Jianmin Su

Collagen XIX is expressed by interneurons and contributes to the formation of hippocampal synapses

Extracellular matrix (ECM) molecules contribute to the formation and maintenance of synapses in the mammalian nervous system. We previously discovered a family of nonfibrillar collagens that organize synaptic differentiation at the neuromuscular junction (NMJ). Although many NMJ‐organizing cues contribute to central nervous system (CNS) synaptogenesis, whether similar roles for collagens exist at central synapses remained unclear. In the present study we discovered that col19a1, the gene encoding nonfibrillar collagen XIX, is expressed by subsets of hippocampal neurons. Colocalization with the interneuron‐specific enzyme glutamate decarboxylase 67 (Gad67), but not other cell‐type‐specific markers, suggests that hippocampal expression of col19a1 is restricted to interneurons. However, not all hippocampal interneurons express col19a1 mRNA; subsets of neuropeptide Y (NPY)‐, somatostatin (Som)‐, and calbindin (Calb)‐immunoreactive interneurons express col19a1, but those containing parvalbumin (Parv) or calretinin (Calr) do not. To assess whether collagen XIX is required for the normal formation of hippocampal synapses, we examined synaptic morphology and composition in targeted mouse mutants lacking collagen XIX. We show here that subsets of synaptotagmin 2 (Syt2)‐containing hippocampal nerve terminals appear malformed in the absence of collagen XIX. The presence of Syt2 in inhibitory hippocampal synapses, the altered distribution of Gad67 in collagen XIX‐deficient subiculum, and abnormal levels of gephyrin in collagen XIX‐deficient hippocampal extracts all suggest inhibitory synapses are affected by the loss of collagen XIX. Together, these data not only reveal that collagen XIX is expressed by central neurons, but show for the first time that a nonfibrillar collagen is necessary for the formation of hippocampal synapses. J. Comp. Neurol. 518:229–253, 2010.

Reelin is required for class-specific retinogeniculate targeting.

Development of visual system circuitry requires the formation of precise synaptic connections between neurons in the retina and brain. For example, axons from retinal ganglion cells (RGCs) form synapses onto neurons within subnuclei of the lateral geniculate nucleus (LGN) [i.e., the dorsal LGN (dLGN), ventral LGN (vLGN), and intergeniculate leaflet (IGL)]. Distinct classes of RGCs project to these subnuclei: the dLGN is innervated by image-forming RGCs, whereas the vLGN and IGL are innervated by non-image-forming RGCs. To explore potential mechanisms regulating class-specific LGN targeting, we sought to identify differentially expressed targeting molecules in these LGN subnuclei. One candidate targeting molecule enriched in the vLGN and IGL during retinogeniculate circuit formation was the extracellular matrix molecule reelin. Anterograde labeling of RGC axons in mutant mice lacking functional reelin (relnrl/rl) revealed reduced patterns of vLGN and IGL innervation and misrouted RGC axons in adjacent non-retino-recipient thalamic nuclei. Using genetic reporter mice, we further demonstrated that mistargeted axons were from non-image-forming, intrinsically photosensitive RGCs (ipRGCs). In contrast to mistargeted ipRGC axons, axons arising from image-forming RGCs and layer VI cortical neurons correctly targeted the dLGN in relnrl/rl mutants. Together, these data reveal that reelin is essential for the targeting of LGN subnuclei by functionally distinct classes of RGCs.

Target-derived matricryptins organize cerebellar synapse formation through α3β1 integrins.

Trans-synaptic organizing cues must be passed between synaptic partners for synapses to properly form. Much of our understanding of this process stems from studies at the neuromuscular junction, where target-derived growth factors, extracellular matrix (ECM) molecules, and matricryptins (proteolytically released fragments of ECM molecules) are all essential for the formation and maintenance of motor nerve terminals. While growth factors and ECM molecules also contribute to the formation of brain synapses, it remains unclear whether synaptic roles exist for matricryptins in the mammalian brain. We report that collagen XVIII and its matricryptin endostatin are generated by cerebellar Purkinje cells and are necessary for the organization of climbing fiber terminals in these neurons. Moreover, endostatin is sufficient to induce climbing fiber terminal formation in vitro by binding and signaling through α3β1 integrins. Taken together, these studies reveal roles for both matricryptins and integrins in the organization of brain synapses.

Multiple Retinal Axons Converge onto Relay Cells in the Adult Mouse Thalamus.

SUMMARY Activity-dependent refinement of neural circuits is a fundamental principle of neural development. This process has been well studied at retinogeniculate synapses—synapses that form between retinal ganglion cells (RGCs) and relay cells within the dorsal lateral geniculate nucleus. Physiological studies suggest that shortly after birth, inputs from ~20 RGCs converge onto relay cells. Subsequently, all but just one to two of these inputs are eliminated. Despite widespread acceptance, this notion is at odds with ultrastructural studies showing numerous retinal terminals clustering onto relay cell dendrites in the adult. Here, we explored this discrepancy using brainbow AAVs and serial block face scanning electron microscopy (SBFSEM). Results with both approaches demonstrate that terminals from numerous RGCs cluster onto relay cell dendrites, challenging the notion that only one to two RGCs innervate each relay cell. These findings force us to re-evaluate our understanding of subcortical visual circuitry.

Toxoplasma gondii Infections Alter GABAergic Synapses and Signaling in the Central Nervous System

ABSTRACT During infections with the protozoan parasite Toxoplasma gondii, gamma-aminobutyric acid (GABA) is utilized as a carbon source for parasite metabolism and also to facilitate parasite dissemination by stimulating dendritic-cell motility. The best-recognized function for GABA, however, is its role in the nervous system as an inhibitory neurotransmitter that regulates the flow and timing of excitatory neurotransmission. When this pathway is altered, seizures develop. Human toxoplasmosis patients suffer from seizures, suggesting that Toxoplasma interferes with GABA signaling in the brain. Here, we show that while excitatory glutamatergic presynaptic proteins appeared normal, infection with type II ME49 Toxoplasma tissue cysts led to global changes in the distribution of glutamic acid decarboxylase 67 (GAD67), a key enzyme that catalyzes GABA synthesis in the brain. Alterations in GAD67 staining were not due to decreased expression but rather to a change from GAD67 clustering at presynaptic termini to a more diffuse localization throughout the neuropil. Consistent with a loss of GAD67 from the synaptic terminals, Toxoplasma-infected mice develop spontaneous seizures and are more susceptible to drugs that induce seizures by antagonizing GABA receptors. Interestingly, GABAergic protein mislocalization and the response to seizure-inducing drugs were observed in mice infected with type II ME49 but not type III CEP strain parasites, indicating a role for a polymorphic parasite factor(s) in regulating GABAergic synapses. Taken together, these data support a model in which seizures and other neurological complications seen in Toxoplasma-infected individuals are due, at least in part, to changes in GABAergic signaling. IMPORTANCE Infections of the central nervous system can cause seizures. While inflammation in the brain has been proposed to initiate the onset of the seizures, relatively little is known about how inflammation impacts the structure and function of the neurons. Here we used a parasite called Toxoplasma gondii that infects the brain and showed that seizures arise due to a defect in signaling of GABA, which is the neurotransmitter primarily responsible for preventing the onset of seizures. Infections of the central nervous system can cause seizures. While inflammation in the brain has been proposed to initiate the onset of the seizures, relatively little is known about how inflammation impacts the structure and function of the neurons. Here we used a parasite called Toxoplasma gondii that infects the brain and showed that seizures arise due to a defect in signaling of GABA, which is the neurotransmitter primarily responsible for preventing the onset of seizures.

A Molecular Mechanism Regulating the Timing of Corticogeniculate Innervation

Neural circuit formation demands precise timing of innervation by different classes of axons. However, the mechanisms underlying such activity remain largely unknown. In the dorsal lateral geniculate nucleus (dLGN), axons from the retina and visual cortex innervate thalamic relay neurons in a highly coordinated manner, with those from the cortex arriving well after those from retina. The differential timing of retino- and corticogeniculate innervation is not a coincidence but is orchestrated by retinal inputs. Here, we identified a chondroitin sulfate proteoglycan (CSPG) that regulates the timing of corticogeniculate innervation. Aggrecan, a repulsive CSPG, is enriched in neonatal dLGN and inhibits cortical axons from prematurely entering the dLGN. Postnatal loss of aggrecan from dLGN coincides with upregulation of aggrecanase expression in the dLGN and corticogeniculate innervation and, it is important to note, is regulated by retinal inputs. Taken together, these studies reveal a molecular mechanism through which one class of axons coordinates the temporal targeting of another class of axons.

Fibroblast Growth Factor 22 Contributes to the Development of Retinal Nerve Terminals in the Dorsal Lateral Geniculate Nucleus

At least three forms of signaling between pre- and postsynaptic partners are necessary during synapse formation. First, “targeting” signals instruct presynaptic axons to recognize and adhere to the correct portion of a postsynaptic target cell. Second, trans-synaptic “organizing” signals induce differentiation in their synaptic partner so that each side of the synapse is specialized for synaptic transmission. Finally, in many regions of the nervous system an excess of synapses are initially formed, therefore “refinement” signals must either stabilize or destabilize the synapse to reinforce or eliminate connections, respectively. Because of both their importance in processing visual information and their accessibility, retinogeniculate synapses have served as a model for studying synaptic development. Molecular signals that drive retinogeniculate “targeting” and “refinement” have been identified, however, little is known about what “organizing” cues are necessary for the differentiation of retinal axons into presynaptic terminals. To identify such “organizing” cues, we used microarray analysis to assess whether any target-derived “synaptic organizers” were enriched in the mouse dorsal lateral geniculate nucleus (dLGN) during retinogeniculate synapse formation. One candidate “organizing” molecule enriched in perinatal dLGN was FGF22, a secreted cue that induces the formation of excitatory nerve terminals in muscle, hippocampus, and cerebellum. In FGF22 knockout mice, the development of retinal terminals in dLGN was impaired. Thus, FGF22 is an important “organizing” cue for the timely development of retinogeniculate synapses.

Cell-specific and developmental expression of lectican-cleaving proteases in mouse hippocampus and neocortex

Mounting evidence has demonstrated that a specialized extracellular matrix exists in the mammalian brain and that this glycoprotein‐rich matrix contributes to many aspects of brain development and function. The most prominent supramolecular assemblies of these extracellular matrix glycoproteins are perineuronal nets, specialized lattice‐like structures that surround the cell bodies and proximal neurites of select classes of interneurons. Perineuronal nets are composed of lecticans, a family of chondroitin sulfate proteoglycans that includes aggrecan, brevican, neurocan, and versican. These lattice‐like structures emerge late in postnatal brain development, coinciding with the ending of critical periods of brain development. Despite our knowledge of the presence of lecticans in perineuronal nets and their importance in regulating synaptic plasticity, we know little about the development or distribution of the extracellular proteases that are responsible for their cleavage and turnover. A subset of a large family of extracellular proteases (called a disintegrin and metalloproteinase with thrombospondin motifs [ADAMTS]) is responsible for endogenously cleaving lecticans. We therefore explored the expression pattern of two aggrecan‐degrading ADAMTS family members, ADAMTS15 and ADAMTS4, in the hippocampus and neocortex. Here, we show that both lectican‐degrading metalloproteases are present in these brain regions and that each exhibits a distinct temporal and spatial expression pattern. Adamts15 mRNA is expressed exclusively by parvalbumin‐expressing interneurons during synaptogenesis, whereas Adamts4 mRNA is exclusively generated by telencephalic oligodendrocytes during myelination. Thus, ADAMTS15 and ADAMTS4 not only exhibit unique cellular expression patterns but their developmental upregulation by these cell types coincides with critical aspects of neural development. J. Comp. Neurol. 523:629–648, 2015.

Contributions of VLDLR and LRP8 in the establishment of retinogeniculate projections.

BackgroundRetinal ganglion cells (RGCs), the output neurons of the retina, project to over 20 distinct brain nuclei, including the lateral geniculate nucleus (LGN), a thalamic region comprised of three functionally distinct subnuclei: the ventral LGN (vLGN), the dorsal LGN (dLGN) and the intergeniculate leaflet (IGL). We previously identified reelin, an extracellular glycoprotein, as a critical factor that directs class-specific targeting of these subnuclei. Reelin is known to bind to two receptors: very-low-density lipoprotein receptor (VLDLR) and low-density lipoprotein receptor-related protein 8 (LRP8), also known as apolipoprotein E receptor 2 (ApoER2). Here we examined the roles of these canonical reelin receptors in retinogeniculate targeting.ResultsTo assess the roles of VLDLR and LRP8 in retinogeniculate targeting, we used intraocular injections of fluorescently conjugated cholera toxin B subunit (CTB) to label all RGC axons in vivo. Retinogeniculate projections in mutant mice lacking either VLDLR or LRP8 appeared similar to controls; however, deletion of both receptors resulted in dramatic defects in the pattern of retinal innervation in LGN. Surprisingly, defects in vldlr−/−;lrp8−/− double mutant mice were remarkably different than those observed in mice lacking reelin. First, we failed to observe retinal axons exiting the medial border of the vLGN and IGL to invade distant regions of non-retino-recipient thalamus. Second, an ectopic region of binocular innervation emerged in the dorsomedial pole of vldlr−/−;lrp8−/− mutant dLGN. Analysis of retinal projection development, retinal terminal sizes and LGN cytoarchitecture in vldlr−/−;lrp8−/− mutants, all suggest that a subset of retinal axons destined for the IGL are misrouted to the dorsomedial pole of dLGN in the absence of VLDLR and LRP8. Such mistargeting is likely the result of abnormal migration of IGL neurons into the dorsomedial pole of dLGN in vldlr−/−;lrp8−/− mutants.ConclusionsIn contrast to our expectations, the development of both the LGN and retinogeniculate projections appeared dramatically different in mutants lacking either reelin or both canonical reelin receptors. These results suggest that there are reelin-independent functions of VLDLR and LRP8 in LGN development, and VLDLR- and LRP8-independent functions of reelin in class-specific axonal targeting.

Nuclei-specific differences in nerve terminal distribution, morphology, and development in mouse visual thalamus

BackgroundMouse visual thalamus has emerged as a powerful model for understanding the mechanisms underlying neural circuit formation and function. Three distinct nuclei within mouse thalamus receive retinal input, the dorsal lateral geniculate nucleus (dLGN), the ventral lateral geniculate nucleus (vLGN), and the intergeniculate nucleus (IGL). However, in each of these nuclei, retinal inputs are vastly outnumbered by nonretinal inputs that arise from cortical and subcortical sources. Although retinal and nonretinal terminals associated within dLGN circuitry have been well characterized, we know little about nerve terminal organization, distribution and development in other nuclei of mouse visual thalamus.ResultsImmunolabeling specific subsets of synapses with antibodies against vesicle-associated neurotransmitter transporters or neurotransmitter synthesizing enzymes revealed significant differences in the composition, distribution and morphology of nonretinal terminals in dLGN, vLGN and IGL. For example, inhibitory terminals are more densely packed in vLGN, and cortical terminals are more densely distributed in dLGN. Overall, synaptic terminal density appears least dense in IGL. Similar nuclei-specific differences were observed for retinal terminals using immunolabeling, genetic labeling, axonal tracing and serial block face scanning electron microscopy: retinal terminals are smaller, less morphologically complex, and more densely distributed in vLGN than in dLGN. Since glutamatergic terminal size often correlates with synaptic function, we used in vitro whole cell recordings and optic tract stimulation in acutely prepared thalamic slices to reveal that excitatory postsynaptic currents (EPSCs) are considerably smaller in vLGN and show distinct responses following paired stimuli. Finally, anterograde labeling of retinal terminals throughout early postnatal development revealed that anatomical differences in retinal nerve terminal structure are not observable as synapses initially formed, but rather developed as retinogeniculate circuits mature.ConclusionsTaken together, these results reveal nuclei-specific differences in nerve terminal composition, distribution, and morphology in mouse visual thalamus. These results raise intriguing questions about the different functions of these nuclei in processing light-derived information, as well as differences in the mechanisms that underlie their unique, nuclei-specific development.