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Genetic single-neuron tracing from the olfactory bulb to higher brain centers in zebrafish
e98
Abstracts / Neuroscience Research 68S (2010) e55–e108
O2-8-1-1 Neuronal circuits responsible for the generation of olfactory cortex and olfactory bulb sharp waves during slow-wave sleep Hiroyuki Manabe 1,2 , Ikue Kusumoto-Yoshida 1,2 , Mizuho Ota 1,2 , Kensaku Mori 1,2 1
2
Department Physiol., Grad. Sch. Med., University of Tokyo, Tokyo JST
We recently found that olfactory cortex generates sharp waves during slowwave sleep in freely behaving rats. Olfactory bulb also generates sharp wave-like activity, which is typically synchronized with olfactory cortex sharp waves. Here, we addressed the question which neuronal circuits generate olfactory cortex and olfactory bulb sharp waves using urethaneanesthetized rats.Olfactory cortex sharp waves occurred during slow-wave state in urethane-anesthetized rats. Many olfactory cortex neurons showed synchronous spike discharges at the descending phase of the olfactory cortex sharp waves. Current source density analysis indicated that olfactory cortex sharp waves were generated by recurrent association fiber synaptic inputs to pyramidal cells in anterior piriform cortex. Pyramidal neurons in the olfactory cortex give rise to massive centrifugal fibers that terminate on granule cells in the olfactory bulb. Sharp wave activity synchronized with olfactory cortex sharp waves occurred in the granule cell layer of the olfactory bulb during slow-wave state. Current source density analysis indicated that olfactory bulb sharp waves were generated by synchronous depolarization of proximal and basal dendrites of granule cells. These results suggest that olfactory cortex sharp waves travel to the granule cells in the olfactory bulb. Burst stimulation of the centrifugal fibers induced long lasting potentiation of the centrifugal fiber synapses on granule cells, suggesting that olfactory cortex sharp waves generate plastic changes in the synapses of granule cells. doi:10.1016/j.neures.2010.07.196
O2-8-1-3 Molecular basis of CO2 sensing in the mouse olfactory system Hiroo Takahashi 1 , Hitoki Nanaura 1 , Sei-ichi Yoshihara 1 , Takeshi Imai 2 , Junzo Hirono 3 , Takaaki Sato 3 , Akio Tsuboi 1 1
Lab for Mol Biol of Neural System, Nara Med Univ, Kashihara 2 Dep of Biophys & Biochem, Grad Sch of Science, Univ of Tokyo, Tokyo 3 Genome Inte, Health Res Inst, AIST, Amagasaki
Olfactory sensory neurons (OSNs) expressing a given odorant receptor (OR) project axons to a pair of fixed glomeruli on the olfactory bulb to form a topographic map. Interestingly, in the ventro-lateral region of the olfactory epithelium, there is a unique subset of OSNs, termed Car2 OSNs, which express carbonic anhydrase 2 (Car2) and guanylate cyclase-D, instead of OR. Although Car2 OSNs is known to respond to CO2 and urinary peptide, a molecular mechanism that regulates the development of these neurons remains unrevealed. Here, we found that Pax6 is expressed specifically in Car2 OSNs. In canonical OSNs, ectopic expression of Pax6 gave rise to inhibition of the canonical OSN-specific gene expression, but to no affection of the Car2 OSN-specific gene expression. These results suggest that Pax6 may act as a negative regulator to prevent the expression of canonical OSN-specific genes in Car2 OSNs. We are in the process of identifying a transcriptional factor to specify the Car2-neuronal lineage. Furthermore, we found that other subsets of OSNs, not expressing Car2, also respond to CO2 in calcium imaging. This indicates that mice sense CO2 with novel subsets of neurons as well as Car2 neurons in nose. doi:10.1016/j.neures.2010.07.198
O2-8-1-4 The feasibility study of novel odor biosensor using dissociate neuronal culture expressing ion channel built-in odor receptors
O2-8-1-2 Genetic single-neuron tracing from the olfactory bulb to higher brain centers in zebrafish
Norio Tanada 1 , Takeshi Sakurai 2 , Hidefumi Mitsuno 2 , Bakkum Douglas 2 , Ryohei Kanzaki 2 , Hirokazu Takahashi 2,3
Nobuhiko Miyasaka , Yoshihiro Yoshihara
1
Lab Neurobiology of Synapse, RIKEN BSI, Wako, Japan In the olfactory system, odor information is initially represented as a topographic, chemotopic map in the olfactory bulb (OB). In zebrafish, bile salts (putative social pheromones) activate anterior and dorsal clusters of glomeruli in the OB, whereas amino acids (potent feeding cues) activate lateral cluster of glomeruli. However, it remains largely unknown how the odor map in the OB is transferred to higher olfactory centers. To answer this question, it is essential to analyze in detail the neuroanatomy of individual OB output neurons with special reference to the dendritic innervation of glomeruli and the axonal trajectory to target regions. We found that two different transcriptional promoters of lhx2a and tbx21 genes can drive expression of fluorescent proteins in distinct subpopulation of OB output neurons without labeling of cells in other brain regions. lhx2atg+ neurons predominantly exhibit dendritic innervations to medial cluster of glomeruli (mG), whereas tbx21tg+ neurons preferentially innervate glomeruli outside the mG. Tracing axons at single-cell resolution with a genetic mosaic labeling technique revealed that (1) individual OB output neurons send axons to multiple target regions in the forebrain, including the telencephalon, the habenula and the hypothalamus; (2) OB output neurons innervating distinct glomerular clusters tend to project axons to different, but partly overlapping, target regions. (3) OB output neurons innervating the same glomerulus do not necessarily display the same axon trajectory; (4) OB output neurons innervating the mG send axons to the right habenular nucleus directly and asymmetrically. These results suggest that the topographic odor map in the OB is not maintained intact, but reorganized in higher olfactory centers. This study will pave the way for understanding of the functional logic of odor information coding employed by higher olfactory centers in vertebrate brains. doi:10.1016/j.neures.2010.07.197
Dept Advanced interdisciplinary studies, Univ of Tokyo, Tokyo 2 RCAST, Univ of Tokyo, Tokyo 3 PRESTO, JST, Tokyo
We propose a highly sensitive and real-time odor biosensor by expressing ion channel built-in odor receptors of insects into dissociated cultures of neurons of rats. The ion channel built-in structure of insect odor receptor easily allows functional expression into cells. The neuronal dissociated cultures of rats have 2 significant advantages: a long lifetime comparable to rats, i.e., a few years; and amplification ability from weak ionic currents of odor receptors into easily detectable action potentials of neurons. To demonstrate the feasibility of this proposed sensor, we attempted to express the pheromone receptors of silkmoth, Bombyx mori, into cultured neurons of rats. Two types of pheromone receptors, BmOR1 and BmOR3, show high selectivity to their pheromone substance, Bombykol (BOL) and Bombykal (BAL), respectively. Each pheromone receptor forms heterodimeric complexes with a co-receptor, BmOR2, and this complex perform as non-selective cationic channel. We attempted to co-express BmOR1/BmOR2 or BmOR3/BmOR2 into neuronal cultures by lipofection. As the result, the transfection efficiency was about 10%, which was sufficient for an odor senor. To test the functional consequences of pheromone receptors, the calcium imaging investigates the neural activity of transfected cells in response to the pheromone. Administration of 100 M pheromone substances increased calcium responses up to 10% - 50% within 40 seconds. These results suggest the feasibility of the proposed sensor. doi:10.1016/j.neures.2010.07.199
O2-8-2-1 In vivo connection imaging and its application To monkey inferotemporal face system Noritaka Ichinohe 1,2,3 Manabu Tanifuji 3
, Takayuki Sato 3 , Kathleen Rockland 2 ,
1 Department of Neuroanatomy, Hirosaki University, Graduate School of Medicine 2 Lab. for Cortical Organization and Systematics, Brain Science Institute, RIKEN 3 Lab. for integrative physiology, BSI, RIKEN, Japan
Anatomical tracing techniques have been the firm technique connected brain sites. However, evaluation of connection on postmortem brain makes it unable to compare response property between connected two sites. We have developed a new use for the tracer CTB-Alexa555, for imaging cortical connections in the living macaque monkey brain. On 7-14 days after tracer
Abstracts / Neuroscience Research 68S (2010) e55–e108
O2-8-1-1 Neuronal circuits responsible for the generation of olfactory cortex and olfactory bulb sharp waves during slow-wave sleep Hiroyuki Manabe 1,2 , Ikue Kusumoto-Yoshida 1,2 , Mizuho Ota 1,2 , Kensaku Mori 1,2 1
2
Department Physiol., Grad. Sch. Med., University of Tokyo, Tokyo JST
We recently found that olfactory cortex generates sharp waves during slowwave sleep in freely behaving rats. Olfactory bulb also generates sharp wave-like activity, which is typically synchronized with olfactory cortex sharp waves. Here, we addressed the question which neuronal circuits generate olfactory cortex and olfactory bulb sharp waves using urethaneanesthetized rats.Olfactory cortex sharp waves occurred during slow-wave state in urethane-anesthetized rats. Many olfactory cortex neurons showed synchronous spike discharges at the descending phase of the olfactory cortex sharp waves. Current source density analysis indicated that olfactory cortex sharp waves were generated by recurrent association fiber synaptic inputs to pyramidal cells in anterior piriform cortex. Pyramidal neurons in the olfactory cortex give rise to massive centrifugal fibers that terminate on granule cells in the olfactory bulb. Sharp wave activity synchronized with olfactory cortex sharp waves occurred in the granule cell layer of the olfactory bulb during slow-wave state. Current source density analysis indicated that olfactory bulb sharp waves were generated by synchronous depolarization of proximal and basal dendrites of granule cells. These results suggest that olfactory cortex sharp waves travel to the granule cells in the olfactory bulb. Burst stimulation of the centrifugal fibers induced long lasting potentiation of the centrifugal fiber synapses on granule cells, suggesting that olfactory cortex sharp waves generate plastic changes in the synapses of granule cells. doi:10.1016/j.neures.2010.07.196
O2-8-1-3 Molecular basis of CO2 sensing in the mouse olfactory system Hiroo Takahashi 1 , Hitoki Nanaura 1 , Sei-ichi Yoshihara 1 , Takeshi Imai 2 , Junzo Hirono 3 , Takaaki Sato 3 , Akio Tsuboi 1 1
Lab for Mol Biol of Neural System, Nara Med Univ, Kashihara 2 Dep of Biophys & Biochem, Grad Sch of Science, Univ of Tokyo, Tokyo 3 Genome Inte, Health Res Inst, AIST, Amagasaki
Olfactory sensory neurons (OSNs) expressing a given odorant receptor (OR) project axons to a pair of fixed glomeruli on the olfactory bulb to form a topographic map. Interestingly, in the ventro-lateral region of the olfactory epithelium, there is a unique subset of OSNs, termed Car2 OSNs, which express carbonic anhydrase 2 (Car2) and guanylate cyclase-D, instead of OR. Although Car2 OSNs is known to respond to CO2 and urinary peptide, a molecular mechanism that regulates the development of these neurons remains unrevealed. Here, we found that Pax6 is expressed specifically in Car2 OSNs. In canonical OSNs, ectopic expression of Pax6 gave rise to inhibition of the canonical OSN-specific gene expression, but to no affection of the Car2 OSN-specific gene expression. These results suggest that Pax6 may act as a negative regulator to prevent the expression of canonical OSN-specific genes in Car2 OSNs. We are in the process of identifying a transcriptional factor to specify the Car2-neuronal lineage. Furthermore, we found that other subsets of OSNs, not expressing Car2, also respond to CO2 in calcium imaging. This indicates that mice sense CO2 with novel subsets of neurons as well as Car2 neurons in nose. doi:10.1016/j.neures.2010.07.198
O2-8-1-4 The feasibility study of novel odor biosensor using dissociate neuronal culture expressing ion channel built-in odor receptors
O2-8-1-2 Genetic single-neuron tracing from the olfactory bulb to higher brain centers in zebrafish
Norio Tanada 1 , Takeshi Sakurai 2 , Hidefumi Mitsuno 2 , Bakkum Douglas 2 , Ryohei Kanzaki 2 , Hirokazu Takahashi 2,3
Nobuhiko Miyasaka , Yoshihiro Yoshihara
1
Lab Neurobiology of Synapse, RIKEN BSI, Wako, Japan In the olfactory system, odor information is initially represented as a topographic, chemotopic map in the olfactory bulb (OB). In zebrafish, bile salts (putative social pheromones) activate anterior and dorsal clusters of glomeruli in the OB, whereas amino acids (potent feeding cues) activate lateral cluster of glomeruli. However, it remains largely unknown how the odor map in the OB is transferred to higher olfactory centers. To answer this question, it is essential to analyze in detail the neuroanatomy of individual OB output neurons with special reference to the dendritic innervation of glomeruli and the axonal trajectory to target regions. We found that two different transcriptional promoters of lhx2a and tbx21 genes can drive expression of fluorescent proteins in distinct subpopulation of OB output neurons without labeling of cells in other brain regions. lhx2atg+ neurons predominantly exhibit dendritic innervations to medial cluster of glomeruli (mG), whereas tbx21tg+ neurons preferentially innervate glomeruli outside the mG. Tracing axons at single-cell resolution with a genetic mosaic labeling technique revealed that (1) individual OB output neurons send axons to multiple target regions in the forebrain, including the telencephalon, the habenula and the hypothalamus; (2) OB output neurons innervating distinct glomerular clusters tend to project axons to different, but partly overlapping, target regions. (3) OB output neurons innervating the same glomerulus do not necessarily display the same axon trajectory; (4) OB output neurons innervating the mG send axons to the right habenular nucleus directly and asymmetrically. These results suggest that the topographic odor map in the OB is not maintained intact, but reorganized in higher olfactory centers. This study will pave the way for understanding of the functional logic of odor information coding employed by higher olfactory centers in vertebrate brains. doi:10.1016/j.neures.2010.07.197
Dept Advanced interdisciplinary studies, Univ of Tokyo, Tokyo 2 RCAST, Univ of Tokyo, Tokyo 3 PRESTO, JST, Tokyo
We propose a highly sensitive and real-time odor biosensor by expressing ion channel built-in odor receptors of insects into dissociated cultures of neurons of rats. The ion channel built-in structure of insect odor receptor easily allows functional expression into cells. The neuronal dissociated cultures of rats have 2 significant advantages: a long lifetime comparable to rats, i.e., a few years; and amplification ability from weak ionic currents of odor receptors into easily detectable action potentials of neurons. To demonstrate the feasibility of this proposed sensor, we attempted to express the pheromone receptors of silkmoth, Bombyx mori, into cultured neurons of rats. Two types of pheromone receptors, BmOR1 and BmOR3, show high selectivity to their pheromone substance, Bombykol (BOL) and Bombykal (BAL), respectively. Each pheromone receptor forms heterodimeric complexes with a co-receptor, BmOR2, and this complex perform as non-selective cationic channel. We attempted to co-express BmOR1/BmOR2 or BmOR3/BmOR2 into neuronal cultures by lipofection. As the result, the transfection efficiency was about 10%, which was sufficient for an odor senor. To test the functional consequences of pheromone receptors, the calcium imaging investigates the neural activity of transfected cells in response to the pheromone. Administration of 100 M pheromone substances increased calcium responses up to 10% - 50% within 40 seconds. These results suggest the feasibility of the proposed sensor. doi:10.1016/j.neures.2010.07.199
O2-8-2-1 In vivo connection imaging and its application To monkey inferotemporal face system Noritaka Ichinohe 1,2,3 Manabu Tanifuji 3
, Takayuki Sato 3 , Kathleen Rockland 2 ,
1 Department of Neuroanatomy, Hirosaki University, Graduate School of Medicine 2 Lab. for Cortical Organization and Systematics, Brain Science Institute, RIKEN 3 Lab. for integrative physiology, BSI, RIKEN, Japan
Anatomical tracing techniques have been the firm technique connected brain sites. However, evaluation of connection on postmortem brain makes it unable to compare response property between connected two sites. We have developed a new use for the tracer CTB-Alexa555, for imaging cortical connections in the living macaque monkey brain. On 7-14 days after tracer