Abstract: We have developed and demonstrated the use of a dedicated CMOS device for in vivo functional imaging of the mouse brain. In order to achieve this, a 176times144 pixel array image sensor is designed, fabricated and specially packaged using a novel process. By using on-chip fluorescence imaging configuration, we have successfully imaged deep inside the hippocampus of the mouse brain. Functional imaging is verified by using a fluorogenic substrate that detects the presence of serine protease in the brain. Introduction of kainic acid induces the expression of the serine protease. The protease reacts with the substrate which then fluorescence. By imaging and measuring the fluorescence signal, we have successfully measured the brain protease activity and accurately determined its reaction onset. This method represents a novel approach for neural imaging.

Abstract: Long-term potentiation (LTP) is thought to be particularly important in the acquisition of hippocampus-associated memory, in part because it develops quickly and persists for indefinite periods. Extracellular proteolysis has been hypothesized to contribute to LTP by modifying adhesive relations of synapses and thus the morphology of excitatory synapses. Here we report that neuropsin (NP), an extracellular serine protease, is critically involved in the formation of both the potentiation effect and hippocampus-dependent forms of memory. NP-knockout mice were significantly impaired in the Morris water maze and Y-mazes and failed to exhibit early phase LTP induced by a single tetanus. Potentiation was also impaired or completely blocked by in vivo application of a specific inhibitor or a neutralizing monoclonal antibody for NP. Intriguingly, recombinant (r-) NP alone, without tetanic stimulation, elicited either long-lasting potentiation or depression, depending on the applied dose. The r-NP-elicited potentiation was occluded by prior induction of LTP, while theta-burst-elicited LTP was occluded by application of r-NP alone, suggesting that the two forms of plasticity have a common signalling pathway. r-NP-elicited potentiation and depression increased phosphorylation at different sites on the GluR1 subunit of the AMPA receptor that had previously been associated with LTP or long-term depression. Thus, we conclude that NP is necessary for establishment of LTP and has a significant role in memory acquisition.

Abstract: We report that neuropsin is involved in the synaptogenesis/maturation of orphan and small synaptic boutons in the Schaffer-collateral pathway. Most non-synaptic orphan boutons and a number of immature small synaptic boutons expressed the cell adhesion molecule L1 in presynaptic Schaffer-collateral terminals, whereas mature large boutons on mushroom spines were devoid of L1. The number of L1-immunoreactive boutons was markedly higher in neuropsin-deficient mice than in wild-type mice, whereas there were far fewer mature large boutons. L1-immunoreactive boutons were hypertrophied in the mutant mice. When a recombinant active neuropsin was microinjected into the mutant hippocampus, the number of immunoreactive synaptic boutons reverted to wild-type levels after one day. These results strongly suggest that enzymatically active neuropsin allows a maturational change of L1-immunoreactive small boutons, both orphan and synaptic, and this step may be important in synaptic plasticity based on activity-dependent structural change.

Abstract: The capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output conversion efficiency at the synapses. In the adult mouse hippocampus in vivo, we carefully compared the input-output relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e. conforming to the so-called gamma curve. In extreme cases, long-term potentiation and depression coexist in the same synaptic pathway with long-term potentiation dominating over long-term depression at higher levels of presynaptic activity. These findings predict a novel function of synaptic plasticity, i.e. a contrast-enhancing filtering of neural information through a gamma correction-like process.

Abstract: The aim of the present study is to demonstrate the application of complementary metal-oxide semiconductor (CMOS) imaging technology for studying the mouse brain. By using a dedicated CMOS image sensor, we have successfully imaged and measured brain serine protease activity in vivo, in real-time, and for an extended period of time. We have developed a biofluorescence imaging device by packaging the CMOS image sensor which enabled on-chip imaging configuration. In this configuration, no optics are required whereby an excitation filter is applied onto the sensor to replace the filter cube block found in conventional fluorescence microscopes. The fully packaged device measures 350 microm thick x 2.7 mm wide, consists of an array of 176 x 144 pixels, and is small enough for measurement inside a single hemisphere of the mouse brain, while still providing sufficient imaging resolution. In the experiment, intraperitoneally injected kainic acid induced upregulation of serine protease activity in the brain. These events were captured in real time by imaging and measuring the fluorescence from a fluorogenic substrate that detected this activity. The entire device, which weighs less than 1% of the body weight of the mouse, holds promise for studying freely moving animals.

Abstract: Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 "dots"), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress.

Abstract: Previous immunohistochemical analysis revealed a wide distribution of L1cam-positive neural and nonneural structures in adult mouse brain. Although there were numerous punctate immunoreactive nerve terminals, only a few immunoreactive neuronal cell somata were present (Munakata et al. [2003] BMC Neurosci. 4:7). To explore the distribution of L1cam mRNA-containing cells, which are interpreted to be L1cam-producing cells, we performed in situ hybridization histochemistry with an antisense L1cam cRNA probe. L1cam mRNA was distributed widely from the olfactory bulb to the upper cervical cord with an uneven localization pattern in adult brain. All positive cell somata with silver grains after emulsion autoradiography were neuronal, and no grains were detected on nonneural cells in the present study. A high density of signals for neuronal L1cam mRNA was found in the thalamus, mammillary body, and hippocampus. In addition, strong hybridization signals were localized in various nuclei: main and accessory olfactory bulb, compact part of the substantia nigra, pontine gray matter, tegmental reticular nucleus, Edinger-Westphal nucleus, trigeminal motor nucleus, locus coeruleus, mesencephalic trigeminal nucleus, raphe nuclei, facial nucleus, ambiguus nucleus, dorsal motor vagal nucleus, and inferior olivary nucleus. Some long projection neurons such as the pyramidal, mitral, principal neurons of several cranial nuclei, and presumably monoaminergic cells containing noradrenalin, dopamine, and serotonin, expressed high levels of L1cam.

Abstract: The subthalamic nucleus (STN) is a key component of basal ganglia circuitry that mediates a variety of motor functions. The STN neurons send glutamatergic projections to the output structures of basal ganglia, including the substantia nigra pars reticulata (SNr) and the entopeduncular nucleus, and also innervate the globus pallidus (GP). However, the mechanism by which the STN regulates motor functions in the neural circuitry is not fully understood. Here we performed conditional ablation of the STN neurons by using immunotoxin-mediated cell targeting. We then analyzed dopamine (DA)-mediated motor behavior and firing activity of the SNr and GP neurons. Ablation of the STN neurons increased spontaneous movement and reduced hyperactivity in response to DA stimulation. Ablation of these neurons modulated the pattern and rate of spontaneous firing of the SNr neurons, although it did not substantially affect spontaneous firing of the GP neurons. The ablation attenuated DA-induced suppression of the firing rate of the SNr neurons and inhibited DA-induced elevation of the rate of the GP neurons. In addition, pharmacological blockade of GP activation in response to DA stimulation inhibited the suppression of SNr activity and the resultant motor activation. These results suggest that the STN neurons suppress spontaneous behavior through their direct projection to the output neurons and that, in response to DA, they contribute to expression of behavior by acting on the output neurons mainly through the GP-mediated pathways. We conclude that the STN coordinates motor behavior through differential neural pathways depending on the state of DA transmission.

Abstract: A number of molecules have been postulated to be involved in long-term potentiation, an experimental model for learning and short-term memory. Although the molecular mechanisms of the long-term potentiation have been considerably well understood, it is not yet known why and how real memory can last very long with outstanding stability. A mechanical change of synaptic morphology at acquisition, consolidation and retention of memory is hypothesized to explain long-lasting memory. Changes in the synaptic morphology may be due, at least in part, to local extracellular proteolysis of cell adhesion and extracellular matrix molecules. Some extracellular serine proteases of the Clan PA family may modulate synaptic adhesion and associate with long-term potentiation and learning behavior. In the present review, candidate proteases that are involved in the hippocampal memory are overviewed.

Abstract: BACKGROUND: Neuropsin (KLK8), a serine protease of the kallikrein family, is thought to be involved in the function of keratinocytes, i.e. migration, differentiation and desquamation. However, how neuropsin participates is still unknown. OBJECTIVE: To observe the epidermal function of serine protease in neuropsin-deficient mice. METHODS: We irradiated the skin of neuropsin-deficient mice with ultraviolet light to induce acute inflammation and compared the morphology with that of wild-type mice. RESULTS: We observed a phenotypic change in the epidermis. An acute inflammatory dose of ultraviolet light induced a marked increase in neuropsin mRNA expression in the skin. The signal intensity of the mRNA expression was highest on day 2-3 after irradiation, when keratinocytes were aligned irregularly in the recovery period. Morphological comparison between neuropsin -/- and +/+ mice revealed that an irregular alignment of cells in the thickened epidermis was obvious on day 2 after irradiation in the wild-type mice, whereas it was prolonged for at least 2 days in the neuropsin-deficient mice. The stratum corneum of neuropsin-deficient mice was remarkably thicker than that of the wild-type mice at 5, 14 and 21 days after irradiation. The increase, as a response to this stimulus, in involucrin immunoreactivity, a marker for cell envelope assembly, was delayed in the mutant mice. CONCLUSIONS: Thus, neuropsin might be involved early in the process of differentiation, such as in the assembly of the cell envelope, but not in migration and desquamation.