections of the striatum and GABAergic CRHR1‐expressing neurons located within its patch compartment. In addition, the tZ reporter gene revealed novel details of CRHR1 expression in the spinal cord, skin, and eye. CRHR1 expression in the retina prompted the identification of a new physiological role of CRHR1 related to the visual system. Besides its reporter properties, this novel CRHR1 allele comprises the possibility to conditionally restore or delete CRHR1 via Flp and Cre recombinase, respectively. Finally, the allele is suitable for further manipulations of the CRHR1 locus by recombinase‐mediated cassette exchange. Taken together, this novel mouse allele will significantly facilitate the neuroanatomical analysis of CRHR1 circuits and opens up new avenues to address CRHR1 function in more detail. J. Comp. Neurol., 520:3150–3180, 2012. © 2012 Wiley Periodicals, Inc.     

Examination of the SLITRK1 gene in Caucasian patients with Tourette syndrome

OBJECTIVE: To determine whether variants in the Slit and Trk-like 1 gene (SLITRK1) are present in American Caucasian population of patients with Tourette syndrome (TS). METHODS: We sequenced the 3'-untranslated region for var321 and the whole coding region in the SLITRK1 gene in 82 Caucasian patients with TS from North America. RESULTS: None of the 82 samples from patients with TS showed the non-coding sequence variant (var321). Only one patient with familial TS was heterozygous for a novel 708C > T (Ile236Ile) nucleotide variant. CONCLUSIONS: The var321 and mutation(s) in the coding region of the SLITRK1 gene probably are a rare cause of TS in a Caucasian population; therefore, genetic heterogeneity of TS should be considered. Tests designed to detect variant(s) in the SLITRK1 gene probably will not have a diagnostic utility in clinical practice.     

Motor deficits and beta oscillations are dissociable in an alpha‐synuclein model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder characterised by progressive motor symptoms resulting from chronic loss of dopaminergic neurons in the nigrostriatal pathway. The over expression of the protein alpha‐synuclein in the substantia nigra has been used to induce progressive dopaminergic neuronal loss and to reproduce key histopathological and temporal features of PD in animal models. However, the neurophysiological aspects of the alpha‐synuclein PD model have been poorly characterised. Hereby, we performed chronic in vivo electrophysiological recordings in the corticostriatal circuit of rats injected with viral vector to over express alpha‐synuclein in the right substantia nigra. Our model, previously shown to exhibit mild motor deficits, presented moderate dopaminergic cell loss but did not present prominent local field potential oscillations in the beta frequency range (11–30 Hz), considered a hallmark of PD, during the 9 weeks after onset of alpha‐synuclein over expression. Spinal cord stimulation, a potential PD symptomatic therapy, was applied regularly from sixth to ninth week after alpha‐synuclein over expression onset and had an inhibitory effect on the firing rate of corticostriatal neurons in both control and alpha‐synuclein hemispheres. Dopamine synthesis inhibition at the end of the experiment resulted in severe parkinsonian symptoms such as akinesia and increased beta and high‐frequency (>90 Hz) oscillations. These results suggest that the alpha‐synuclein PD model with moderate level of dopaminergic depletion does not reproduce the prominent corticostriatal beta oscillatory activity associated to parkinsonian conditions.     

Regulation of dendrite growth by the Cdc42 activator Zizimin1/Dock9 in hippocampal neurons

Rho family small GTPases are key regulators of morphological changes in neurons. Cdc42, one of the most characterized members of the Rho family of proteins, is involved in axon and dendrite outgrowth through cytoskeletal reorganization. Recent studies have identified Zizimin1, a member of the Dock180‐related family of proteins [also called CDM (Ced‐5/Dock180/Myoblast city)–zizimin homology (CZH) proteins], as a specific guanine‐nucleotide exchange factor (GEF) for Cdc42. However, the physiological function of Zizimin1 is totally unknown. In this study, we investigated the role of Zizimin1 in dendrite development in rat hippocampal neurons. In situ hybridization and Western blot analysis showed that Zizimin1 is strongly expressed in the developing brain including in the hippocampus and cerebral cortex in late developmental stages. Overexpression of wild‐type Zizimin1 promoted dendrite growth, whereas knockdown of Zizimin1 by short hairpin RNA or expression of a mutant Zizimin1 lacking Cdc42 GEF activity suppressed dendrite growth in primary cultured rat hippocampal neurons. Both the N‐terminal CZH1 domain, which is conserved among CZH proteins, and the Pleckstrin homology domain of Zizimin1 are involved in membrane localization, Cdc42 activation, and regulation of dendrite growth. Thus, these results suggest that Zizimin1 plays an important role in dendrite growth in hippocampal neurons through activation of Cdc42. © 2009 Wiley‐Liss, Inc.     

Evidence of cell‐nonautonomous changes in dendrite and dendritic spine morphology in the met‐signaling–deficient mouse forebrain

Human genetic findings and murine neuroanatomical expression mapping have intersected to implicate Met receptor tyrosine kinase signaling in the development of forebrain circuits controlling social and emotional behaviors that are atypical in autism‐spectrum disorders (ASD). To clarify roles for Met signaling during forebrain circuit development in vivo, we generated mutant mice (Emx1^Cre/Met^fx/fx) with an Emx1‐Cre‐driven deletion of signaling‐competent Met in dorsal pallially derived forebrain neurons. Morphometric analyses of Lucifer yellow‐injected pyramidal neurons in postnatal day 40 anterior cingulate cortex (ACC) revealed no statistically significant changes in total dendritic length but a selective reduction in apical arbor length distal to the soma in Emx1^Cre/Met^fx/fx neurons relative to wild type, consistent with a decrease in the total tissue volume sampled by individual arbors in the cortex. The effects on dendritic structure appear to be circuit‐selective, insofar as basal arbor length was increased in Emx1^Cre/Met^fx/fx layer 2/3 neurons. Spine number was not altered on the Emx1^Cre/Met^fx/fx pyramidal cell populations studied, but spine head volume was significantly increased (∼20%). Cell‐nonautonomous, circuit‐level influences of Met signaling on dendritic development were confirmed by studies of medium spiny neurons (MSN), which do not express Met but receive Met‐expressing corticostriatal afferents during development. Emx1^Cre/Met^fx/fx MSN exhibited robust increases in total arbor length (∼20%). As in the neocortex, average spine head volume was also increased (∼12%). These data demonstrate that a developmental loss of presynaptic Met receptor signaling can affect postsynaptic morphogenesis and suggest a mechanism whereby attenuated Met signaling could disrupt both local and long‐range connectivity within circuits relevant to ASD. J. Comp. Neurol. 518:4463–4478, 2010. © 2010 Wiley‐Liss, Inc.     

Developmental regulation of connexins 26, 32, 36, and 43 in trigeminal neurons

The transition from sucking to chewing during postnatal development is accompanied by changes in masticatory muscle activity patterns. We previously demonstrated that changes in numerous parameters of chemical synapses among neurons, and intrinsic membrane properties of neurons, comprising brainstem oral-motor circuits are coincident with changes in masticatory muscle activity patterns. Considering recent findings that implicate a role for gap junctions in early locomotor and respiratory behaviors, our present study focuses on the developmental regulation of connexin proteins in trigeminal neurons as a first step in understanding a role for gap junctions in developing oral-motor circuits used for ingestive behaviors. We conducted immunohistochemistry studies to examine connexin (Cx) 26, 32, 36, and 43 expression in trigeminal motor and mesencephalic trigeminal nuclei during postnatal development at the light and electron microscopic levels. Postnatal days (P) 1, 6, 14, 21, and adult mice were used. Cx32, 36, and 43 expression was developmentally regulated in the trigeminal motor nucleus, while Cx26 expression remained high throughout postnatal development. In the mesencephalic trigeminal nucleus, Cx26, 32, and 43 expression was intense throughout development, with only Cx36 showing a developmental regulation. Ultrastructural examination of neonatal trigeminal motoneurons and mesencephalic trigeminal neurons revealed connexin expression in cell membranes, cytoplasm, and cell nuclei (Cx43, Cx32). Our results show that connexin proteins are differentially regulated between trigeminal motoneurons and mesencephalic trigeminal neurons during development, and suggest a possible role for gap junctions in the development of trigeminal neurons and the function and maturation of oral-motor circuits.     

The expression of tubb2b undergoes a developmental transition in murine cortical neurons

The development of the mammalian brain requires the generation, migration, and differentiation of neurons, cellular processes that are dependent on a dynamic microtubule cytoskeleton. Mutations in tubulin genes, which encode for the structural subunits of microtubules, cause detrimental neurological disorders known as the tubulinopathies. The disease spectra associated with different tubulin genes are overlapping but distinct, an observation believed to reflect functional specification of this multigene family. Perturbation of the β‐tubulin TUBB2B is known to cause polymicrogyria, pachygyria, microcephaly, and axon guidance defects. Here we provide a detailed analysis of the expression pattern of its murine homolog Tubb2b. The generation and characterization of BAC‐transgenic eGFP reporter mouse lines has revealed that it is highly expressed in progenitors and postmitotic neurons during cortical development. This contrasts with the 8‐week‐old cortex, in which Tubb2b expression is restricted to macroglia, and expression is almost completely absent in mature neurons. This developmental transition in neurons is mirrored in the adult hippocampus and the cerebellum but is not a universal feature of Tubb2b; its expression persists in a population of postmitotic neurons in the 8‐week‐old retina. We propose that the dynamic spatial and temporal expression of Tubb2b reflects specific functional requirements of the microtubule cytoskeleton. J. Comp. Neurol. 523:2161–2186, 2015. © 2015 Wiley Periodicals, Inc.