Immunocytochemical localization of tubulin and microtubule-associated protein 2 during the development of hippocampal neurons in culture

Abstract

In dissociated-cell cultures prepared from the embryonic rat hippocampus, neurons establish both axons and dendrites, which differ in geometry, in ultrastructure, and in synaptic polarity. We have used immunocytochemistry with monoclonal antibodies to study the regional distribution of beta-tubulin and micro-tubule-associated protein 2 (MAP2) in hippocampal cultures and their localization during early stages of axonal and dendritic development. After development for a week or more in culture, when axons and dendrites were well-differentiated, the distribution of these two proteins was quite different. Beta-tubulin was present throughout the nerve cell, in soma, dendrites, and axon. It was also present in all classes of non-neuronal cells, astrocytes, fibroblasts, and a presumptive glial progenitor cell. In contrast, MAP2 was preferentially localized to nerve cells; within neurons, MAP2 was present in soma and dendrites, but little or no immunostaining was detectable in axons. Both beta-tubulin and MAP2 were present in nerve cells at the time of plating. From the earliest stages of process extension, beta-tubulin was present in all neuronal processes, both axons and dendrites. Surprisingly, MAP2 was also initially present in both axons and dendrites, extending as far as the axonal growth cone. With subsequent development, MAP2 staining was selectively lost from the axon so that after 1 week in vitro little or no axonal staining remained. Taken together with earlier results (Cáceres et al., 1984a), these data indicate that the establishment of neuronal polarity, as manifested by the molecular differentiation of the axonal and dendritic cytoskeleton, occurs largely under endogenous control, even under culture conditions in which cell interactions are greatly restricted.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the Role of MAP1B in Axon Formation

“…reflects a lower degree of maturation (Cáceres et al, 1986) rather than a compensatory mechanism. To distinguish between these possibilities, a comparison of MAP2 immunofluorescence was performed in WT and MAP1B-deficient neurons having axons of equivalent length.…”

“…Homozygous MAP1B-deficient embryos were distinguished from controls by their abnormal limb posture (Gonzalez-Billault et al, 2000). Dissociated cultures of hippocampal pyramidal cells from embryonic mice brain tissue were then prepared as described previously (Cáceres et al, 1986;Paglini et al, 1998). Cultures were performed with the hippocampi of E18-19 embryos because at this time point hippocampal pyramidal neurons that develop in situ are beginning to extend neurites.…”

NGF induces neonatal rat sensory neurons to extend dendrites in culture after removal of satellite cells

“…In the same solution, the primary antibodies were added whereas tau proteins are predominantly distributed in axons (HM-2, 1:5000; R4, l:lO,OOO; AP-20, 1:500; SM131, 1:20,000) for 1 hr at room temperature. After rinsing, the secondary antibodies (goat anti- (Kosik and Finch, 1987 (Carceres et al, 1986;Kosik and Finch, 1987). In contrast, nodose neurons had no apparent staining in their axons at and thereby prevent dendrite formation on these neurons.…”

Microtubule Organization and Microtubule-Associated Proteins (MAPs)

“…In contrast, HMWMAP2 is selectively localized in the cell body and dendrites of neurons (Bernhardt and Matus 1982;Caceres et al 1984Caceres et al , 1986Chung et al 1996;Lev and White 1997;Scheetz and Dubin 1994;Shafit-Zagardo and Kalcheva 1998) (Fig. 3.1).…”