Toshiaki Nakashiba

Transgenic Inhibition of Synaptic Transmission Reveals Role of CA3 Output in Hippocampal Learning

The hippocampus is an area of the brain involved in learning and memory. It contains parallel excitatory pathways referred to as the trisynaptic pathway (which carries information as follows: entorhinal cortex → dentate gyrus → CA3 → CA1 → entorhinal cortex) and the monosynaptic pathway (entorhinal cortex → CA1 → entorhinal cortex). We developed a generally applicable tetanus toxin–based method for transgenic mice that permits inducible and reversible inhibition of synaptic transmission and applied it to the trisynaptic pathway while preserving transmission in the monosynaptic pathway. We found that synaptic output from CA3 in the trisynaptic pathway is dispensable and the short monosynaptic pathway is sufficient for incremental spatial learning. In contrast, the full trisynaptic pathway containing CA3 is required for rapid one-trial contextual learning, for pattern completion–based memory recall, and for spatial tuning of CA1 cells.

Entorhinal cortex layer III input to the hippocampus is crucial for temporal association memory.

A specific neural circuit integrates temporally dispersed stimuli into a coherent memory episode. Associating temporally discontinuous elements is crucial for the formation of episodic and working memories that depend on the hippocampal-entorhinal network. However, the neural circuits subserving these associations have remained unknown. The layer III inputs of the entorhinal cortex to the hippocampus may contribute to this process. To test this hypothesis, we generated a transgenic mouse in which these inputs are specifically inhibited. The mutant mice displayed significant impairments in spatial working-memory tasks and in the encoding phase of trace fear-conditioning. These results indicate a critical role of the entorhinal cortex layer III inputs to the hippocampus in temporal association memory.

Hippocampal CA3 Output Is Crucial for Ripple-Associated Reactivation and Consolidation of Memory

A widely held memory consolidation theory posits that memory of events and space is initially stored in the hippocampus (HPC) in a time-limited manner and is consolidated in the neocortex for permanent storage. Although posttraining HPC lesions result in temporally graded amnesia, the precise HPC circuits and mechanisms involved in remote memory storage remain poorly understood. To investigate the role of the trisynaptic pathway in the consolidation process we employed the CA3-TeTX transgenic mouse, in which CA3 output can be specifically and inducibly controlled. We found that posttraining blockade of CA3 output for up to 4 weeks impairs the consolidation of contextual fear memory. Moreover, in vivo hippocampal recordings revealed a reduced intrinsic frequency of CA1 ripples and a significant decrease in the experience-dependent, ripple-associated coordinated reactivation of CA1 cell pairs. Collectively, these results suggest that the posttraining integrity of the trisynaptic pathway and the ripple-associated reactivation of hippocampal memory engram are crucial for memory consolidation.

Complementary expression and neurite outgrowth activity of netrin-G subfamily members

Classical members of the UNC6/netrin family are secreted proteins which play a role as long-range cues for directing growth cones. We here identified in mice a novel member netrin-G2 which constitute a subfamily with netrin-G1 among the UNC6/netrin family. Both of these netrin-Gs are characterized by glycosyl phosphatidyl-inositol linkage onto cells, molecular variants presumably generated by alternative splicing and lack of any appreciable affinity to receptors for classical netrins. These genes are preferentially expressed in the central nervous system with complementary distribution in most brain areas, that is netrin-G1 in the dorsal thalamus, olfactory bulb and inferior colliculus, and netrin-G2 in the cerebral cortex, habenular nucleus and superior colliculus. Consistently, immunohistochemical analysis revealed that netrin-G1 molecules are present on thalamocortical but not corticothalamic axons. Thalamic and neocortical neurons extended long neurites on immobilized recombinant netrin-G1 or netrin-G2 in vitro. Immobilized anti-netrin-G1 antibodies altered shapes of cultured thalamic neurons. We propose that netrin-Gs provide short-range cues for axonal and/or dendritic behavior through bi-directional signaling.

Laminar organization of the developing lateral olfactory tract revealed by differential expression of cell recognition molecules

The projection neurons in the olfactory bulb (mitral and tufted cells) send axons through the lateral olfactory tract (LOT) onto several structures of the olfactory cortex. However, little is known of the molecular and cellular mechanisms underlying establishment of functional connectivity from the bulb to the cortex. Here, we investigated the developmental process of LOT formation by observing expression patterns of cell recognition molecules in embryonic mice. We immunohistochemically identified a dozen molecules expressed in the developing LOT and some of them were localized to subsets of mitral cell axons. Combinatorial immunostaining for these molecules revealed that the developing LOT consists of three laminas: superficial, middle, and deep. Detailed immunohistochemical, in situ hybridization, and 5‐bromodeoxyuridine labeling analyses suggested that the laminar organization reflects: 1) the segregated pathways from the accessory and main olfactory bulbs, and 2) the different maturity of mitral cell axons. Mitral cell axons of the accessory olfactory bulb were localized to the deep lamina, segregated from those of the main olfactory bulb. In the main olfactory pathway, axons of mature mitral cells, whose somata is located in the apical sublayer of the mitral cell layer, were localized to the middle lamina within LOT, while those of immature mitral cells that located in the basal sublayer were complementarily localized to the superficial lamina. These results suggest that newly generated immature axons are added to the most superficial lamina of LOT successively, leading to the formation of piled laminas with different maturational stages of the mitral cell axons. J. Comp. Neurol. 479:243–256, 2004.

Control of CA3 Output by Feedforward Inhibition Despite Developmental Changes in the Excitation-Inhibition Balance

In somatosensory cortex, the relative balance of excitation and inhibition determines how effectively feedforward inhibition enforces the temporal fidelity of action potentials. Within the CA3 region of the hippocampus, glutamatergic mossy fiber (MF) synapses onto CA3 pyramidal cells (PCs) provide strong monosynaptic excitation that exhibit prominent facilitation during repetitive activity. We demonstrate in the juvenile CA3 that MF-driven polysynaptic IPSCs facilitate to maintain a fixed EPSC-IPSC ratio during short-term plasticity. In contrast, in young adult mice this MF-driven polysynaptic inhibitory input can facilitate or depress in response to short trains of activity. Transgenic mice lacking the feedback inhibitory loop continue to exhibit both facilitating and depressing polysynaptic IPSCs, indicating that this robust inhibition is not caused by the secondary engagement of feedback inhibition. Surprisingly, eliminating MF-driven inhibition onto CA3 pyramidal cells by blockade of GABAA receptors did not lead to a loss of temporal precision of the first action potential observed after a stimulus but triggered in many cases a long excitatory plateau potential capable of triggering repetitive action potential firing. These observations indicate that, unlike other regions of the brain, the temporal precision of single MF-driven action potentials is dictated primarily by the kinetics of MF EPSPs, not feedforward inhibition. Instead, feedforward inhibition provides a robust regulation of CA3 PC excitability across development to prevent excessive depolarization by the monosynaptic EPSP and multiple action potential firings.

A novel gene, Btcl1, encoding CUB and LDLa domains is expressed in restricted areas of mouse brain.

A variety of secreted and membrane proteins play key roles in the formation of neuronal circuits in the central nervous system. Using the signal sequence trap method, we isolated and characterized a novel gene, Btcl1 (brain-specific transmembrane protein containing two CUB and an LDLa domains). BTCL1 has significant homology with neuropilin-1 and -2 in their CUB domains. Domain structure of BTCL1 indicates that BTCL1 belongs to a new class of brain-specific CUB domain-containing protein. On Northern blot analysis, Btcl1 mRNA was observed as a single transcript of 3.7 kb specifically in the brain. In situ hybridization analysis revealed that Btcl1 mRNA was highly expressed in the hippocampal CA3 region, olfactory bulb, and neocortex in the adult brain. Expression pattern of mRNA and structural similarity with neuropilin suggest that BTCL1 plays a role in the development and/or maintenance of neuronal circuitry.

Netrin-G/NGL Complexes Encode Functional Synaptic Diversification

Synaptic cell adhesion molecules are increasingly gaining attention for conferring specific properties to individual synapses. Netrin-G1 and netrin-G2 are trans-synaptic adhesion molecules that distribute on distinct axons, and their presence restricts the expression of their cognate receptors, NGL1 and NGL2, respectively, to specific subdendritic segments of target neurons. However, the neural circuits and functional roles of netrin-G isoform complexes remain unclear. Here, we use netrin-G-KO and NGL-KO mice to reveal that netrin-G1/NGL1 and netrin-G2/NGL2 interactions specify excitatory synapses in independent hippocampal pathways. In the hippocampal CA1 area, netrin-G1/NGL1 and netrin-G2/NGL2 were expressed in the temporoammonic and Schaffer collateral pathways, respectively. The lack of presynaptic netrin-Gs led to the dispersion of NGLs from postsynaptic membranes. In accord, netrin-G mutant synapses displayed opposing phenotypes in long-term and short-term plasticity through discrete biochemical pathways. The plasticity phenotypes in netrin-G-KOs were phenocopied in NGL-KOs, with a corresponding loss of netrin-Gs from presynaptic membranes. Our findings show that netrin-G/NGL interactions differentially control synaptic plasticity in distinct circuits via retrograde signaling mechanisms and explain how synaptic inputs are diversified to control neuronal activity.

Monoclonal antibodies discriminating netrin-G1 and netrin-G2 neuronal pathways

Netrin-G1 and netrin-G2, belonging to a vertebrate-specific subfamily of the netrin family, distribute on axons of distinct neuronal pathways. To add to the array of molecular probes available for labeling unique neuronal circuits, we generated monoclonal antibodies against the netrin-G1 and netrin-G2 proteins. The monoclonal antibody clones 171A18 and 30B15 differentially labeled specific neuronal circuits, the so-called netrin-G1 or netrin-G2 circuits in mice, respectively. Epitope mapping revealed linear epitopes for these monoclonal antibodies, which are common among splicing variants, and suggested that the anti-netrin-G1 monoclonal antibodies are applicable to various species including humans.

Genetic factors underlying attention and impulsivity: mouse models of attention-deficit/hyperactivity disorder

Increasing evidence suggests complex genetic factors for attention-deficit/hyperactivity disorder (ADHD). Animal models with definitive genetic characteristics are indispensable for gaining an understanding of the molecular, cellular, and neural circuit mechanisms underlying ADHD. Toward this aim, mice have several advantages because of their well-controlled genetic backgrounds and the relative ease with which functions of defined neuronal circuits can be manipulated. Dopamine signaling dysfunction was once the major pathogenic focus of interest in ADHD research, but hypotheses have expanded to include functionally distinct molecules. Forward and reverse genetic approaches have produced diverse mouse genetic models for genes involved in monoaminergic signaling, synaptic plasticity, and neuronal circuit formation. Data suggest crucial roles of gene–gene interactions and gene–environment interactions in the pathophysiology of ADHD.