Abstract. We have indirectly analyzed the role of tau in generating the highly organized microtubule (MT) array of the axon. Axons contain MT arrays of uniform polarity orientation, plus ends distal to the cell body (Heidemann, S. R., J. M. Landers, and M. A. Hamborg. 1981 . J. Cell Biol. 91 :661-673). Surprisingly, these MTs do not radiate from a single discrete nucleating structure in the cell body (Sharp, G. A., K. Webet, and M. Osborn . 1982 . Eur. J. Cell Biol. 29 : 97-103), but rather stop and start at multiple sites along the length of the axon (Bray, D., and M. B.

We have used an indirect method to compare the dynamic properties of microtubules (MTs) in the main shaft and distal regions of the axon. Individual MTs are staggered along the length of the axon and consist of a labile domain situated at the plus end of a stable domain (Baas and Black, 1990). As a result of this organization and the plus-end-distal orientation of axonal MTs, the most distal region of the axon consists entirely of labile domains, while the main shaft consists of a mixture of labile and stable domains. In this study, we wished to determine whether the labile domains extending into the distal axon differ in their dynamic properties from the labile domains terminating in the main shaft. To address this issue, we used immunoelectron microscopy to compare the tyrosination state of the labile domains terminating in these 2 axon regions. Because detyrosination is a polymerspecific modification of alpha-tubulin that accumulates with time, the levels of tyrosinated alpha-tubulin will be a reflection of the age, and hence dynamic properties, of the polymer. To maximize our chances of visualizing potential differences, we varied the concentration of the primary antibody in these experiments. Our studies indicate that the stable domains are generally deficient in tyrosinated alpha-tubulin, while the labile domains contain clearly detectable levels. Within the labile domain, the subsection closer to the plus end of the MT contains relatively higher levels of tyrosinated alpha-tubulin than does the subsection farther from the plus end, suggesting that the levels of tyrosinated alpha-tubulin in the labile domain may gradually increase as one moves away from the stable domain toward the plus end of the MT. Although these observations apply to the labile domains in both regions of the axon, the labile domains extending into the distal region contain comparatively higher levels of tyrosinated alpha-tubulin than do the labile domains terminating in the main shaft. These results are consistent with the view that highly dynamic MT polymer is present throughout the axon, but that the polymer nearest the advancing growth cone is particularly dynamic.

We have sought to determine the principal site(s) in the neuron where axonal microtubules (Mts) are stabilized. To accomplish this, we compared the proximal and distal regions of the axon and the axon shaft with regard to their content of newly stabilized MT polymer, using the following criteria. Stable polymer was identified by its resistance to nocodazole, and newly stabilized polymer was distinguished from older stable polymer by the staining of the former but not the latter for tyrosinated a-tubulin.Our results indicate that roughly 36.4%, 5.4%, and 2.4% of the total MT mass in the proximal and distal regions of the axon and the axon shaft is newly stabilized, respectively. Thus, while MT stabilization occurs throughout the axon, the proximal region is by far the most active with regard to this process.[Key words: neuron, axon growth, microtubule stability, tyrosinated a-tub&n]The microtubule (MT) array of the axon consists of two types of polymer, stable and labile, that differ in their rates of druginduced depolymerization, solubility properties, and presumably, rates of subunit turnover (Black et al., 1984;Brady et al., 1984;Morris and Lasek, 1984;Ferreira et al., 1989;Baas and Black, 1990; Baas et al., 1991;Baas and Ahmad, 1992). The proportion of stable polymer is particularly high in axons compared to non-neuronal cells, and this presumably relates to the unique morphologic constraints inherent in the elongate geometry of the axon. In addition, it is now well established that the stable MT polymer has an essential role in regulating MT dynamics in the axon. The plus ends of stable MTs represent the exclusive sites for MT assembly in the axon, and hence serve as nucleating structures for MT assembly in the axon (Baas and Ahmad, 1992; see also Baas and Black, 1990). MTs are intrinsically labile structures, and stability is conferred upon them subsequent to their assembly (Kirschner and Mitchison, 1986 , 1982;Ferreira et al., 1989;Lim et al., 1989;Arregui et al., 1991). Despite the functional significance of the conversion from labile to stable polymer, a process termed MT stabilization, surprisingly little is known about how or where it occurs.In the present study, we have sought to determine the principal site(s) in the neuron where axonal MTs are stabilized. Different possibilities for where axonal MTs are stabilized have decidedly different implications for the mechanisms by which the entire MT array of the axon is elaborated during axon growth. Some models for axon growth stress that MT assembly and stabilization occur principally behind the advancing growth cone (Bamburg et al., 1986;Mitchison and Kirschner, 1988) while other models stress that MTs are assembled and stabilized principally proximally, and are then translocated distally by an active transport mechanism (Lasek, 1982(Lasek, , 1986(Lasek, , 1988 Reinsch et al., 199 1). Determination of sites of MT stabilization will assist in evaluating the merits of these very different models.In our previous studies on the stability properties of the ...

To understand the roles of various microtubule-associated proteins (MAPs) in the development of axons and dendrites, we have expressed individual neuronal MAPs in normally rounded Sf9 host cells. We previously reported that expression of tau protein in these cells results in the elaboration of long processes containing dense bundles of microtubules (MTs). These bundles generally terminate in the hillock region of the cell body, and almost all of the MTs within the bundles are oriented with their plus ends distal to the cell body. Here we report the expression of a construct that approximates the MAP2C sequence and also induces the elaboration of processes with dense bundles of predominantly plus-end-distal MTs. Whereas tau generally results in a single process, there is a significantly greater tendency for the MAP2C-like construct to induce multiple processes. In contrast to the tau processes, the MT bundle in these processes extends far into the cell body. This latter observation suggests that MAP2C and tau have different effects on MT assembly and/or transport events in the cell. Although both of these MAPs can organize MTs that are competent to participate in process formation, the detailed organization of MTs induced by each of the two constructs is distinctive, and these differences may be relevant to axonal and dendritic differentiation.Microtubules (MTs) are key architectural elements that serve as the substrate for bidirectional transport of cytoplasmic organelles. In the axon, MTs are tightly packed and are all oriented with their plus ends distal to the cell body (1, 2), whereas dendritic MTs are spaced farther apart and are oriented in both directions (2,3

Application of a cationic surfactant, benzalkonium chloride, to the serosa of rat jejunum results in an increase in thickness of both longitudinal and circular smooth muscle layers. The increase in thickness is due primarily to an increase in the number of smooth muscle cells (hyperplasia). Little cellular hypertrophy was observed. The time sequence of surfactant-induced effects on the muscle layers was determined. Within 24 h, total destruction of the longitudinal muscle and partial destruction of the circular muscle was evident. The myenteric plexus was also necrotic; however, the submucosal plexus remained intact. By 48 h after surfactant treatment, the smooth muscle cells remaining in the circular muscle layer had begun to divide, as indicated by the presence of mitotic figures and incorporation of 3H-thymidine. A repopulation of the longitudinal muscle layer began at this time, apparently the result of migration of cells arising in the circular muscle layer. By 5 days post-treatment, both muscle layers had regenerated to their original states. The myenteric plexus was totally absent. The denervated smooth muscle cells proceeded to divide until approximately day 15, resulting in hyperplasia of both muscle layers. Between 15 and 105 days, the number of muscle cells in the circular layer progressively declined, eventually returned to the value seen in control tissue. In contrast, the number of smooth muscle cells in the longitudinal layer remained elevated through the period of study (165 days). We hypothesize that the smooth muscle hyperplasia observed after serosal benzalkonium chloride application results from loss of the myenteric nerves.

The histaminergic innervation of the thalamic dorsal and ventral lateral geniculate nuclei and the perigeniculate nucleus of the cat was examined immunohistochemically by means of an antibody to histamine.We find histamine-immunoreactive neurons in the cat brain are concentrated in the ventrolateral portion of the posterior hypothalamus, confirming a previous report. However, this cell group also spreads into medial, dorsal, and extreme lateral regions of the posterior hypothalamus and extends as far rostral as the optic chiasm.Histamine-labeled fibers cover all regions of the lateral geniculate complex, but the density of labeling varies. The ventral lateral geniculate nucleus (vLGN) is most densely labeled, the A laminae of the dorsal lateral geniculate are sparsely labeled, and the geniculate C laminae and the perigeniculate nucleus show intermediate amounts of label. Thus, histaminergic fibers demonstrate a predilection for zones innervated by the W-cell system. Labeled fibers exhibit few branchings and numerousen passantswellings, lending a beaded appearance. The vLGN showed more instances of fibers with larger-sized swellings (up to 2 μm).Following injections of biotinylated tracers into the hypothalamus, we find labeled fibers throughout the lateral geniculate complex. The anterogradely labeled fibers resemblethe histaminergic fibers in morphology, distribution, and relative bouton size. Thus, the hypothalamus appears to be the source of the histaminergic fibers in the lateral geniculate complex.Histamine-labeled fibers in the dorsal lateral geniculate nucleus (dLGN) exhibit uncommon ultrastructural morphology. Many extremely large, round, or elliptical vesicles fill the fiber swellings. Swellings are directly apposed to a variety of other dendritic and axonal profiles, but thus far no convincing synaptic contacts have been seen. The distribution and appearance of these histaminergic fibers resembles those reported for serotonergic fibers.Our results support the idea that histamine works nonsynaptically as a neuromodulator in the lateral geniculate complex, affecting the level of visual arousal.

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The tumorigenic potential of dulaglutide was evaluated in rats and transgenic mice. Rats were injected sc twice weekly for 93 weeks with dulaglutide 0, 0.05, 0.5, 1.5, or 5 mg/kg corresponding to 0, 0.5, 7, 20, and 58 times, respectively, the maximum recommended human dose based on plasma area under the curve. Transgenic mice were dosed sc twice weekly with dulaglutide 0, 0.3, 1, or 3 mg/kg for 26 weeks. Dulaglutide effects were limited to the thyroid C-cells. In rats, diffuse C-cell hyperplasia and adenomas were statistically increased at 0.5 mg/kg or greater (P ≤ .01 at 5 mg/kg), and C-cell carcinomas were numerically increased at 5 mg/kg. Focal C-cell hyperplasia was higher compared with controls in females given 0.5, 1.5, and 5 mg/kg. In transgenic mice, no dulaglutide-related C-cell hyperplasia or neoplasia was observed at any dose; however, minimal cytoplasmic hypertrophy of C cells was observed in all dulaglutide groups. Systemic exposures decreased over time in mice, possibly due to an antidrug antibody response. In a 52-week study designed to quantitate C-cell mass and plasma calcitonin responses, rats received twice-weekly sc injections of dulaglutide 0 or 5 mg/kg. Dulaglutide increased focal C-cell hyperplasia; however, quantitative increases in C-cell mass did not occur. Consistent with the lack of morphometric changes in C-cell mass, dulaglutide did not affect the incidence of diffuse C-cell hyperplasia or basal or calcium-stimulated plasma calcitonin, suggesting that diffuse increases in C-cell mass did not occur during the initial 52 weeks of the rat carcinogenicity study.