Mutations of the adenomatous polyposis coli (APC) tumor suppressor gene have been linked to familial polyposis, an inherited predisposition to colon cancer, and a high percentage of sporadic colon adenomas. Although this gene is best known for its role in development of bowel neoplasms, in recent studies we have found that APC mRNA levels are greatly enriched in brain compared with peripheral tissues. To help define its role in the nervous system, in this study we have determined its cellular localization immunohistochemically in adult rat brain sections and have detected intense APC immunoreactivity in oligodendrocytes. Since prominent APC immunostaining is detected in cell bodies of mature oligodendrocytes, these antibodies may provide a useful addition to available oligodendrocyte markers. Although the cellular function of APC remains undefined, previous biochemical studies have demonstrated that APC is associated with catenins, cytoplasmic proteins involved in regulating cell-cell adhesion. We propose that, in addition to its critical role in ensuring normal maturation of colonic epithelial cells, the APC tumor suppressor protein also regulates the adhesive properties of oligodendrocytes. (C) 1996 Wiley-Liss, Inc.

As immediate early genes (IEGs) are thought to play a critical role in mediating stimulus-induced neuronal plasticity, several laboratories have characterized the IEG response induced by cocaine to help define the changes in gene expression that may underlie its long-lasting behavioral effects. Although activation of several transcription factor IEGs has been described, little is known about which "effector" IEGs, if any, are also induced. In the present study, we have examined whether cocaine administration affects expression of a recently identified "effector" IEG, referred to as arc (activity-regulated, cytoskeleton-associated). This IEG encodes a protein with homology to spectrin that appears to be associated with the actin cytoskeleton. Using in situ hybridization, we have found that systemic cocaine administration elicits a robust, transient rise in arc mRNA levels in striatum, which is suppressed by D1 dopamine receptor blockade, reserpine treatment, or striatal 6-hydroxydopamine lesions. D2 receptor antagonist triggered arc expression when administered alone. Immunohistochemical studies indicated that Arc protein induced by cocaine is expressed in neuronal cell bodies and dendrites. As Arc appears to be component of the neuronal cytoskeleton, it may be involved in structural alterations underlying neuronal plasticity triggered by cocaine.

The aquaporins transport water through membranes of numerous tissues, but the molecular mechanisms for sensing changes in extracellular osmolality and regulating water balance in brain are unknown. We have isolated a brain aquaporin by homology cloning. Like aquaporin 1 (AQP1, also known as CHIP, channel-forming integral membrane protein of 28 kDa), the deduced polypeptide has six putative transmembrane domains but lacks cysteines at the known mercury-sensitive sites. Two initiation sites were identified encoding polypeptides of 301 and 323 amino acids; expression of each in Xenopus oocytes conferred a 20-fold increase in osmotic water permeability not blocked by 1 mM HgCl2, even after substitution of cysteine at the predicted mercury-sensitive site. Northern analysis and RNase protection demonstrated the mRNA to be abundant in mature rat brain but only weakly detectable in eye, kidney, intestine, and lung. In situ hybridization of brain localized the mRNA to ependymal cells lining the aqueduct, glial cells forming the edge of the cerebral cortex and brainstem, vasopressin-secretory neurons in supraoptic and paraventricular nuclei of hypothalamus, and Purkinje cells of cerebellum. Its distinctive expression pattern implicates this fourth mammalian member of the aquaporin water channel family (designated gene symbol, AQP4) as the osmoreceptor which regulates body water balance and mediates water flow within the central nervous system.

The adenomatous polyposis coli (APC) gene is a tumor suppressor gene that is mutated in human familial adenomatous polyposis, an autosomal dominant condition with predisposition to colorectal carcinoma and brain tumors. Although tumor suppressor genes appear to play a general role in regulating cellular proliferation, the normal biological function of the APC gene product is unknown. In the present study, we cloned fragments of the rat homolog of the APC gene and examined its tissue distribution by Northern blot analysis. These studies demonstrated particularly high levels of APC mRNA in brain. To gain clues to the role of the APC gene in brain function, we examined the neuroanatomical distribution of APC mRNA using in situ hybridization. In the adult, prominent expression of APC mRNA was observed in the olfactory bulb, hippocampus, and cerebellum, with low levels of hybridization in other regions of adult rat brain. In contrast, during embryonic and early postnatal development (1-2 weeks), high levels of APC expression were found throughout the brain and then decreased to adult levels by 6 weeks after birth, except in the olfactory bulb where the high levels of APC mRNA found in development persist in the adult. During development of cortex, cerebellum, and retina, APC mRNA expression was particularly prominent in layers containing newly formed postmitotic neurons, with lower levels observed in the proliferative zones where neurogenesis occurs. The high levels of APC expression from early neurogenesis until late stages of neuronal maturation suggest that APC may contribute to suppressing neuronal proliferation during this period of intense growth.

Calcium/calmodulin-dependent protein kinase II (CaMK) and p42 mitogen-activated protein kinase (MAPK) are enriched in neurons and possess the capacity to become persistently active, or autonomous, following removal of the activating stimulus. Since persistent kinase activation may be a mechanism for information storage, we have used primary cultures of cortical neurons to investigate whether kinase autonomy can be triggered by bursts of spontaneous synaptic activity. We and others have found that both these kinases respond to synaptic stimulation, but differ markedly in their kinetics of activation and inactivation, as well as in their sensitivity to NMDA receptor blockade. While 90% of maximal CaMK activation was observed after only 10 sec of synaptic bursting, MAPK activity was unaffected at this early time and rose to only 30% of maximal after 2 min of stimulation. Following blockade of synaptic stimulation, CaMK activity decreased by 50% in 10-30 sec, while MAPK activity decayed by 50% within 6-10 min. Although MAPK exhibited relatively slow activation, short periods of synaptic activity could trigger the MAPK activation process, which persisted in the absence of synaptic stimulation. Comparison of the effect of NMDA receptor blockade on synaptic activation of these kinases revealed that CaMK activity is preferentially suppressed. As previous immunocytochemical studies indicate that CaMK is concentrated in dendritic processes in the vicinity of synapses, we measured synaptic calcium transients in fine dendritic processes (approximately 1 microns diameter) to assess their sensitivity to NMDA receptor blockade. Calcium transients in these fine processes were reduced by up to 90% by NMDA receptor blockade, possibly accounting for the profound sensitivity of CaMK to this treatment. The sharp contrast between the regulation of CaMK and MAPK by synaptic activity indicates that they may mediate neuronal responses to different patterns of afferent stimulation. The relatively slow activation and inactivation of MAPK suggests that it may be able to integrate information from multiple, infrequent bursts of synaptic activity.

Recent studies suggest a role for rapid induction of transcription factors in stimulus-induced neuronal plasticity in the mammalian brain. Synaptic activation of transcription factors has been analyzed in the hippocampus using the long-term potentiation or enhancement (LTP/LTE) paradigm. Using this approach, several studies have identified transcription factors that are induced in hippocampal granule cells by NMDA receptor-dependent mechanisms; however, the link between long-term plasticity and activation of these genes has been called into question by reports suggesting that the thresholds for LTE and gene activation differ. To address this issue, we have used a chronic in vivo recording technique to monitor mRNA responses of several transcription factor genes to two different patterns of LTE-inducing electrical stimulation of entorhinal cortical afferents to hippocampus. One pattern consisted of 10 repetitions of a 20 or 25 msec train of pulses at 400 Hz (80 or 100 pulses total). This "10-train" pattern has been used in previous studies of LTE and produces robust synaptic enhancement lasting at least 3 d (Barnes, 1979). The other stimulation pattern consisted of 50 repetitions of a 20 msec train delivered at 400 Hz (400 pulses total), which is similar to parameters used in other studies reporting induction of c-fos in association with LTE (Dragunow et al., 1989; Jeffery et al., 1990; Abraham et al., 1992). Our results indicate that expression of zif268, monitored by in situ hybridization and immunostaining, is strongly induced by the 10-train stimulus pattern to levels similar to those induced by seizure activity. JunB mRNA levels are also modestly increased by the 10-train stimulus pattern; however, increases in JunB immunostaining were not detected. Neither c-fos nor c-jun mRNA were detectably induced by this stimulus. In contrast, the 50-train stimulus pattern resulted in a robust induction of c-fos and c-jun mRNA, in addition to zif268 and junB. Transcription factor responses to either stimulus pattern were blocked by the noncompetitive NMDA receptor antagonist MK-801. Identical transcription factor responses were observed in adult (6-12-month-old) and aged (23-26-month-old) rats, suggesting that synaptic mechanisms involved in these responses are preserved in aged animals. Analysis of LTE following either the 10- or 50-train stimulus patterns revealed identical magnitudes of initial induction and decay kinetics (approximately 3 d) and indicates that the 10-train stimulus pattern is sufficient to produce maximal synaptic enhancement.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute administration of cocaine increases expression of the transcription factor genes c-fos and zif268 in the striatum. This response is thought to be mediated via D1 dopamine (DA) receptors, as it is blocked by the selective D1 receptor antagonist SCH 23390. However, the directly acting D1 receptor agonists, apomorphine and SKF 38393, do not mimic cocaine's activation of these genes raising the possibility that D1 receptor activation is necessary, but not sufficient, to trigger transcription factor expression. Because cocaine blocks uptake of norepinephrine (NE) and serotonin (5-HT), as well as DA, we examined whether cocaine's ability to inhibit NE and 5-HT uptake may contribute to its induction of c-fos and zif268 expression in striatum. In examining the effects of selective monoamine uptake inhibitors, we observed that fluoxetine or citalopram, selective inhibitors of 5-HT uptake, potentiated the ability of mazindol, a DA and NE uptake inhibitor, to induce zif268 and c-fos expression, even though these 5-HT uptake inhibitors had no effect when administered alone. In contrast, the selective NE uptake inhibitor, desipramine, administered alone, or in combination with fluoxetine, did not increase expression of zif268 or c-fos. Furthermore, selective denervation of 5-HT projections by p-chloroamphetamine treatment attenuated the increase in zif268 and c-fos expression induced by cocaine in the striatum. In contrast, selective lesions of NE projections with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride failed to block cocaine's activation of these genes in the striatum. Taken together, these findings indicate that cocaine's ability to induce striatal expression of c-fos and zif268 is mediated by its effects on both the 5-HT and DA systems.

Recent studies have demonstrated that seizure activity causes a dramatic increase in neuropeptide expression in specific regions of the rat hippocampus. In this study we investigated the effect of electroconvulsive treatment (ECT) on the expression of three posttranslational processing enzymes involved in the production of many bioactive peptides from their inactive precursors. Peptidylglycine alpha-amidating monooxygenase (PAM) converts peptidylglycine substrates into alpha-amidated products and prohormone convertases 1 and 2 perform the tissue-specific endoproteolytic cleavage of many prohormones. After a single ECT, in situ hybridization demonstrated a rapid increase in the level of PAM mRNA in the dentate granule cells of the hippocampus, reaching peak levels between 1 and 4 h and then returning to near baseline levels within 24 h. Northern blot analysis confirmed the changes in PAM mRNA expression seen by using in situ hybridization. Similar rapid changes in PAM mRNA expression were seen after repeated ECT, suggesting that chronic ECT did not affect the regulation of PAM expression in the hippocampus. Immunohistochemical staining demonstrated an increase in PAM protein in the molecular layer of the dentate gyrus at 4 and 8 h after a single ECT. Based on in situ hybridization, levels of mRNA for the prohormone convertases 1 and 2 were also increased in dentate granule cells after a single ECT. Prohormone convertase 2 mRNA levels exhibited a slower response to ECT, not reaching maximal levels until 8 h after ECT. The response of the dentate granule cells of the hippocampus to ECT provides a model system for studying the rapid, coordinate regulation of peptide-processing enzymes.

The transcription factor Zif268 displays high basal levels of expression in cortex that appear to be dependent on physiological synaptic activity. We report that selective lesions of the noradrenergic system induced by DSP4 markedly suppress basal zif268 mRNA levels in cortex. Accordingly, the noradrenergic system which projects extensively to the cortex and is tonically active may play a key role in maintaining normal patterns of gene expression in target neurons.

Rapid activation of transcription factor genes is thought to play a key role in stimulus-induced neuronal plasticity. To help understand the genomic response that may underlie long-term effects of cocaine and amphetamine, we have investigated the effect of these agents on Zif268, a transcription regulatory factor that is expressed at high levels in brain neurons. Like c-fos, zif268 is markedly activated in striatum by cocaine and amphetamine. This response appears to involve the dopamine system, since it is abolished by SCH23390, a selective D1 dopamine receptor antagonist, or by 6-hydroxydopamine lesions. To assess the role of other monoamine systems in regulating the expression of these transcription factors, we have examined the effects of selective monoamine uptake blockers as well as agents that lesion the norepinephrine and serotonin systems. These studies indicate that, in addition to the dopamine system, the norepinephrine and serotonin systems also play prominent roles in the activation of zif268 and c-fos by cocaine and amphetamine.

Recent studies have shown that dopamine receptor agonists induce expression of Fos-like immunoreactivity in rat striatal neurons. The protooncogene c-fos belongs to a family of immediate early genes that are rapidly induced in fibroblasts by growth factors. In light of previous findings that several immediate early gene mRNAs that encode proven or putative transcription factors are differentially regulated by neuronal stimulation in vivo, we have examined the effect of dopaminergic agents on mRNA levels of several such genes using in situ hybridization and northern blot analysis. d-Amphetamine (2.5-10 mg/kg i.p.) causes a rapid but transient dose-dependent increase in zif268 and jun-B mRNA levels in striatum that was abolished by striatal 6-hydroxydopamine lesions or by pretreatment with the specific D1 receptor antagonist SCH-23390 but not by specific D2 receptor antagonists. Apomorphine, a dopamine agonist that acts at both D1 and D2 receptors, and SKF-38393, a specific D1 receptor agonist, produce similar mRNA changes in rats pretreated with either 6-hydroxydopamine or reserpine, whereas LY-171,555, a specific D2 receptor agonist, has no effect. Direct dopamine agonist effects on these immediate early gene mRNA levels are also blocked by D1 but not by D2 antagonists. We observed similar, although less robust, changes in c-fos and fos-B mRNA levels. These results demonstrate that striatal D1 dopamine receptors are coupled to activation of multiple transcription factor genes, including zif268 and jun-B as well as members of the fos family.

Transcription regulatory factors are rapidly induced in brain by a wide variety of stimuli and may be important in coordinating changes in gene expression underlying neuronal plasticity. In addition to the transient activation profile typically displayed by many of these genes, zif268 exhibits prominent basal expression in the brain that is dependent on synaptic activity. Accordingly, zif268 may play a key role in regulating neuronal gene expression induced by naturally occurring stimuli. Acute cocaine administration (15 mg/kg i.p.) produces a robust and transient induction of several transcription factors in the brain, including zif268. In the present study we report that, in contrast to the acute effects of a single dose, chronic cocaine treatment (15 mg/kg i.p., twice daily for a total of 10 injections), produces a widespread suppression of basal zif268 mRNA levels in rat forebrain between 8 and 24 hr after the last cocaine injection. This reduction is not evident after a single injection of cocaine or comparable chronic treatment with a structural analog, procaine, that does not share cocaine's uptake inhibitor properties. The suppression of basal zif268 expression may provide a mechanism for producing widespread effects of chronic cocaine administration on neuronal gene expression.

The nerve growth factor-induced clone C (NGFI-C) gene encodes a zinc-finger transcription factor that is rapidly induced by nerve growth factor in rat pheochromocytoma PC12 cells and by seizure in brain. NGFI-C is closely related to the previously described early response genes, nerve growth factor-induced clone A (NGFI-A or EGR1), EGR2, and EGR3. These four early response (immediate early) proteins all contain very similar zinc-finger DNA binding domains; in addition, analysis of the non-zinc-finger region revealed that they share an additional five highly homologous subdomains, four of which are within the amino terminus. The 5' flanking region of NGFI-C contains several cAMP response elements but does not contain any serum-response elements or CArG boxes [CC(A/T)6GG], cis-acting elements commonly involved in early response gene regulation. NGFI-C mRNA was detected in neural tissues of postnatal animals, but no expression was found in rat embryos. In situ hybridization demonstrated that NGFI-C is rapidly induced in the dentate gyrus of the hippocampus after seizure, but in contrast to NGFI-A, increases in NGFI-C mRNA were not detected in the overlying cortex. By using fluorescence in situ hybridization, NGFI-C was localized to human chromosome 2p13. This region contains a constitutive fragile site that is associated with chromosomal breakpoints and translocations characteristic of some chronic lymphocytic leukemias.

Zif268 and krox-20 are transcription regulatory factors that contain highly homologous zinc finger DNA-binding domains. Recent studies have demonstrated that zif268 expression is rapidly regulated in brain by neuronal stimulation. We now report that, like zif268, krox-20 is rapidly and transiently activated by electroconvulsive shock treatment (ECT), D1 dopamine receptor activation, and opiate withdrawal. These studies indicate that, as found for the leucine zipper family of transcription factors, multiple members of the zinc finger family of transcription factors are induced by neuronal stimulation.

Transcription factors are rapidly and transiently induced in brain by excitatory stimuli and may be important in coordinating changes in gene expression underlying neuronal plasticity. In contrast to their transient induction after stimulation, certain transcription factors display stable, relatively high basal levels of expression in brain. Here we demonstrate that this "constitutive" expression of the transcription factor zif268 in cortex is driven by natural synaptic activity. Blockade of afferent visual activity with intraocular injections of tetrodotoxin results in rapid, dramatic reductions of Zif268 mRNA and immunoreactivity in visual cortex. Moreover, dark-adaptation for several days lowers zif268 expression in visual cortex, and expression rapidly returns to control levels upon subsequent light exposure. Several other transcription factors, which are induced in cortical neurons by excitatory stimuli, appear less responsive to changes in natural sensory input. These studies suggest that transcription factors play a role not only in responses to artificial stimuli but also in the normal maintenance of cortical physiology. Anatomic markers for zif268 may be useful in mapping normal cortical activity in brain.