Preparation and Readout of Multielectron High-Spin States in a Gate-Defined GaAs/AlGaAs Quantum Dot.

We report the preparation and readout of multielectron high-spin states, a three-electron quartet, and a four-electron quintet, in a gate-defined GaAs/AlGaAs single quantum dot using spin filtering by quantum Hall edge states coupled to the dot. The readout scheme consists of mapping from multielectron to two-electron spin states and a subsequent two-electron spin readout, thus obviating the need to resolve dense multielectron energy levels. Using this technique, we measure the relaxations of the high-spin states and find them to be an order of magnitude faster than those of low-spin states. Numerical calculations of spin relaxation rates using the exact diagonalization method agree with the experiment. The technique developed here offers a new tool for the study and application of high-spin states in quantum dots.

Single-Shot Ternary Readout of Two-Electron Spin States in a Quantum Dot Using Spin Filtering by Quantum Hall Edge States.

We report on the single-shot readout of three two-electron spin states-a singlet and two triplet substates-whose z components of spin angular momentum are 0 and +1, in a gate-defined GaAs single quantum dot. The three spin states are distinguished by detecting spin-dependent tunnel rates that arise from two mechanisms: spin filtering by spin-resolved edge states and spin-orbital correlation with orbital-dependent tunneling. The three states form one ground state and two excited states, and we observe the spin relaxation dynamics among the three spin states.

Nondestructive real-time measurement of charge and spin dynamics of photoelectrons in a double quantum dot.

We demonstrate one and two photoelectron trapping and the subsequent dynamics associated with interdot transfer in double quantum dots over a time scale much shorter than the typical spin lifetime. Identification of photoelectron trapping is achieved via resonant interdot tunneling of the photoelectrons in the excited states. The interdot transfer enables detection of single photoelectrons in a nondestructive manner. When two photoelectrons are trapped at almost the same time we observed that the interdot resonant tunneling is strongly affected by the Coulomb interaction between the electrons. Finally the influence of the two-electron singlet-triplet state hybridization has been detected using the interdot tunneling of a photoelectron.

Single-shot detection of electrons generated by individual photons in a tunable lateral quantum dot.

We demonstrate single-shot detection of single electrons generated by single photons using an electrically tunable quantum dot and a quantum point contact charge detector. By tuning the quantum dot in a Coulomb blockade before the photoexcitation, we observe the trapping and subsequent resetting of single photogenerated electrons. The photogenerated electrons can be stored in the dot for a tunable time range from shorter to longer than the spin-flip time T1. We combine this trap-reset technique with spin-dependent tunneling under magnetic fields to observe the spin-dependent photon detection within the T1.

Accelerated Nerve Regeneration in Mice by upregulated expression of interleukin (IL) 6 and IL-6 receptor after trauma.

In this study we aimed to examine a role for interleukin 6 (IL-6) and its receptor (IL-6R) in peripheral nerve regeneration in vivo. We first observed that cultured mouse embryonic dorsal root ganglia exhibited dramatic neurite extension by simultaneous addition of IL-6 and soluble IL-6R (sIL-6R), a complex that is known to interact with and activate a signal transducing receptor component, gp130. After injury in the hypoglossal nerve in adult mice by ligation, immunoreactivity to IL-6 was upregulated in Schwann cells at the lesional site as well as in the cell bodies of hypoglossal neurons in the brain stem. In the latter, upregulation of the immunoreactivity to IL-6R was also observed. Regeneration of axotomized hypoglossal nerve in vivo was significantly retarded by the administration of anti-IL-6R antibody. Surprisingly, accelerated regeneration of the axotomized nerve was achieved in transgenic mice constitutively expressing both IL-6 and IL-6R, as compared with nontransgenic controls. These results suggest that the IL-6 signal may play an important role in nerve regeneration after trauma in vivo.

Localization and ontogeny of damage-induced neuronal endopeptidase mRNA-expressing neurons in the rat nervous system.

Neuropeptides are crucial mediators in nervous and endocrine systems. Processing and degradation, the major regulatory mechanisms, of enzymes are essential for the control of these peptidergic intercellular signaling systems. Damage-induced neuronal endopeptidase (or endothelin converting enzyme-like1), a member of the neprilysin family, has recently been identified as an M13 zinc metalloprotease. Damage-induced neuronal endopeptidase mRNA expression is strikingly restricted to neurons, and is remarkably induced in response to various types of neuronal injuries, although its function and substrate remain unknown. To clarify the role of damage-induced neuronal endopeptidase, we examined the localization and ontogeny of damage-induced neuronal endopeptidase mRNA expression in the rat nervous system using in situ hybridization. Damage-induced neuronal endopeptidase mRNA was detected at embryonic day 12, and its expression restricted to the ventral region of the neural tube. Subsequently, expression was also apparent in primordia of the striatum, hypothalamus, and cranial motor nuclei during neural development. This specific distribution was relatively maintained in the adult brain, although expression levels became weaker. Expression of damage-induced neuronal endopeptidase was absent in the cerebral cortex, hippocampus, and cerebellum. In addition to prominent expression in CNS, intestinal and sensory ganglia and retina demonstrated transient intense damage-induced neuronal endopeptidase mRNA expression during the embryonic period that then declined, and disappeared after birth. The results indicated that damage-induced neuronal endopeptidase might play an important role in embryonic neural development, in particular in peripheral ganglia derived from the neural crest, and in some neurons originating from the basal plate such as the hypothalamus and cranial motor neurons.

Nerve injury induces the expression of EXT2, a glycosyltransferase required for heparan sulfate synthesis.

Heparan sulfate proteoglycans, which bear long chains of heparan sulfate glycosaminoglycan, play significant roles during embryogenesis, including the formation of the CNS. However, their involvement in nerve regeneration has not yet been clarified. Here, we found that the mRNA expression of EXT2, one of the crucial enzymes for heparan sulfate-glycosaminoglycan synthesis, was markedly up-regulated in injured hypoglossal motor neurons after axotomy. In addition, immunohistochemical staining with an antibody specific for heparan sulfate-glycosaminoglycan chains demonstrated increased expression of heparan sulfate-glycosaminoglycan chains in the injured nucleus. Furthermore, the mRNA expressions of glypican-1 and syndecan-1, which are both well-known heparan sulfate proteoglycans, were prominently up-regulated in injured motor neurons. These results suggest that the biosynthesis of heparan sulfate chains promoted by EXT2 is activated in injured motor neurons, and that glypican-1 and syndecan-1 are potent candidates for heparan sulfate proteoglycans involved in peripheral nerve regeneration.

Increase of transcription factor EB (TFEB) and lysosomes in rat DRG neurons and their transportation to the central nerve terminal in dorsal horn after nerve injury.

In the spinal dorsal horn (DH), nerve injury activates microglia and induces neuropathic pain. Several studies clarified an involvement of adenosine triphosphate (ATP) in the microglial activation. However, the origin of ATP together with the release mechanism is unclear. Recent in vitro study revealed that an ATP marker, quinacrine, in lysosomes was released from neurite terminal of dorsal root ganglion (DRG) neurons to extracellular space via lysosomal exocytosis. Here, we demonstrate a possibility that the lysosomal ingredient including ATP released from DRG neurons by lysosomal-exocytosis is an additional source of the glial activation in DH after nerve injury. After rat L5 spinal nerve ligation (SNL), mRNA for transcription factor EB (TFEB), a transcription factor controlling lysosomal activation and exocytosis, was induced in the DRG. Simultaneously both lysosomal protein, LAMP1- and vesicular nuclear transporter (VNUT)-positive vesicles were increased in L5 DRG neurons and ipsilateral DH. The quinacrine staining in DH was increased and co-localized with LAMP1 immunoreactivity after nerve injury. In DH, LAMP1-positive vesicles were also co-localized with a peripheral nerve marker, Isolectin B4 (IB4) lectin. Injection of the adenovirus encoding mCherry-LAMP1 into DRG showed that mCherry-positive lysosomes are transported to the central nerve terminal in DH. These findings suggest that activation of lysosome synthesis including ATP packaging in DRG, the central transportation of the lysosome, and subsequent its exocytosis from the central nerve terminal of DRG neurons in response to nerve injury could be a partial mechanism for activation of microglia in DH. This lysosome-mediated microglia activation mechanism may provide another clue to control nociception and pain.

Requirement of Ras for the activation of mitogen-activated protein kinase by calcium influx, cAMP, and neurotrophin in hippocampal neurons.

Mitogen-activated protein (MAP) kinase plays important roles in the establishment of long-term potentiation both in vitro and in living animals. MAP kinase is activated in response to a broad range of stimuli, including calcium influx through NMDA receptor and L-type calcium channel, cAMP, and neurotrophins. To investigate the role of Ras in the activation of MAP kinase and cAMP response element-binding protein (CREB) in hippocampal neurons, we inhibited Ras function by overexpressing a Ras GTPase-activating protein, Gap1(m), or dominant negative Ras by means of adenovirus vectors. Gap1(m) expression almost completely suppressed MAP kinase activation in response to NMDA, calcium ionophore, membrane depolarization, forskolin, and brain-derived neurotrophic factor (BDNF). Dominant negative Ras also showed similar effects. On the other hand, Rap1GAP did not significantly inhibit the forskolin-induced activation of MAP kinase. In contrast to MAP kinase activation, the inactivation of Ras activity did not inhibit significantly NMDA-induced CREB phosphorylation, whereas BDNF-induced CREB phosphorylation was inhibited almost completely. These results demonstrate that Ras transduces signals elicited by a broad range of stimuli to MAP kinase in hippocampal neurons and further suggest that CREB phosphorylation depends on multiple pathways.

The small GTP-binding protein TC10 promotes nerve elongation in neuronal cells, and its expression is induced during nerve regeneration in rats.

We have made a rat cDNA library using nerve-transected hypoglossal nuclei. Using this library, we performed expressed-sequence tag analysis coupled with in situ hybridization to identify genes whose expression is altered in response to nerve injury. In this gene screening, a member of Rho family GTPases, TC10, which had not yet been characterized in neuronal cells, was identified. TC10 mRNA expression was very low in normal motor neurons; however, axotomy induced its expression dramatically. Other family members such as RhoA, Rac1, and Cdc42 were moderately expressed in normal motor neurons and showed slight upregulation after axotomy. The expression level of TC10 mRNA was low in the embryonic brain and gradually increased with development. However, the expression of TC10 mRNA in the adult brain was lower and more restricted than that of RhoA, Rac1, and Cdc42. Cultured dorsal root ganglia exhibited dramatic neurite extension secondary to adenovirus-mediated expression of TC10. It can be concluded that although TC10 expression is lower in developing and mature motor neurons compared with other Rho family members, TC10 expression is induced by nerve injury to play a crucial role in nerve regeneration, particularly neurite elongation, in cooperation with other family members.

Akt/protein kinase B prevents injury-induced motoneuron death and accelerates axonal regeneration.

Motoneurons require neurotrophic factors for their survival and axonal projection during development, as well as nerve regeneration. By using the axotomy-induced neuronal death paradigm and adenovirus-mediated gene transfer, we attempted to gain insight into the functional significances of major growth factor receptor downstream cascades, Ras-extracellular signal-regulated kinase (Ras-ERK) pathway and phosphatidylinositol-3 kinase-Akt (PI3K-Akt) pathway. After neonatal hypoglossal nerve transection, the constitutively active Akt-overexpressing neurons could survive as well as those overexpressing Bcl-2, whereas the constitutively active ERK kinase (MEK)-overexpressing ones failed to survive. A dominant negative Akt experiment demonstrated that inhibition of Akt pathway hastened axotomy-induced neuronal death in the neonate. In addition, the dominant active Akt-overexpressing adult hypoglossal neurons showed accelerated axonal regeneration after axotomy. These results suggest that Akt plays dual roles in motoneuronal survival and nerve regeneration in vivo and that PI3K-Akt pathway is probably more vital in neuronal survival after injury than Ras-ERK pathway.

Mutant loricrin is not crosslinked into the cornified cell envelope but is translocated into the nucleus in loricrin keratoderma.

Loricrin is a major constituent of the epidermal cornified cell envelope. We have recently identified heterozygous loricrin gene mutations in two dominantly inherited skin diseases, the ichthyotic variant of Vohwinkel syndrome and progressive symmetric erythrokeratoderma, collectively termed loricrin keratoderma. In order to see whether the mutant loricrin molecules predicted by DNA sequencing are expressed in vivo and to define their pathologic effects, we raised antibodies against synthetic peptides corresponding to the mutated sequences of loricrin. Immunoblotting of horny cell extracts from loricrin keratoderma patients showed specific bands for mutant loricrin. Immunohistochemistry of loricrin keratoderma skin biopsies showed positive immunoreactivity to the mutant loricrin antibodies in the nuclei of differentiated epidermal keratinocytes. The immunostaining was localized to the nucleoli of the lower granular cell layer. As keratinocyte differentiation progressed the immunoreactivity moved gradually into the nucleoplasm leaving nucleoli mostly nonimmunoreactive. No substantial staining was observed along the cornified cell envelope. This study confirmed that mutant loricrin was expressed in the loricrin keratoderma skin. Mutant loricrin, as a dominant negative disrupter, is not likely to affect cornified cell envelope crosslinking directly, but seems to interfere with nuclear/nucleolar functions of differentiating keratinocytes. In addition, detection of the mutant loricrin in scraped horny layer could provide a simple noninvasive screening test for loricrin keratoderma. J Invest Dermatol 115:1088-1094 2000

Localization of oxytocin receptor messenger ribonucleic acid in the rat brain.

The expression of oxytocin receptor (OT-R) mRNA in the rat central nervous system was examined by in situ hybridization histochemistry using cRNA probe. Wide distribution of cells expressing OT-R mRNA was observed not only in the hypothalamus, but also in other regions. There were high levels of OT-R mRNA in the anterior olfactory nuclei, tenia tecta, olfactory tubercle, rostral most region of the frontal cortex, piriform cortex, layers 2 and 3 of the neocortex, bed nucleus of the stria terminalis, anterior medial preoptic nucleus (AV3V region), magnocellular preoptic nucleus, supraoptic nucleus, paraventricular hypothalamic nucleus, retrochiasmatic nucleus, ventromedial hypothalamic nucleus, paraventricular thalamic nucleus, central amygdaloid nucleus, medial amygdaloid nucleus, posterior cortical amygdaloid nucleus, amygdalohippocampal area, subiculum, prepositus hypoglossal nucleus, and dorsal motor nucleus of vagus. In most regions of the brain, our findings concurred with those obtained by receptor binding autoradiography using a ligand specific to OT. However, in the inferior olive nucleus, OT-R mRNA was not detected despite an abundance of binding sites showed by receptor binding autography. Despite this discrepancy OT appears to have central nervous system functions in addition to its hormonal functions.

Endotoxin activates a chemokinergic neuronal pathway in the hypothalamo-pituitary system.

Cytokines play a critical role in the cascade of events that cause septic shok. One regulatory system suggested to be important in controlling inflammatory response is the neuroendocrine axis. One of the chemokines is cytokine induced neutrophil chemoattractant (CINC), which was first described as an immuno-modulator of peripheral tissue in inflammatory responses. To assess further the contribution of the chemokine to the central nervous system, we performed immunohistochemistry on rat brains and found strong CINC-like immunoreactivity in the posterior pituitary gland. Treatment with bacterial endotoxin (lipopolysaccharide; LPS) markedly enhanced CINC-like immunoreactivity in the posterior pituitary. Before the LPS challenge, signal for CINC mRNA was undetectable in the paraventricular hypothalamic nucleus (PVN). The LPS challenge induced strong hybridization signals of CINC mRNA in the parvocellular and magnocellular subdivision of the PVN within 15 minutes (min) and peaked at 30 min. The LPS challenge provoked no observable change in the supraoptic nucleus. These studies demonstrate the presence of an endotoxin-sensitive chemokinergic neuronal pathway in the hypothalamo-neurohypophysial system and this newly-described pathway will provide a novel information to understand another possible neuralimmune mechanism.

Increased F1/GAP-43 mRNA accumulation in gerbil hippocampus after brain ischemia.

To assess whether ischemia could induce GAP-43 mRNA expression, we performed in situ hybridization in gerbil brains that had been subjected to 5 min of global ischemia. In control dentate granule cells, little hybridization was detected in contrast to the intense signal generated by pyramidal neurons of the adult hippocampal formation. After ischemia, we detected a robust GAP-43 signal over hippocampal granule cells at 3 h of reperfusion, persisting through 7 days, and disappearing by 14 days. This demonstrated GAP-43 gene induction after ischemia, and suggests that GAP-43 may be involved in reactive events, including fiber sprouting and synaptic reorganization, that follow ischemia.

Distribution of Tyrosine Hydroxylase mRNA in the Rat Central Nervous System Visualized by Alkaline Phosphatase in situ Hybridization Histochemistry.

The expression of tyrosine hydroxylase mRNA in the rat brain was examined using a novel alkaline phosphatase labelled antisense oligodeoxynucleotide probe. The alkaline phosphatase labelled probe revealed the presence of tyrosine hydroxylase mRNA in all the major cell groups and cell bodies previously described as containing catecholamine fluorescence or known to contain tyrosine hydroxylase immunoreactivity. Using standardized development protocols qualitative comparisons between the amount of mRNA signal in different adrenergic, noradrenergic or dopaminergic cell groups could be made. These studies showed that of the three known catecholaminergic cell types the level of tyrosine hydroxylase mRNA signal was high in the noradrenergic and dopaminergic cells, but much lower in the adrenergic cell groups. The sensitivity of this nonradioactive method of in situ hybridization is excellent and has considerable potential for studies of coexistence or coexpression of two mRNA signals for the localization of mRNA signals at the electron-microscope level.

Postnatal ontogeny of cells expressing prepro-neurotensin/neuromedin N mRNA in the rat forebrain and midbrain: a hybridization histochemical study involving isotope-labeled and enzyme-labeled probes.

The postnatal ontogeny of cells expressing prepro-neurotensin/neuromedin N messenger RNA (prepro-NT/NN mRNA) in the rat forebrain and midbrain was investigated by in situ hybridization histochemistry. According to the pattern of expression during development, the cells which express prepro-NT/NN mRNA can be roughly divided into 2 groups. In type I cells, prepro-NT/NN mRNA expression reaches a maximum in terms of content during the postnatal period. After this early peak, cells of this type express the same or less prepro-NT/NN mRNA, reaching a plateau at an adult level that still contains a high level of expression. In type II cells, prepro-NT/NN mRNA appears during the postnatal period, and the expression decreases dramatically after the first postnatal week, being almost undetectable by a few weeks after birth. Type I cells were observed in the following areas: the piriform cortex, field CA1 of Ammon's horn, subiculum, vertical, and horizontal limbs of the diagonal band of Broca, intermediate part of the lateral septal nucleus, bed nucleus of the stria terminalis, medial preoptic area, lateral hypothalamus, caudal part of the caudate putamen, medial, cortical, and central amygdaloid nuclei, ventral tegmental area, deep mesencephalic nucleus, cuneiform nucleus, dorsal raphe nucleus, laterodorsal tegmental nucleus, parabrachial nucleus, and oral part of the pontine reticular nucleus. Cells of type II were observed in the following areas: the mitral cell layer of the olfactory bulb, rostral part of the caudate putamen, (anterior) cingulate cortex, and retrosplenial cortex (posterior cingulate cortex).

Localization of two calcium binding proteins, calbindin (28 kD) and parvalbumin (12 kD), in the vertebrate retina.

We used immunocytochemistry to locate two calcium binding proteins, calbindin (CaB) and parvalbumin (PV), in the retina of goldfish, frog, chick, rat, guinea pig, dog, and man. The location of CaB depended on the type of dominant photoreceptor cells in birds and mammals. In cone-dominant retinas such as those of the chick, CaB-like immunoreactivity was found in the cones, cone bipolars, and ganglion cells. Amacrine cells 5-12 microns across were also labeled. In rod-dominant retinas, such as those of the rat, guinea pig, and dog, horizontal cells, small amacrine cells (about 6 microns across), and cells in the ganglion cell layer were labeled. In the human retina, which has both cones and rods in abundance, cones, cone bipolars, ganglion cells, horizontal cells, and small and large amacrine cells were labeled. In the frog and goldfish, the level of CaB-like immunoreactivity was low. In the frog, a few cones, amacrine cells, and cells in the ganglion cell layer were labeled. No immunoreactive structures were seen in the goldfish retina. PV-like immunoreactivity was found in chicks, rats, and dogs. No such immunoreactive structures were seen in the other species. In the chick, only amacrine cells were labeled. In the rat, amacrine cells and several displaced amacrine cells were labeled. In the dog, in addition to amacrine cells and displaced amacrine cells, horizontal cells were strongly labeled. Thus, PV-like immunoreactivity was found in those elements relating to the modulation of the main pathway of the visual transmission system.

Postnatal development of preproenkephalin mRNA containing neurons in the rat lower brainstem.

Postnatal developmental changes of preproenkephalin (PPE) gene expression in rat brainstem neurons were studied by in situ hybridization histochemistry. On the basis of PPE mRNA expression, brainstem neurons were categorized into three types: 1) type I neurons were characterized by constant or increasing expression of PPE mRNA during postnatal development; 2) type II neurons started to express PPE mRNA several days after birth and continued to do so thereafter; and 3) type III neurons showed transient expression of PPE mRNA or stopped expressing the mRNA during early postnatal development. Type I PPE neurons were observed in diverse brainstem structures including the mesencephalic and pontine central gray matter, various reticular and raphe nuclei, the ventral tegmental area of Tsai, the interpeduncular nucleus, the nucleus of the brachium of the inferior colliculus, the ventral and dorsal tegmental nuclei of Gudden, the sphenoid nucleus, the laterodorsal tegmental nucleus, Barrington's nucleus, the parabrachial region, the lateral lemniscus and its related nuclei, the trapezoid nucleus, the rostral and ventromedial periolivary nuclei, the mesencephalic trigeminal and principal sensory trigeminal nuclei, the locus coeruleus, the subcoeruleus nucleus, the medial and spinal vestibular nuclei, the dorsal and ventral cochlear nuclei, the medial and lateral cerebellar nuclei, the Roller nucleus, and the intermedius nucleus of the medulla. Type II PPE neurons were found in the superior colliculus, the inferior colliculus, the central part of the dorsal tegmental nucleus, and as Golgi neurons in the granular layer of the cerebellum. Type III PPE neurons were located in the substantia nigra, the red nucleus, the superior olive, the motor trigeminal nucleus, the facial nucleus, the inferior olive, the dorsal motor nucleus of the vagus, and the hypoglossal nucleus. Such region-specific expression of the PPE gene during postnatal ontogeny suggests that rat brainstem PPE neurons may be involved in a variety of developmental events, such as cell proliferation, differentiation, and migration.

Ontogeny of cholecystokinin-8-containing neuron system of the rat: an immunohistochemical analysis. I. Forebrain and upper brainstem.

Ontogeny of the cholecystokinin-8 (CCK) neuron system in the forebrain and upper brainstem of the rat was investigated by means of indirect immunofluorescence. CCK cells and fibers first appeared in the developing ventral tegmental area and in the primordium of the medial forebrain bundle, respectively, at gestational day 15 (12-14-mm embryos). From that time, CCK cells appeared in various areas of the forebrain and upper brainstem until birth and reached the maximum content at postnatal day 10. After postnatal day 10, although CCK cells tended to decrease slightly in number, colchicine treatment in the adult rats brought out numerous CCK cells in the same areas. In contrast, although CCK fibers developed only slightly during the fetal period, marked development was seen after birth, particularly between postnatal days 5 and 10. After that time, as the rats grew, CCK fibers continued to increase in number and formed a meshwork of varying density in various areas of the forebrain and upper brainstem.