Yoshiaki Kita

Development of Cerebellar Neurons and Glias Revealed by in Utero Electroporation : Golgi-Like Labeling of Cerebellar Neurons and Glias

Cerebellar cortical functions rely on precisely arranged cytoarchitectures composed of several distinct types of neurons and glias. Studies have indicated that cerebellar excitatory and inhibitory neurons have distinct spatial origins, the upper rhombic lip (uRL) and ventricular zone (VZ), respectively, and that different types of neurons have different birthdates. However, the spatiotemporal relationship between uRL/VZ progenitors and their final phenotype remains poorly understood due to technical limitations. To address this issue, we performed in utero electroporation (IUE) of fluorescent protein plasmids using mouse embryos to label uRL/VZ progenitors at specific developmental stages, and observed labeled cells at maturity. To overcome any potential dilution of the plasmids caused by progenitor division, we also utilized constructs that enable permanent labeling of cells. Cerebellar neurons and glias were labeled in a Golgi-like manner enabling ready identification of labeled cells. Five types of cerebellar neurons, namely Purkinje, Golgi, Lugaro and unipolar brush cells, large-diameter deep nuclei (DN) neurons, and DN astrocytes were labeled by conventional plasmids, whereas plasmids that enable permanent labeling additionally labeled stellate, basket, and granule cells as well as three types of glias. IUE allows us to label uRL/VZ progenitors at different developmental stages. We found that the five types of neurons and DN astrocytes were labeled in an IUE stage-dependent manner, while stellate, basket, granule cells and three types of glias were labeled regardless of the IUE stage. Thus, the results indicate the IUE is an efficient method to track the development of cerebellar cells from uRL/VZ progenitors facing the ventricular lumen. They also indicate that while the generation of the five types of neurons by uRL/VZ progenitors is regulated in a time-dependent manner, the progenitor pool retains multipotency throughout embryonic development.

Plasma hepatocyte growth factor levels are increased in systemic inflammatory response syndrome

Interleukin-1 (IL-1), a cytokine released from macrophages by endotoxin stimulation, has been shown to upregulate the genetic expression of the hepatocyte growth factor (HGF). The present study was conducted to determine whether plasma HGF is increased in patients with systemic inflammatory response syndrome (SIRS). The plasma levels of HGF, endotoxin, and beta-glucan were measured in 41 surgical patients without hepatic diseases, 18 of whom had been diagnosed with sepsis, and 33, with nonseptic SIRS. The plasma HGF was found to be significantly increased in the 18 patients with sepsis, at 0.69±0.47 ng/ml (mean ± SD), and in the 23 patients with nonseptic SIRS, at 0.49±0.37 ng/ml, compared to values in 40 normal controls, at 0.10±0.03 ng/ml (P<0.001). No significant correlations were observed between the plasma levels of HGF and endotoxin (r=0.02) or beta-glucan (r=−0.05) in any of the patients; however, plasma HGF was significantly correlated with the WBC count (r=0.34, P<0.05) and with total bilirubin (r=0.45, P<0.01). Plasma HGF was also strongly correlated with alanine transaminase (ALT) in 8 patients with ALT levels higher than 50 U/I (r=0.70), but there was no such correlation in 33 patients with ALT levels of 50 U/I or less (r=0.30). Thus, although the clinicopathologic significance of HGF is not well understood, the present findings indicate that plasma HGF increases in response to infection or inflammation.

Pringle maneuver during hepatic resection induces inflammatory cytokines.

To The Editor: The Pringle maneuver (1) and hemihepatic vascular occlusion (2) allow consistent reduction of intraoperative bleeding during hepatic resection. Understanding of tolerance of the normal and cirrhotic liver to normothermic ischemia has increased substantially in recent years (2-4). However, ischemia and reperfusion associated with procedures such as the Pringle maneuver still may be responsible for potentially hazardous hepatic injury. In an animal model, hepatic ischemia and reperfusion result in release of hepaticaily derived tumor necrosis factor-c~ (TNF-~) (5) and interleukin-8 (IL-8) (6), which mediates neutrophildependent microvascular injury. To investigate whether hepatic ischemia induces the generation of such cytokines in humans, we measured the perioperative plasma concentrations of inflammatory cytokines (IL-6, TNF-c~, and IL-8) in 13 hepatectomized patients with or without the Pringle maneuver. Patients who underwent hepatectomy with the Pringle maneuver from April 1995 to June 1995 in our hospital were classified as group A. Their total warm ischemia time was 48 _ 17 min (mean _ SD). Diseases were hepatocellular carcinoma (HCC) in two, bile duct carcinoma (BDC) in one, cholangiocellular carcinoma in one, and metastatic liver carcinoma in two. Diagnoses for group B (without the Pringle maneuver) were HCC in three, BDC in two, metastatic liver carcinoma in one, and focal fatty liver in one. Plasma levels of IL-6, IL-8, and TNF-c~ were determined by sandwich ELISA preoperatively, immediately after hepatectomy, 3 hr after hepatectomy, and on postoperative days 1 and 2. Serum levels of AST, ALT, and total bilirubin were also measured before and after hepatectomy (postoperative days 1, 2, 3, 5, and 7). No significant difference was found in age, intraoperative bleeding, operation time, and weight of the resected liver between these two groups. Plasma TNF-c~ was not detected in any samples. In group A, plasma levels of both IL-6 and IL-8 were significantly elevated immediately after and 3 hr after hepatectomy compared with those in group B (unpaired Students t test; Figure 1). Meanwhile, no significant difference was observed in serum ALT, AST, and total bilirubin levels between the two A m E

Specific labeling of climbing fibers shows early synaptic interactions with immature Purkinje cells in the prenatal cerebellum

During development, growing axons must locate target cells to form synapses. This is not easy, since target cells are also growing and even actively migrating. In some brain regions, such axons have been reported to wait for the timing when target cells become mature, without invading their target region. However, in the cerebellum climbing fibers (CFs), major afferent axons, arrive near their target neurons, Purkinje cells, when the neurons are still actively migrating. We, therefore, examined whether synaptic contacts are established at such early stages. To specifically label CFs, we introduced by in utero electroporation a mixture of genes encoding for Ptf1a‐enhancer‐driven Cre recombinase and Cre‐dependent fluorescent protein into the mouse hindbrain at embryonic day (E) 10.5 and observed them during development. The earliest stages at which labeled CFs were observed in the cerebellar primordium were E15.5–E16.5. These fibers were fasciculated in the dorsal region and entered the cerebellar primordium. Some fibers defasciculated and reached the caudal region. At E17.5 and E18.5, fasciculated fibers were also found in the mantle region, and some grew toward the surface of the primordium to penetrate a mass of Purkinje cells. Interestingly, as early as E16.5, labeled fibers were found to run in close apposition to Purkinje cell dendrites and to express a presynaptic marker. These observations suggest that CFs form synapses with Purkinje cells as soon as the fibers enter the cerebellum.

In vivo imaging of cortical interneurons migrating in the intermediate/subventricular zones.

We developed an imaging system that enables migrating cortical interneurons (CIs) through the lower intermediate zone/subventricular zone (IZ/SVZ) in mouse embryos. CIs were labeled by in utero electroporation performed at embryonic day (E) 11.5 and were observed, through the skull of living embryos, detached from the dam with the umbilical cord remain attached. To identify imaged cell locations, we used GAD67-GFP mice and GFP fluorescence was photo-bleached after the recording. We found that CIs in the IZ/SVZ migrated medially straight toward the midline on the tangential plane, while those in the marginal zone migrated in all directions.

Spatio-temporal origin of cerebellar neurons and glias revealed by in utero electroporation

Neuronal clusters called nuclei play essential roles in information processing in the CNS. Thus, understanding the mechanisms of nucleogenesis is of crucial importance. Previously, we found that pontine neurons (PNs) in early stages of nucleogenesis show pausing behavior and a transition of migration from tangential to radial at the region where nuclei are to be formed, suggesting that a key mechanism for nucleogenesis is present in this region. Here we examined morphological changes of PNs during the entire course of nucleogenesis. To analyze the morphology of individually distinguishable PNs, we co-electroporated cre and gfp gene flanked by loxp sites into the lower rhombic lip of mouse embryos. We found that PNs arriving at the future nuclear region extended many fine processes, while those on their way towards this region did not. The morphology of PNs in the nuclear region became more complex with increasing number of branches as development proceeded. These findings imply that both intrinsic and extrinsic mechanisms operate for the maturation of PNs associated with nucleogenesis.