Tetsuo Yamamori

Mutually exclusive expression of odorant receptor transgenes.

To study the mutually exclusive expression of odorant receptor (OR) genes, we generated transgenic mice that carried the murine OR gene MOR28. Expression of the transgene and the endogenous MOR28 was distinguished by using two different markers, β-galactosidase and green fluorescent protein (GFP), respectively. Double staining of the olfactory epithelium revealed that the two genes were rarely expressed simultaneously in individual olfactory neurons. A similar exclusion was also observed between differently tagged but identical transgenes integrated into the same locus of one particular chromosome. Although allelic inactivation has been reported for the choice between the maternal and paternal alleles, this is the first demonstration of mutually exclusive activation among non-allelic OR gene members with identical coding and regulatory sequences. Such an unusual mode of gene expression, monoallelic and mutually exclusive, has previously been shown only for the antigen-receptor genes of the immune system.

Differential expression of γ-aminobutyric acid type B receptor-1a and -1b mRNA variants in GABA and non-GABAergic neurons of the rat brain

To understand the heterogeneity of γ‐aminobutyric acid type B receptor (GABABR)‐ mediated events, we investigated expression of GABABR1a and 1b mRNA variants in GABA and non‐GABAergic neurons of the rat central nervous system (CNS), by using nonradioactive in situ hybridization histochemistry and, in combination with GABA immunocytochemistry, double labeling. In situ hybridization with a pan probe, which recognizes a common sequence of both GABABR1a and GABABR1b mRNA variants, demonstrated widespread expression of GABABR1 mRNA at various levels in the CNS. Both GABABR1a and GABABR1b were expressed in the neocortex, hippocampus, dorsal thalamus, habenula, and septum, but only GABABR1a was detected in cerebellar granule cells, in caudate putamen, and most hindbrain structures. A majority of GABA neurons in cerebral cortex showed hybridization signals for both GABABR1a and GABABR1b, whereas those in most subcortical structures expressed either or neither of the two. GABA neurons in thalamic reticular nucleus and caudate putamen hybridized primarily for GABABR1a. Purkinje cells in the cerebellar cortex expressed predominantly GABABR1b. GABA neurons in dorsal lateral geniculate nucleus did not display significant levels of either GABABR1a or GABABR1b mRNAs. These data suggested widespread availability of GABABR‐mediated inhibition in the CNS. The differential but overlapping expression of GABABR1 mRNA variants in different neurons and brain structures may contribute to the heterogeneity of GABABR‐mediated inhibition. Some GABA neurons possessed, but others might lack the molecular machinery for GABABR‐mediated disinhibition, autoinhibition, or both. J. Comp. Neurol. 416:475–495, 2000.

Genetic dissection of the circuit for hand dexterity in primates

It is generally accepted that the direct connection from the motor cortex to spinal motor neurons is responsible for dexterous hand movements in primates. However, the role of the ‘phylogenetically older’ indirect pathways from the motor cortex to motor neurons, mediated by spinal interneurons, remains elusive. Here we used a novel double-infection technique to interrupt the transmission through the propriospinal neurons (PNs), which act as a relay of the indirect pathway in macaque monkeys (Macaca fuscata and Macaca mulatta). The PNs were double infected by injection of a highly efficient retrograde gene-transfer vector into their target area and subsequent injection of adeno-associated viral vector at the location of cell somata. This method enabled reversible expression of green fluorescent protein (GFP)-tagged tetanus neurotoxin, thereby permitting the selective and temporal blockade of the motor cortex–PN–motor neuron pathway. This treatment impaired reach and grasp movements, revealing a critical role for the PN-mediated pathway in the control of hand dexterity. Anti-GFP immunohistochemistry visualized the cell bodies and axonal trajectories of the blocked PNs, which confirmed their anatomical connection to motor neurons. This pathway-selective and reversible technique for blocking neural transmission does not depend on cell-specific promoters or transgenic techniques, and is a new and powerful tool for functional dissection in system-level neuroscience studies.

Serotonin Modulates Fast-Spiking Interneuron and Synchronous Activity in the Rat Prefrontal Cortex through 5-HT1A and 5-HT2A Receptors

Alterations of the serotonergic system in the prefrontal cortex (PFC) are implicated in psychiatric disorders such as schizophrenia and depression. Although abnormal synchronous activity is observed in the PFC of these patients, little is known about the role of serotonin (5-HT) in cortical synchrony. We found that 5-HT, released by electrical stimulation of the dorsal raphe nucleus (DRN) in anesthetized rats, regulates the frequency and the amplitude of slow (<2 Hz) waves in the PFC via 5-HT2A receptors (5-HT2ARs). 5-HT also modulates prefrontal gamma (30–80 Hz) rhythms through both 5-HT1ARs and 5-HT2ARs, but not 5-HT2CRs, inducing an overall decrease in the amplitude of gamma oscillations. Because fast-spiking interneurons (FSi) are involved in the generation of gamma waves, we examined serotonergic modulation of FSi activity in vivo. Most FSi are inhibited by serotonin through 5-HT1ARs, while a minority is activated by 5-HT2ARs, and not 5-HT2CRs. In situ hybridization histochemistry confirmed that distinct populations of FSi in the PFC express 5-HT1ARs and 5-HT2ARs, and that the number of FSi expressing 5-HT2CRs is negligible. We conclude that 5-HT exerts a potent control on slow and gamma oscillations in the PFC. On the one hand, it shapes the frequency and amplitude of slow waves through 5-HT2ARs. On the other hand, it finely tunes the amplitude of gamma oscillations through 5-HT2AR- and 5-HT1AR-expressing FSi, although it primarily downregulates gamma waves via the latter population. These results may provide insight into impaired serotonergic control of network activity in psychiatric illnesses such as schizophrenia and depression.

Comparative analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral cortex.

Here we investigated the transduction characteristics of adeno-associated viral vector (AAV) serotypes 1, 2, 5, 8 and 9 in the marmoset cerebral cortex. Using three constructs that each has hrGFP under ubiquitous (CMV), or neuron-specific (CaMKII and Synapsin I (SynI)) promoters, we investigated (1) the extent of viral spread, (2) cell type tropism, and (3) neuronal transduction efficiency of each serotype. AAV2 was clearly distinct from other serotypes in small spreading and neuronal tropism. We did not observe significant differences in viral spread among other serotypes. Regarding the cell tropism, AAV1, 5, 8 and 9 exhibited mostly glial expression for CMV construct. However, when the CaMKII construct was tested, cortical neurons were efficiently transduced (>∼70% in layer 3) by all serotypes, suggesting that glial expression obscured neuronal expression for CMV construct. For both SynI and CaMKII constructs, we observed generally high-level expression in large pyramidal cells especially in layer 5, as well as in parvalbumin-positive interneurons. The expression from the CaMKII construct was more uniformly observed in excitatory cells compared with SynI construct. Injection of the same viral preparations in mouse and macaque cortex resulted in essentially the same result with some species-specific differences.

Transiently increased colocalization of vesicular glutamate transporters 1 and 2 at single axon terminals during postnatal development of mouse neocortex: a quantitative analysis with correlation coefficient

Vesicular glutamate transporter 1 (VGLUT1) and VGLUT2 show complementary distribution in neocortex; VGLUT1 is expressed mainly in axon terminals of neocortical neurons, whereas VGLUT2 is located chiefly in thalamocortical axon terminals. However, we recently reported a frequent colocalization of VGLUT1 and VGLUT2 at a subset of axon terminals in postnatal developing neocortex. We here quantified the frequency of colocalization between VGLUT1 and VGLUT2 immunoreactivities at single axon terminals by using the correlation coefficient (CC) as an indicator in order to determine the time course and spatial extent of the colocalization during postnatal development of mouse neocortex. The colocalization was more frequent in the primary somatosensory (S1) area than in both the primary visual (V1) and the motor areas; of area S1 cortical layers, colocalization was most evident in layer IV barrels at postnatal day (P) 7 and in adulthood. CC in layer IV showed a peak at P7 in area S1, and at P10 in area V1 though the latter peak was much smaller than the former. These results suggest that thalamocortical axon terminals contained not only VGLUT2 but also VGLUT1, especially at P7–10. Double fluorescence in situ hybridization confirmed coexpression of VGLUT1 and VGLUT2 mRNAs at P7 in the somatosensory thalamic nuclei and later in the thalamic dorsal lateral geniculate nucleus. As VGLUT1 is often used in axon terminals that show synaptic plasticity in adult brain, the present findings suggest that VGLUT1 is used in thalamocortical axons transiently during the postnatal period when plasticity is required.

The occ1 gene is preferentially expressed in the primary visual cortex in an activity‐dependent manner: a pattern of gene expression related to the cytoarchitectonic area in adult macaque neocortex

Marker molecules to visualize specific subsets of neurons are useful for studying the functional organization of the neocortex. One approach to identify such molecular markers is to examine the differences in molecular properties among morphologically and physiologically distinct neuronal cell types. We used differential display to compare mRNA expression in the anatomically and functionally distinct areas of the adult macaque neocortex. We found that a gene, designated occ1, was preferentially transcribed in the posterior region of the neocortex, especially in area 17. Complete sequence analysis revealed that occ1 encodes a macaque homolog of a secretable protein, TSC‐36/follistatin‐related protein (FRP). In situ hybridization histochemistry confirmed the characteristic neocortical expression pattern of occ1 and showed that occ1 transcription is high in layers II, III, IVA and IVC of area 17. In addition, occ1 transcription was observed selectively in cells of the magnocellular layers in the lateral geniculate nucleus (LGN). Dual labeling immunohistochemistry showed that the occ1‐positive neurons in area 17 include both γ‐aminobutyric acid (GABA)‐positive aspiny inhibitory cells and the α‐subunit of type II calcium/calmodulin‐dependent protein kinase (CaMKII α)‐positive spiny excitatory cells. With brief periods of monocular deprivation, the occ1 mRNA level decreased markedly in deprived ocular dominance columns of area 17. From this we conclude that the expression of occ1 mRNA is present in a subset of neurons that are preferentially localized in particular laminae of area 17 and consist of various morphological and physiological neuronal types, and, furthermore, occ1 transcription is subject to visually driven activity‐dependent regulation.

Gene expression patterns in visual cortex during the critical period: Synaptic stabilization and reversal by visual deprivation

The mapping of eye-specific, geniculocortical inputs to primary visual cortex (V1) is highly sensitive to the balance of correlated activity between the two eyes during a restricted postnatal critical period for ocular dominance plasticity. This critical period is likely to have amplified expression of genes and proteins that mediate synaptic plasticity. DNA microarray analysis of transcription in mouse V1 before, during, and after the critical period identified 31 genes that were up-regulated and 22 that were down-regulated during the critical period. The highest-ranked up-regulated gene, cardiac troponin C, codes for a neuronal calcium-binding protein that regulates actin binding and whose expression is activity-dependent and relatively selective for layer-4 star pyramidal neurons. The highest-ranked down-regulated gene, synCAM, also has actin-based function. Actin-binding function, G protein signaling, transcription, and myelination are prominently represented in the critical period transcriptome. Monocular deprivation during the critical period reverses the expression of nearly all critical period genes. The profile of regulated genes suggests that synaptic stability is a principle driver of critical period gene expression and that alteration in visual activity drives homeostatic restoration of stability.

Enriched Expression of Serotonin 1B and 2A Receptor Genes in Macaque Visual Cortex and their Bidirectional Modulatory Effects on Neuronal Responses

To study the molecular mechanism how cortical areas are specialized in adult primates, we searched for area-specific genes in macaque monkeys and found striking enrichment of serotonin (5-hydroxytryptamine, 5-HT) 1B receptor mRNA, and to a lesser extent, of 5-HT2A receptor mRNA, in the primary visual area (V1). In situ hybridization analyses revealed that both mRNA species were highly concentrated in the geniculorecipient layers IVA and IVC, where they were coexpressed in the same neurons. Monocular inactivation by tetrodotoxin injection resulted in a strong and rapid (<3 h) downregulation of these mRNAs, suggesting the retinal activity dependency of their expression. Consistent with the high expression level in V1, clear modulatory effects of 5-HT1B and 5-HT2A receptor agonists on the responses of V1 neurons were observed in in vivo electrophysiological experiments. The modulatory effect of the 5-HT1B agonist was dependent on the firing rate of the recorded neurons: The effect tended to be facilitative for neurons with a high firing rate, and suppressive for those with a low firing rate. The 5-HT2A agonist showed opposite effects. These results suggest that this serotonergic system controls the visual response in V1 for optimization of information processing toward the incoming visual inputs.

Dichromatism in macaque monkeys.

Old World primates have trichromatic vision because they have three types of cone photoreceptor, each of which is maximally sensitive to short, middle or long wavelengths of light1. Although a proportion of human males (about 8% of caucasians, for example) have X-chromosome-linked colour-vision abnormalities2, no non-human Old World primates have been found to be colour-vision defective3,4. We have tested 3,153 macaque monkeys but found only three dichromats, a frequency that is much lower than in humans.Habitat loss and fragmentation remain the greatest threats to the worlds biodiversity. The local extinction of plant species from habitat fragments is common, although the reasons for this are not fully understood. Fragmentation is known to influence both birth- and death-related processes, but the disruption of plant reproduction, especially pollination and seed production, is thought to be particularly important. The effects of fragmentation on post-pollination processes such as seed dispersal and germination have rarely been explored experimentally. Here I show that seeds planted in forest fragments are less likely to germinate than those in continuous forest. This finding can have negative demographic consequences because it reduces the emergence of seedlings.