Hippocampus Topographic Circuit Assembly

 

Nature volume 554, pages 328–333 (15 February 2018)

Teneurin-3 controls topographic circuit assembly in the hippocampus

Dominic S. Berns, et.al.

Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA

Department of Biology, Stanford University, Stanford, California 94305, USA.

Neurosciences Graduate Program, Stanford University, Stanford, California 94305, USA

[paraphrase]

Brain functions rely on specific patterns of connectivity. Teneurins are evolutionarily conserved transmembrane proteins that instruct synaptic partner matching in Drosophila and are required for vertebrate visual system development. The roles of vertebrate teneurins in connectivity beyond the visual system remain largely unknown and their mechanisms of action have not been demonstrated. Here we show that mouse teneurin-3 is expressed in multiple topographically interconnected areas of the hippocampal region, including proximal CA1, distal subiculum, and medial entorhinal cortex. Viral-genetic analyses reveal that teneurin-3 is required in both CA1 and subicular neurons for the precise targeting of proximal CA1 axons to distal subiculum. Furthermore, teneurin-3 promotes homophilic adhesion in vitro in a splicing isoform-dependent manner. These findings demonstrate striking genetic heterogeneity across multiple hippocampal areas and suggest that teneurin-3 may orchestrate the assembly of a complex distributed circuit in the mammalian brain via matching expression and homophilic attraction.

The hippocampal region is critical for the acquisition of declarative memory and the neural representation of space. The connections between hippocampal subregions and adjacent cortex are topographically organized along both the dorsal–ventral and proximal–distal axes. Along the proximal–distal axis, proximal CA1, distal subiculum, and medial entorhinal cortex (MEC) neurons are specifically interconnected, as are distal CA1, proximal subiculum, and lateral entorhinal cortex (LEC) neurons. These two parallel circuits may be preferentially used for processing spatial and object-related information, respectively. Genetic heterogeneity that could contribute to the observed anatomical and functional differentiation along the proximal–distal axis in CA1 has been reported. However, the mechanisms that control the exquisite wiring specificity remain unknown.

Since the proposal of the chemoaffinity hypothesis for establishing specific neuronal connections, many cell surface and secreted proteins have been discovered that guide developing axons to target regions and recognize specific synaptic partners. Members of the teneurin family of type II transmembrane proteins exhibit matching expression in pre- and postsynaptic partners and instruct synaptic partner choice in the Drosophila olfactory and neuromuscular systems, probably through homophilic attraction. Teneurins are evolutionarily conserved, with four members in mammals that are dynamically expressed during brain development. Human teneurins are risk loci in bipolar disorder and schizophrenia, and are implicated in other neurological disorders. Teneurin-3 (Ten3) is required for proper dendrite morphogenesis and axon targeting in the vertebrate visual system. Although Ten3 has been hypothesized to function as a homophilic attractant, no cellular or molecular mechanisms have been demonstrated. Furthermore, conflicting evidence exists as to whether vertebrate teneurins interact in trans in a homophilic manner, and heterophilic trans interactions with the adhesion-type G-protein-coupled receptors latrophilins have been demonstrated. Here, we examine the role of Ten3 in the development of specific connections within the hippocampal region, and shed new light on its mechanism of action during mammalian neural development.

Using a custom antibody against a cytoplasmic epitope, we found that Ten3 was expressed in highly specific regions of the postnatal brain. In particular, Ten3 was expressed in restricted domains of the developing hippocampal region, including proximal CA1, distal subiculum, and MEC. A second Ten3 antibody against an extracellular epitope recapitulated this staining pattern. Staining with both antibodies was abolished in Ten3 knockout mice (Ten3Δ4/Δ4). Ten3 was most prominent in synaptic layers, including stratum lacunosum-moleculare of CA1 and the molecular layer of subiculum, consistent with Ten3 being present in the synaptic cleft. Ten3 was also present in axons, dendrites, and cell bodies. In situ hybridization revealed that Ten3 mRNA was expressed in all regions where Ten3 protein was observed. In both CA1 and subiculum, Ten3 mRNA showed a graded distribution along the proximal–distal axis, peaking in proximal CA1 and distal subiculum.

Remarkably, Ten3 protein and mRNA expression patterns corresponded to the known topography of multiple connections in the hippocampal region. MEC neurons send axons to proximal CA1 and distal subiculum, proximal CA1 neurons project to distal subiculum and MEC, and distal subicular neurons project to MEC. All of these regions highly expressed Ten3 protein and mRNA. By contrast, LEC neurons are interconnected with distal CA1 and proximal subiculum, all of which expressed low or no Ten3. To further examine the relationship between Ten3 expression and topographic projections, we injected an anterograde viral tracer into MEC, and found that MEC axons and Ten3 protein clearly overlapped in the molecular layers of proximal CA1 and distal subiculum. By contrast, LEC axons projected to distal CA1 and proximal subiculum, regions of low Ten3 expression.

In summary, Ten3 expression matches with topographic connectivity between entorhinal cortex, CA1, and subiculum. Ten3 protein and mRNA were also specifically expressed in subregions of the presubiculum, parasubiculum, medial mammillary nucleus, and anteroventral thalamic nucleus that are topographically connected with subiculum or entorhinal cortex. Given the function of Drosophila teneurins in synaptic partner matching, we hypothesized that Ten3 may act as a homophilic attractant to control the development of these precise wiring patterns.

A striking feature of neural development is the formation of highly precise connections between neurons. Sensory and motor circuits have been extensively used to characterize the molecular control of wiring specificity, but relatively little is known about how neurons in complex high-order circuits find appropriate partners. Here, we have shown that Ten3 acts in both pre- and postsynaptic neurons in the hippocampus to control the assembly of a precise topographic projection. Loss-of-function phenotypes support a homophilic attraction mechanism: when Ten3 is lost from CA1 neurons, proximal CA1 axons spread throughout the entire subiculum, instead of projecting only to distal, Ten3-high targets; when Ten3 is lost from a subset of distal subicular cells, Ten3-high proximal CA1 axons do not target these areas and instead innervate nearby Ten3-high regions. Our in vitro data further show that Ten3 can interact homophilically in trans, supporting a model in which Ten3 on CA1 axons interacts with Ten3 on subicular targets, leading to contact-mediated attraction or stabilization of proximal CA1 axons by distal subicular target cells. This mechanism of action resembles that of the Drosophila teneurins in the development of olfactory and neuromuscular connections, suggesting an evolutionarily conserved mode of teneurin function in neural circuit assembly from insects to mammals. However, whereas Drosophila teneurins instruct matching of discrete types of pre- and postsynaptic cell, the graded expression in both CA1 and subiculum suggests that mouse Ten3 directs continuous topographic mapping along the proximal–distal axis.

Finally, our findings reveal genetic heterogeneity within many areas of the hippocampal region. Although our genetic analyses focused on the CA1→subiculum projection, Ten3-high to Ten3-high connectivity was also observed in the entorhinal→hippocampal projections, and probably exists in additional hippocampus-associated projections. The matching expression of Ten3 in multiple topographically connected subregions, combined with our loss-of-function and in vitro data, suggests that Ten3 may control the assembly of a widely distributed circuit in mammalian brains.

 

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