Sachihiro C. Suzuki

Neuronal circuits are subdivided by differential expression of type-II classic cadherins in postnatal mouse brains

A number of type-II classic cadherin cell-cell adhesion molecules are expressed in the brain. To investigate their roles in brain morphogenesis, we selected three type-II cadherins, cadherin-6 (cad6), -8 (cad8) and -11 (cad11), and mapped their expressions in the forebrain and other restricted regions of postnatal mouse brains. In the cerebral cortex, each cortical area previously defined was delineated by a specific combinatorial expression of these cadherins. The thalamus and other subcortical regions of the forebrain were also subdivided by differential expression of the three cadherins; e.g., the medial geniculate body expressed only cad6; the ventral posterior thalamic nucleus, cad6/cad11; and the anteroventral thalamic nucleus, cad6/cad8. Likewise, in the olivocerebellar system, each subdivision of the inferior olive expressed a unique set of the three cadherins, and the cerebellar cortex had parasagittal stripes of cad8/cad11 expressions. Close analysis of these cadherin expression patterns revealed that they are correlated with neuronal connection patterns. Examples of these correlations include that cad6 delineates the auditory projection system, cad6/cad8/ cad11 are expressed by part of the Papez circuit, and cad6/cad8 are expressed by subdivisions of the olivo-nuclear circuit. Together with the recent finding that the cadherin adhesion system is localized in synaptic junctions, our findings support the notion that cadherin-mediated cell-cell adhesion plays a role in selective interneuronal connections during neural network formation.

Loss of Cadherin-11 Adhesion Receptor Enhances Plastic Changes in Hippocampal Synapses and Modifies Behavioral Responses

Cadherins organize symmetrical junctions between the pre- and postsynaptic membranes in central synapses. One of them, cadherin-11 (cad11), is expressed in the limbic system of the brain, most strongly in the hippocampus. Immunohistochemical studies of the hippocampus showed that cad11 proteins were densely distributed in its synaptic neuropil zones; in cultured hippocampal neurons, their distribution often overlapped with that of synaptophysin, and also occasionally with that of GluR1 at spines. To assess the role of cad11 in synaptic formation and/or function, we analyzed brains of cad11-deficient mice. In these mice, long-term potentiation (LTP) in the CA1 region of the hippocampus was, unexpectedly, enhanced; and the level of LTP saturation was increased. In behavioral tests, the mutant mice showed reduced fear- or anxiety-related responses. These results suggest that the cad11-mediated junctions may modulate synaptic efficacy, confining its dynamic changes to a limited range, or these junctions are required for normal development of synaptic organization in the hippocampus.

Cadherin-6 in the developing mouse brain: Expression along restricted connection systems and synaptic localization suggest a potential role in neuronal circuitry†

Multiple subtypes of the cadherin homophilic cell‐cell adhesion molecule are expressed differentially in developing and mature brains, each being expressed in restricted neuronal groups. Cadherin‐6 (cad6) is one of such cadherins. Recent studies of cad6 mRNA expression in the postnatal mouse forebrain showed that it occurs in neurons constituting a specific subset of thalamocortical connections. Here we analyzed the localization of cad6 mRNA as well as its protein in the entire central nervous system and also in cranial ganglia of mice at late embryonic to postnatal stages. Our results showed that cad6 is expressed by a limited population of neurons or their precursors, which are synaptically connected to one another, throughout the perinatal stages, and that this expression delineates restricted neuronal circuits from the central to peripheral nervous systems, which include subpathways of the auditory, somatosensory, solitary, vestibular, and olivocerebellar systems. cad6 proteins were detected in these cad6 mRNA‐positive neurons on the surface of their cell bodies or dendrites as well as in the cytoplasm. Confocal microscopic analysis revealed that the cad6 protein distribution overlapped that of synaptotagmin in synapse forming areas, suggesting that homotypic cad6 interactions are involved in synaptic connections between neurons expressing this protein. These findings support the idea that cadherin‐mediated cell‐cell adhesions take part in specific interneuronal connections. Dev. Dyn. 1998;211:338–351.

Cadherins in neuronal morphogenesis and function.

Classic cadherins represent a family of calcium‐dependent homophilic cell–cell adhesion molecules. They confer strong adhesiveness to animal cells when they are anchored to the actin cytoskeleton via their cytoplasmic binding partners, catenins. The cadherin/catenin adhesion system plays key roles in the morphogenesis and function of the vertebrate and invertebrate nervous systems. In early vertebrate development, cadherins are involved in multiple events of brain morphogenesis including the formation and maintenance of the neuroepithelium, neurite extension and migration of neuronal cells. In the invertebrate nervous system, classic cadherin‐mediated cell–cell interaction plays important roles in wiring among neurons. For synaptogenesis, the cadherin/catenin system not only stabilizes cell–cell contacts at excitatory synapses but also assembles synaptic molecules at synaptic sites. Furthermore, this system is involved in synaptic plasticity. Recent studies on the role of individual cadherin subtypes at synapses indicate that individual cadherin subtypes play their own unique role to regulate synaptic activities.

Cadherin-8 is required for the first relay synapses to receive functional inputs from primary sensory afferents for cold sensation.

Classic cadherins, comprising multiple subtypes, mediate selective cell–cell adhesion based on their subtype-specific binding nature. Each subtype in the brain is expressed by restricted groups of functionally connected nuclei and laminas. However, whether each subtype has any specific role in neural circuitry remains largely unknown. Here, we show that cadherin-8 (cad8), a type-II classic cadherin, is important for cold sensation, whose circuitry is established by projection of sensory neurons into the spinal cord. Cad8 was expressed by a subset of neurons in the dorsal horn (DH) of the spinal cord, as well as by a small number of neurons in the dorsal root ganglia (DRGs), and the majority of cad8-positive DRG neurons coexpressed cold temperature/menthol receptor (TRPM8). We generated cad8 knock-out mice and analyzed lacZ markers expressed by the targeted cad8 locus using heterozygous mice. LacZ/cad8-expressing sensory neurons and DH neurons were connected together, and cad8 protein was localized around the synaptic junctions formed between them. This relation was, however, not disrupted in cad8−/− mice. We performed whole-cell patch-clamp recordings from DH neurons in spinal cord slices, in combination with menthol stimulation as a tool to excite central terminals of primary afferents expressing TRPM8. LacZ-expressing DH neurons exhibited fast and slow miniature EPSCs. Menthol selectively increased the frequency of the slow mEPSCs in cad8+/− slices, but this effect was abolished in cad8−/− slices. The cad8−/− mice also showed a reduced sensitivity to cold temperature. These results demonstrate that cad8 is essential for establishing the physiological coupling between cold-sensitive sensory neurons and their target DH neurons.

Impairment of afferent synaptic inputs for mechanical and cold sensation to substantia gelatinosa neurons of the rat spinal dorsal horn through cadherin-8 expressing synaptic glomeruli

s / Neuroscience Research 58S (2007) S1–S244 S71 P1-aØ1 Effects univibrissae rearing on response properties of barrel cortical neurons in C-fiber-depleted rats S. Azizollahi1, A. Shamsizade1,2, V. Shey Bani1, S. Arabzadeh1, M. Afarinesh1 1 Neuroscience Research Center, Kerman University of Medical Science, Iran; 2 Department of Physics, Rafsanjan University of Medical Science, Iran C-fiber depletion increases low threshold somatosensory mechanoreceptive fields and decreases lateral inhibition in barrel cortex. In this study, we investigated the role of intact C-fibers in response properties of spared barrel neurons (layer IV) following experience dependent plasticity in barrel cortex. C-fiber depletion was induced by neonatal treatment with capsaicin (50 mg/kg I.P.). Plucking of all whiskers but D2 started at first post-partum day. Neuronal ON and OFF responses were computed following principal whisker (PW) and adjacent whisker (AW) deflections. In both C-fiber depleted and vehicle treated rats, whisker plucking increased ON responses to spared PW deflection. Neuronal ON responses to deflection of deprived AW were decreased in vehicle treated rats but in C-fiber depleted rats they did not decrease. Neuronal OFF responses showed similar result to ON responses. These results suggest that neonatal C-fiber depletion can modulate response properties of spared barrel neurons. P1-aØ2 Low concentration of nicotine triggers a novel regulatory pathway in neuromuscular synapses Vasiliy V. Fedorin, Olga P. Balezina Department of Physiology, Moscow State University, Moscow, Russia Nicotine in 10−8 M concentration was found to significantly reduce amplitude and quantal content of evoked end-plate potentials generated in short trains at stimulation frequencies of 4, 7 and 50 Hz in cut neuromuscular mouse diaphragm preparation. Nicotine-induced inhibition was prevented by -cobratoxin, an antagonist of 7-type neuronal nicotinic acetylcholine receptor (nAChR), and by apamin, small conductance calcium-activated potassium channel (SK-channel) antagonist, as well as found to be Ca2+-dependent. This suggests a presence of a yet undescribed pool of presynaptic 7 nACHRs, that can participate in a regulatory negative feedback pathway: activation of 7 nAChRs by acetylcholine or other agonists, such as nicotine, results in a local Ca2+ influx, coupled with activation of SK-channels. Resulting K+ outflow hyperpolarizes presynaptic nerve terminal, reducing acetylcholine secretion in synaptic active zones. The suggested mechanism could be aimed at lowering mediator loss during prolonged high-frequency synaptic activity. Research funds: RFBR 02-04-48126 P1-aØ3 Single action potential-evoked Ca2+ transients at the calyx of Held presynaptic terminal Yukihiro Nakamura1, David Digregorio2, Tomoyuki Takahashi1,3 1 Faculty of Life and Medical Sciences, Doshisha University, Kyoto, Japan; 2 CNRS UMR8118 Laboratoire de Physiologie Cerebrale, Universite Paris 5, France; 3 Okinawa Institute of Science and Technology, Japan Ca2+ transient at presynaptic nerve terminals triggers transmitter release. Amplitude, kinetics and distribution of Ca2+ transient regulate release modality, but the property of Ca2+ transient within the nerve terminal remains elusive. To investigate this issue, we tried imaging for a single action potential-evoked Ca2+ transient at a giant presynaptic terminal, the calyx of Held, using Oregon Green BAPTA 5N. Confocal spot measurement enabled detection of Ca2+ gradients within the terminal. Ca2+ transients were mainly observed at the synaptic site of the terminal, but the spatial distribution was sparser in older animals (P13–14 versus P7–8). Amplitude of the transient was smaller in older animals. The rise time of transients was equivalent to action potential duration, whereas decay time was regulated by Ca2+ buffer concentration at the both age groups. These differences may underlie the developmental changes in synaptic efficacy at this synapse. P1-aØ4 Regulation of the differentiation of excitatory synapses by the SALM family of cell adhesion-like molecules Jaewon Ko1, Seho Kim1, Hye Sun Chung2, Hyun Kim2, Eunjoon Kim1 1 Department of Biological Sciences, KAIST, Daejeon, South Korea; 2 Department of Anatomy, College of Medicine, Korea University, 126-1, 5-Ka, Seoul 136-705, Korea Synaptic cell adhesion molecules (CAMs) are known to play key roles in various aspects of synaptic structures and functions. We herein report the identification of a family of cell adhesion-like molecules termed SALM that interacts with the abundant postsynaptic density (PSD) protein PSD95. SALM2, a SALM isoform, distributes to excitatory, but not inhibitory, synaptic sites. Overexpression of SALM2 increases the number of excitatory synapses and dendritic spines. Bead-induced direct aggregation of SALM2 results in coclustering of PSD-95 and other postsynaptic proteins, including GKAP and AMPA receptors. Knockdown of SALM2 by RNA interference reduces the number of excitatory synapses and dendritic spines and the frequency, but not amplitude, of miniature excitatory postsynaptic currents. These results suggest that SALM2 is an important regulator of the differentiation of excitatory synapses. P1-aØ6 Impairment of afferent synaptic inputs for mechanical and cold sensation to substantia gelatinosa neurons of the rat spinal dorsal horn through cadherin-8 expressing synaptic glomeruli Hidemasa Furue1, Sachihiro S.C. Suzuki2, Kohei Koga1, Mitsuo Nohmi3, Masatoshi Takeichi2, Megumu Yoshimura1 1 Department Integrative Physiology, Grad. Sch. Med. Sci., Kyushu University, Fukuoka, Japan; 2 Center for Developmental Biology, RIKEN, Kobe, Japan; 3 Anal Research Center for Experimental Science, Saga University, Saga, Japan In vivo and slice patch-clamp recordings were made from spinal substantia gelatinosa neurons in wild and cad8−/− mice to compare the modality-dependent synaptic inputs with or without cad8. SG neurons with cad8 received synaptic inputs from menthol-sensitive afferents and exhibited a barrage of EPSCs in response to cutaneous pinch stimuli. In cad8−/− mice, the amplitude of C fiber-evoked EPSCs and the frequency of miniature EPSCs from menthol-sensitive (TRPM8 expressing) afferents were decreased. Behavioral analysis showed reduced sensitivities for mechanical and cold noxious stimuli in cad8−/− mice. The present results suggest that cad8 has a crucial role for forming functional synaptic connections between SG neurons and primary afferent fibers for mechanical and cold noxious sensation. Research funds: KAKENHI P1-aØ7 Optical imaging of glutamate release at individual hippocampal synapses Hirokazu Sakamoto, Shigeyuki Namiki, Sho Iinuma, Kenzo Hirose Department of Cell Physiology, Nagoya University, Nagoya, Japan At individual glutamatergic synapses, the amount of released glutamate per action potential determines the synaptic strength. However, the direct measurement of synaptically released glutamate has not been done. To measure glutamate release at single synapses, we developed a novel fluorescent glutamate probe called EOS (Glutamate Optical Sensor). Electrically evoked glutamate release was successfully detected at synapses in cultured hippocampal neurons. We found that released glutamate varied in amount among synapses. Also, changes in release probability by manipulating extracellular calcium concentration were associated with changes in the amount of glutamate released at individual synapses. Furthermore, phorbol ester increased the amount of glutamate released at individual synapses. These findings suggest that single hippocampal synapses contain several release sites. In summary, we evaluated the functional release properties of each individual synapses by using EOS.

1110 Cadherin expression associated with neural networks

Little is known about what kind of adhesion molecules are involved in the formation and maintenance of synaptic junctions. In the present study we investigated the localization of cadherin-associated proteins, olN-catenin and p-catenin, in interneuronal adhesion sites by immunoelectron microscopy with gold labeling. These cadherin-associated proteins were found to localize at synaptic junctions. The immunostained signals were contiguous to the active zones and often fused with them, but not localized throughout the active zones. These results suggest that catenins are components of the synaptic junctions, perhaps localizing at so called puncta a&aerentia. Our findings thus indicate the existence of at least two types of cell-cell adhesion sites in synapses: One is the cadherin/catenin-based puncta adhaerentia, and the other is the active zone.