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Activity-dependent regulation of dendritic BC1 RNA in hippocampal neurons in culture.

Muslimov IA, Banker G, Brosius J, Tiedge H - J. Cell Biol. (1998)

Bottom Line: Inhibition of neuronal activity in hippocampal neurons resulted in a substantial but reversible reduction of somatodendritic BC1 expression.We conclude that expression of BC1 RNA in somatic and dendritic domains of hippocampal neurons is regulated in development, and is dependent upon neuronal activity.These results establish (for the first time to our knowledge) that an RNA polymerase III transcript can be subject to control through physiological activity in nerve cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York 11203, USA.

ABSTRACT
Several neuronal RNAs have been identified in dendrites, and it has been suggested that the dendritic location of these RNAs may be relevant to the spatiotemporal regulation of mosaic postsynaptic protein repertoires through transsynaptic activity. Such regulation would require that dendritic RNAs themselves, or at least some of them, be subject to physiological control. We have therefore examined the functional regulation of somatodendritic expression levels of dendritic BC1 RNA in hippocampal neurons in culture. BC1 RNA, an RNA polymerase III transcript that is a component of a ribonucleoprotein particle, became first detectable in somatodendritic domains of developing hippocampal neurons at times of initial synapse formation. BC1 RNA was identified only in such neurons that had established synapses on cell bodies and/or developing dendritic arbors. When synaptic contact formation was initiated later in low-density cultures, BC1 expression was coordinately delayed. Inhibition of neuronal activity in hippocampal neurons resulted in a substantial but reversible reduction of somatodendritic BC1 expression. We conclude that expression of BC1 RNA in somatic and dendritic domains of hippocampal neurons is regulated in development, and is dependent upon neuronal activity. These results establish (for the first time to our knowledge) that an RNA polymerase III transcript can be subject to control through physiological activity in nerve cells.

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Regulation of BC1 expression by neuronal activity.  Hippocampal neurons were grown in culture for 14 d in the absence of TTX (A–C), in the presence of 1 μM TTX (D–F), or in  the presence of 1 μM TTX for the first 9 d, and in the absence of  TTX for the following 5 d (G–I). Left (A–G): BC1 RNA, dark-field  photomicrographs; middle (B–H), BC1 RNA, phase contrast  photomicrographs, corresponding to dark-field photomicrographs  in left column; right (C–I): 7SL RNA, dark-field photomicrographs. Dark field photomicrograph D (BC1 RNA in the presence of TTX) was overexposed to reveal absence of any significant labeling over cells or neurites. All cultures were grown at  medium density. Bar, 50 μm. (J) Cultured hippocampal neurons  on coverslips were exposed to autoradiographic film. Autoradiographs show BC1- and 7SL-labeling signals for cells that were grown for  14 d in the absence of TTX (1), in the presence of 1 μM TTX (2), or in the presence of 1 μM TTX for the first 9 d and in the absence of  TTX for the following 5 d (3). (4) Sense strand control.
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Figure 5: Regulation of BC1 expression by neuronal activity. Hippocampal neurons were grown in culture for 14 d in the absence of TTX (A–C), in the presence of 1 μM TTX (D–F), or in the presence of 1 μM TTX for the first 9 d, and in the absence of TTX for the following 5 d (G–I). Left (A–G): BC1 RNA, dark-field photomicrographs; middle (B–H), BC1 RNA, phase contrast photomicrographs, corresponding to dark-field photomicrographs in left column; right (C–I): 7SL RNA, dark-field photomicrographs. Dark field photomicrograph D (BC1 RNA in the presence of TTX) was overexposed to reveal absence of any significant labeling over cells or neurites. All cultures were grown at medium density. Bar, 50 μm. (J) Cultured hippocampal neurons on coverslips were exposed to autoradiographic film. Autoradiographs show BC1- and 7SL-labeling signals for cells that were grown for 14 d in the absence of TTX (1), in the presence of 1 μM TTX (2), or in the presence of 1 μM TTX for the first 9 d and in the absence of TTX for the following 5 d (3). (4) Sense strand control.

Mentions: We used TTX, a potent inhibitor of voltage-gated Na+-channels, in cultured hippocampal neurons during the first 2 wk of development. Chronic application of TTX, which has been used extensively in hippocampal neurons in culture (see for example Craig et al., 1994; Verderio et al., 1994; Benson and Cohen, 1996), has been shown to result in complete suppression of action potentials (Craig et al., 1994), thereby preventing evoked synaptic transmission. Thus, if BC1 expression is functionally dependent upon such transmission, application of TTX can be expected to result in decreased levels of the RNA. We maintained hippocampal neurons in culture at medium density in the presence of TTX for 14 d. Such application of TTX resulted in a dramatic reduction of somatodendritic BC1 levels relative to neurons that were maintained in culture for the same amount of time in the absence of TTX (Fig. 5). After 14 d in vitro, when BC1 levels were robust in somata and dendrites of neurons in control cultures, TTX cultures showed low-level BC1 labeling that reached, on average, no more than 10% of BC1 labeling levels in control cultures. A similarly substantial TTX-induced reduction of BC1 levels could already be observed at an earlier stage of BC1 expression after 9 d in culture (not shown). The reduction in BC1 expression levels was not uniform: whereas many cells in the 14-d TTX cultures showed no labeling at all, others exhibited low-level somatic labeling, while in still others, labeling was moderate in the soma and extended into proximal dendrites. Table I provides a summary of these data on the basis of a quantitative analysis of silver grain densities in the cultures examined. From the table, it appears that the ratio between dendritic and somatic BC1 signal intensities in TTX-treated vs. untreated cultures is constant. This result may be taken to indicate that regulation of BC1 RNA through electrical activity is mediated mainly through regulation of expression in the nucleus, as this can be expected to result in similar effects on somatic and dendritic BC1 levels, respectively. On the other hand, because BC1 levels in TTX-treated cultures are only marginally above background, making the calculation of ratios difficult, it cannot be ruled out that dendritic BC1 levels may also be regulated locally, in addition to regulation through gene expression.


Activity-dependent regulation of dendritic BC1 RNA in hippocampal neurons in culture.

Muslimov IA, Banker G, Brosius J, Tiedge H - J. Cell Biol. (1998)

Regulation of BC1 expression by neuronal activity.  Hippocampal neurons were grown in culture for 14 d in the absence of TTX (A–C), in the presence of 1 μM TTX (D–F), or in  the presence of 1 μM TTX for the first 9 d, and in the absence of  TTX for the following 5 d (G–I). Left (A–G): BC1 RNA, dark-field  photomicrographs; middle (B–H), BC1 RNA, phase contrast  photomicrographs, corresponding to dark-field photomicrographs  in left column; right (C–I): 7SL RNA, dark-field photomicrographs. Dark field photomicrograph D (BC1 RNA in the presence of TTX) was overexposed to reveal absence of any significant labeling over cells or neurites. All cultures were grown at  medium density. Bar, 50 μm. (J) Cultured hippocampal neurons  on coverslips were exposed to autoradiographic film. Autoradiographs show BC1- and 7SL-labeling signals for cells that were grown for  14 d in the absence of TTX (1), in the presence of 1 μM TTX (2), or in the presence of 1 μM TTX for the first 9 d and in the absence of  TTX for the following 5 d (3). (4) Sense strand control.
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Figure 5: Regulation of BC1 expression by neuronal activity. Hippocampal neurons were grown in culture for 14 d in the absence of TTX (A–C), in the presence of 1 μM TTX (D–F), or in the presence of 1 μM TTX for the first 9 d, and in the absence of TTX for the following 5 d (G–I). Left (A–G): BC1 RNA, dark-field photomicrographs; middle (B–H), BC1 RNA, phase contrast photomicrographs, corresponding to dark-field photomicrographs in left column; right (C–I): 7SL RNA, dark-field photomicrographs. Dark field photomicrograph D (BC1 RNA in the presence of TTX) was overexposed to reveal absence of any significant labeling over cells or neurites. All cultures were grown at medium density. Bar, 50 μm. (J) Cultured hippocampal neurons on coverslips were exposed to autoradiographic film. Autoradiographs show BC1- and 7SL-labeling signals for cells that were grown for 14 d in the absence of TTX (1), in the presence of 1 μM TTX (2), or in the presence of 1 μM TTX for the first 9 d and in the absence of TTX for the following 5 d (3). (4) Sense strand control.
Mentions: We used TTX, a potent inhibitor of voltage-gated Na+-channels, in cultured hippocampal neurons during the first 2 wk of development. Chronic application of TTX, which has been used extensively in hippocampal neurons in culture (see for example Craig et al., 1994; Verderio et al., 1994; Benson and Cohen, 1996), has been shown to result in complete suppression of action potentials (Craig et al., 1994), thereby preventing evoked synaptic transmission. Thus, if BC1 expression is functionally dependent upon such transmission, application of TTX can be expected to result in decreased levels of the RNA. We maintained hippocampal neurons in culture at medium density in the presence of TTX for 14 d. Such application of TTX resulted in a dramatic reduction of somatodendritic BC1 levels relative to neurons that were maintained in culture for the same amount of time in the absence of TTX (Fig. 5). After 14 d in vitro, when BC1 levels were robust in somata and dendrites of neurons in control cultures, TTX cultures showed low-level BC1 labeling that reached, on average, no more than 10% of BC1 labeling levels in control cultures. A similarly substantial TTX-induced reduction of BC1 levels could already be observed at an earlier stage of BC1 expression after 9 d in culture (not shown). The reduction in BC1 expression levels was not uniform: whereas many cells in the 14-d TTX cultures showed no labeling at all, others exhibited low-level somatic labeling, while in still others, labeling was moderate in the soma and extended into proximal dendrites. Table I provides a summary of these data on the basis of a quantitative analysis of silver grain densities in the cultures examined. From the table, it appears that the ratio between dendritic and somatic BC1 signal intensities in TTX-treated vs. untreated cultures is constant. This result may be taken to indicate that regulation of BC1 RNA through electrical activity is mediated mainly through regulation of expression in the nucleus, as this can be expected to result in similar effects on somatic and dendritic BC1 levels, respectively. On the other hand, because BC1 levels in TTX-treated cultures are only marginally above background, making the calculation of ratios difficult, it cannot be ruled out that dendritic BC1 levels may also be regulated locally, in addition to regulation through gene expression.

Bottom Line: Inhibition of neuronal activity in hippocampal neurons resulted in a substantial but reversible reduction of somatodendritic BC1 expression.We conclude that expression of BC1 RNA in somatic and dendritic domains of hippocampal neurons is regulated in development, and is dependent upon neuronal activity.These results establish (for the first time to our knowledge) that an RNA polymerase III transcript can be subject to control through physiological activity in nerve cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York 11203, USA.

ABSTRACT
Several neuronal RNAs have been identified in dendrites, and it has been suggested that the dendritic location of these RNAs may be relevant to the spatiotemporal regulation of mosaic postsynaptic protein repertoires through transsynaptic activity. Such regulation would require that dendritic RNAs themselves, or at least some of them, be subject to physiological control. We have therefore examined the functional regulation of somatodendritic expression levels of dendritic BC1 RNA in hippocampal neurons in culture. BC1 RNA, an RNA polymerase III transcript that is a component of a ribonucleoprotein particle, became first detectable in somatodendritic domains of developing hippocampal neurons at times of initial synapse formation. BC1 RNA was identified only in such neurons that had established synapses on cell bodies and/or developing dendritic arbors. When synaptic contact formation was initiated later in low-density cultures, BC1 expression was coordinately delayed. Inhibition of neuronal activity in hippocampal neurons resulted in a substantial but reversible reduction of somatodendritic BC1 expression. We conclude that expression of BC1 RNA in somatic and dendritic domains of hippocampal neurons is regulated in development, and is dependent upon neuronal activity. These results establish (for the first time to our knowledge) that an RNA polymerase III transcript can be subject to control through physiological activity in nerve cells.

Show MeSH
Related in: MedlinePlus