Singing, but not seizure, induces synaptotagmin IV in zebra finch song circuit nuclei.

Synaptotagmins are a family of proteins that function in membrane fusion events, including synaptic vesicle exocytosis. Within this family, synaptotagmin IV (Syt IV) is unique in being a depolarization-induced immediate early gene (IEG). Experimental perturbation of Syt IV modulates neurotransmitter release in mice, flies, and PC12 cells, and modulates learning in mice. Despite these features, induction of Syt IV expression by a natural behavior has not been previously reported. We used the zebra finch, a songbird species, to investigate Syt IV because song is a naturally learned behavior whose neuroanatomical basis is largely identified. We observed that, similar to rodents, Syt IV is inducible in songbirds. This induction was selective and depended on the nature of neuronal depolarization. Generalized seizures caused by the GABA(A) receptor antagonist, metrazole, induced the IEG, ZENK, in zebra finch brain. However, these same seizures failed to induce Syt IV in song control areas. In contrast, when nontreated birds sang, three song control areas showed striking Syt IV induction. Further, this induction appeared sensitive to the social context in which song was sung. Together, these data suggest that neural activity during singing can drive Syt IV expression within song circuitry whereas generalized seizure activity fails to do so even though song control areas are depolarized. Our findings indicate that, within this neural circuit for a procedurally learned sensorimotor behavior, Syt IV is selective and requires precisely patterned neural activity and/or neuromodulation associated with singing.

NID67, a small putative membrane protein, is preferentially induced by NGF in PC12 pheochromocytoma cells.

In an effort to identify genes involved in neuronal differentiation, we have used representational difference analysis (RDA) to clone cDNAs that are preferentially induced by nerve growth factor (NGF) vs. epidermal growth factor (EGF) in PC12 pheochromocytoma cells. We now report the cloning of a previously unknown primary response gene, NID67. In addition to a robust induction by NGF and FGF, both of which cause PC12 cells to differentiate, NID67 is strongly induced by forskolin, A23187 and ATP. EGF, TPA and KCl induce NID67 only weakly. NID67 mRNA is most abundant in heart, ovary and adrenal. Modest levels are present in most brain regions, testis, thyroid, thymus, pituitary, kidney and intestine; little NID67 is present in skeletal muscle and cerebellum. The NID67 cDNA contains a 180 bp open reading frame (ORF) that encodes a 60 amino acid protein. The central 29 amino acids are very hydrophobic and very likely comprise a transmembrane domain. Mouse and human NID67 cDNAs contain an ORF similar to NID67; the rat and human protein sequences are 85% identical whereas the rat and mouse sequences are 92% identical. In vitro transcription and translation reactions confirmed that the ORF we identified produces a 6000 Da protein product. Several small membrane proteins are similar to NID67; they contain a transmembrane domain and little more. All of these proteins participate in forming or regulating ion channels. NID67 may play a similar role in cellular physiology.

Expression of the urokinase plasminogen activator receptor is transiently required during "priming" of PC12 cells in nerve growth factor-directed cellular differentiation.

We previously identified the urokinase plasminogen activator receptor (UPAR) as a gene induced by nerve growth factor (NGF), but not by epidermal growth factor (EGF), in PC12 cells (Farias-Eisner et al. [2000] J. Neurosci. 20:230-239). Antisense oligonucleotides for the UPAR mRNA or an antibody directed against UPAR protein, added simultaneously with NGF, block NGF-induced morphological and biochemical differentiation of PC12 cells. In this report, we show that anti-UPAR antibody blocks morphological differentiation and the expression of two NGF-specific secondary response genes, collagenase-1 and transin, in PC12 cells only during the first 2 hr following NGF exposure. These data suggest that induced UPAR expression is required only over a short period of time following exposure to NGF for the differentiation program in PC12 cells to proceed. For two models of "primed" PC12 cells, we found that UPAR expression and function are not required for NGF-induced differentiation. UPAR and the secondary response genes collagenase-1 and transin are not induced in "primed" PC12 cells in response to NGF, and anti-UPAR antibody does not block morphological differentiation in these cells. Our data suggests that UPAR is required only transiently during the "priming" of PC12 cells in NGF-induced PC12 cell differentiation.

Synaptotagmin IV: biochemistry, genetics, behavior, and possible links to human psychiatric disease.

We isolated the rat synaptotagmin IV (Syt IV) cDNA in a screen for sequences that are specifically induced in neuronal cells. The Syts are a large family of genes thought to mediate synaptic function. Syt IV is brain-specific, induced in hippocampus by depolarization, and predominantly vesicular. To assess the function role of Syt IV in vivo, we generated Syt IV(-/-) mutant mice. Syt IV (-/-) mice are viable and appear normal, indicating this gene is not essential for survival or gross development. However, Syt IV (-/-) mutants, when compared to wild-type littermates, have deficits in fine motor coordination and hippocampus-dependent memory, suggesting Syt IV has a role in normal brain function. The human Syt IV ortholog maps to a region of chromosome 18 previously associated with the human psychiatric disorders, schizophrenia and bipolar disease. These results suggest that Syt IV is required in certain types of neurons for optimal functionality, that perturbations in the levels of Syt IV can result in memory loss in mice, and that Syt IV alterations may lead to psychiatric disease in humans.

Searching for depolarization-induced genes that modulate synaptic plasticity and neurotrophin-induced genes that mediate neuronal differentiation.

We identify and characterize two classes of immediate-early genes: (i) genes, induced by depolarization in neurons, that play a role in depolarization-induced neuronal plasticity and (ii) genes, induced in neuronal precursors by neurotrophins, that play a causal role in neurotrophin-directed neuronal differentiation. We use rat PC12 pheochromocytoma cells to identify (i) genes preferentially induced by [depolarization or forskolin] versus [Nerve Growth Factor (NGF) or Epidermal Growth Factor (EGF)] and (ii) genes preferentially induced by NGF versus EGF. We describe (i) a collection of genes preferentially induced by depolarization/forskolin in PC12 cells and by kainic acid in vivo, and (ii) a collection of genes preferentially induced by NGF. The synaptotagmin IV gene encodes a synaptic vesicle protein whose level is modulated by depolarization. NGF preferentially induces the urokinase-plasminogen activator receptor in PC12 cells. Antisense oligonucleotide and anti-UPAR antibody experiments demonstrate that NGF-induced UPAR expression is required for NGF-driven PC12 cell differentiation.

rTLE3, a newly identified transducin-like enhancer of split, is induced by depolarization in brain.

The transducin-like enhancers of split are a family of mammalian proteins that share sequence homology with the Drosophila protein Groucho. Using representational difference analysis, we isolated the cDNA for a previously unidentified gene, rTLE3 (rat transducin-like enhancer of split 3), as a sequence induced by depolarization and forskolin, but not by neurotrophins or growth factors, in PC12 pheochromocytoma cells. rTLE3 encodes the protein rTLE3, a 764-amino acid orthologue of mouse and human TLE3. R-esp2, the gene encoding the closest related rat protein, is not induced by any of the four treatments in PC12 cells. rTLE3 and R-esp2 have different patterns of expression in the adult rat CNS and other tissues. After systemic administration of kainic acid, rTLE3 is induced specifically in the dentate gyrus of the hippocampus. We propose that members of the transducin-like enhancer of split family of proteins may have distinct functions in the mature CNS, in addition to their functions during development.

The salt-inducible kinase, SIK, is induced by depolarization in brain.

Membrane depolarization of neurons is thought to lead to changes in gene expression that modulate neuronal plasticity. We used representational difference analysis to identify a group of cDNAs that are induced by membrane depolarization or by forskolin, but not by neurotrophins or growth factors, in PC12 pheochromocytoma cells. One of these genes, SIK (salt-inducible kinase), is a member of the sucrose-nonfermenting 1 protein kinase/AMP-activated protein kinase protein kinase family that was also recently identified from the adrenal gland of rats treated with high-salt diets. SIK mRNA is induced up to eightfold in specific regions of the hippocampus and cortex in rats, following systemic kainic acid administration and seizure induction.

The urokinase plasminogen activator receptor (UPAR) is preferentially induced by nerve growth factor in PC12 pheochromocytoma cells and is required for NGF-driven differentiation.

Nerve growth factor (NGF)-driven differentiation of PC12 pheochromocytoma cells is a well studied model used both to identify molecular, biochemical, and physiological correlates of neurotrophin-driven neuronal differentiation and to determine the causal nature of specific events in this differentiation process. Although epidermal growth factor (EGF) elicits many of the same early biochemical and molecular changes in PC12 cells observed in response to NGF, EGF does not induce molecular or morphological differentiation of PC12 cells. The identification of genes whose expression is differentially regulated by NGF versus EGF in PC12 cells has, therefore, been considered a source of potential insight into the molecular specificity of neurotrophin-driven neuronal differentiation. A "second generation" representational difference analysis procedure now identifies the urokinase plasminogen activator receptor (UPAR) as a gene that is much more extensively induced by NGF than by EGF in PC12 cells. Both an antisense oligonucleotide for the UPAR mRNA and an antibody directed against UPAR protein block NGF-induced morphological and biochemical differentiation of PC12 cells; NGF-induced UPAR expression is required for subsequent NGF-driven differentiation.

Seizure activity induces PIM-1 expression in brain.

We recently identified KID-1, a previously undescribed protein kinase induced by depolarization in PC12 cells and brain (Feldman et al., 1998). KID-1 shares a high degree of sequence homology with PIM-1, a proto-oncogene previously reported to be expressed in hematopoietic and germ cells. We examined PIM-1 expression in stimulated PC12 cells, brains of kainic acid-treated rats, and a number of tissues from untreated rats. We now report that forskolin, but not depolarization or growth factors, induces PIM-1 expression in PC12 cells. PIM-1 is an immediate early gene induced in response to forskolin stimulation. We detect PIM-1 mRNA in a number of unstimulated tissues and at low levels in unstimulated brain. Systemic kainic acid administration to adult rats induces PIM-1 expression in the dentate gyrus region of the hippocampus.

KID-1, a protein kinase induced by depolarization in brain.

Membrane depolarization leads to changes in gene expression that modulate neuronal plasticity. Using representational difference analysis, we have identified a previously undiscovered cDNA, KID-1 (kinase induced by depolarization), that is induced by membrane depolarization or forskolin, but not by neurotrophins or growth factors, in PC12 pheochromocytoma cells. KID-1 is an immediate early gene that shares a high degree of sequence similarity with the family of PIM-1 serine/threonine protein kinases. Recombinant KID-1 fusion protein is able to catalyze both histone phosphorylation and autophosphorylation. KID-1 mRNA is present in a number of unstimulated tissues, including brain. In response to kainic acid and electroconvulsive shock-induced seizures, KID-1 is induced in specific regions of the hippocampus and cortex.

Identification of genes preferentially induced by nerve growth factor versus epidermal growth factor in PC12 pheochromocytoma cells by means of representational difference analysis.

Neurotrophins induce neuronal differentiation by binding to a subclass of ligand-stimulated protein tyrosine kinase receptors and activating signal transduction pathways that mediate altered patterns of gene expression. Nerve growth factor (NGF) induced differentiation of PC12 pheochromocytoma cells is one of the major model systems used to study neuronal differentiation in response to neurotrophins. Although epidermal growth factor (EGF) does not induce PC12 cell differentiation, NGF and EGF activate many of the same signal transduction pathways and induce transcription of many of the same genes in PC12 cells. We have now employed cDNA representational difference analysis to identify four genes (activity-regulated cytoskeletal protein, collagenase 1, plasminogen activator inhibitor-1, and VH6/MKP-3) as genes preferentially induced withing 4 hours by NGF in PC12 cells.

Two synaptotagmin genes, Syt1 and Syt4, are differentially regulated in adult brain and during postnatal development following kainic acid-induced seizures.

The synaptotagmins together with other vesicle proteins are thought to be essential for the docking and/or fusion of synaptic vesicles with the plasma membrane that occurs following depolarization and calcium influx in presynatic terminals. Syt4, the fourth identified member of the synaptotagmin family, is inducible in PC12 cells by depolarization and secretagogues, and in limbic regions of the adult rat brain by kainic acid-induced seizures. In the present study, we examined the time course of the seizure-induced changes in the expression of Syt4 and Syt1, both in adult animals and during the postnatal period. Syt4 was transiently induced in several structures of the adult rat brain following seizure activity with peak inductions between 4 and 8 h and overal return to control values by 30 h. No induction was observed following seizure activity in 7-day-old animals. The brain regions most sensitive to increased induction were, in decreasing order of sensitivity, hippocampal pyramidal cells dentate granule cells and piriform cortex pyramidal cells. The brain areas showing the greatest Syt4 stimulation in adults were also the areas in which Syt4 was induced by seizures earlier in development. In contrast, Syt1 mRNA was depressed in adult brains following seizure activity, particularly in the dentate granule cells. Our results suggest that the differential regulation of different synaptotagmin genes following excessive neuronal activity might participate in rapid adaptation of subsequent transmitter release.

Synaptotagmin IV is an immediate early gene induced by depolarization in PC12 cells and in brain.

Subtractive library construction and differential screening were used to identify a cDNA for a cell type-specific immediate early gene induced in rat PC12 pheochromocytoma cells. Sequencing identified the protein product of this gene as rat synaptotagmin IV (SytIV). Synaptotagmins are synaptic vesicle proteins thought to play a role in depolarization-induced, calcium-mediated exocytosis and neurotransmitter release. SytIV mRNA accumulation is transiently induced in PC12 cells by potassium depolarization, calcium ionophore, ATP, and forskolin. In contrast, growth factors and phorbol 12-myristate 13-acetate induce little or no SytIV mRNA accumulation. Kainic acid-induced seizures in rats are followed by accumulation of SytIV message in the hippocampus and piriform cortex. The SytIV gene may provide a direct link between depolarization-induced neuronal gene expression and subsequent modulation of synaptic structure and function.

Differential localization of two glutamic acid decarboxylases (GAD65 and GAD67) in adult monkey visual cortex.

Adult monkey primary visual cortex contains a diverse population of stellate neurons that utilize the neurotransmitter gamma aminobutyric acid (GABA). Two glutamic acid decarboxylase (GAD) enzymes that synthesize GABA, GAD65 and GAD67, were localized within these stellate neurons by in situ hybridization of 35S or digoxigenin (DIG) labeled riboprobes. Double labels were done by using 35S GAD67 riboprobe and GABA immunocytochemistry on the same section to verify that the neuronal population identified by immunocytochemistry was the same one studied in the in situ hybridization experiments. We find that GAD65 mRNA and GAD67 mRNA are widely distributed in the cortex, with four bands of heavily labeled neurons in upper layer 2, lower 3, 4C, and 6. GAD67 labeled neurons were more obvious in layer 4C beta, while GAD65 containing neurons were common in layer 1 and white matter. Northern blots and in situ hybridization on sections with both 35S and DIG riboprobes indicate that cortical neurons typically contain more GAD67 mRNA. Cell counts show that 18% of all cortical neurons contain GAD67 mRNA and 13% contain GAD65 mRNA, suggesting that a small population of GABA neurons might lack GAD65. Cell bodies that contain high amounts of GAD65 mRNA are prominent in layers deep 3, 4B, 4C alpha, and 6 and often are the largest cells in their respective layers. Double labels demonstrate that 96% of all GABA+ neurons contain GAD67 mRNA. Neurons heavily labeled for GABA tend to have smaller cell bodies and contain less GAD67 mRNA, while lightly labeled GABA neurons are larger and contain more GAD67 mRNA. These data indicate that most GABA neurons in monkey striate cortex contain both GAD enzymes. Although the differences in GABA content, cell size, laminar distribution, and GAD mRNA concentration suggest different requirements for GAD67 and GAD65 in cortical circuits, our experiments do not reveal what different roles these two enzymes subserve within GABAergic stellate neurons.

Testosterone regulates pro-opiomelanocortin gene expression in the primate brain.

Endogenous opioid peptides such as beta-endorphin, derived from proopiomelanocortin (POMC), have been widely implicated as serving an important role in the neuroendocrine regulation of the primate reproductive axis. In both human and nonhuman primates, POMC neurons are thought to mediate, at least in part, the negative feedback action of sex steroids on GnRH. Sex steroids, such as testosterone, are thought to inhibit GnRH secretion by enhancing the inhibitory activity of beta-endorphin; however, the cellular mechanisms by which steroid hormones regulate the activity of POMC neurons in the primate brain are unknown. In this study, we tested the hypothesis that testosterone stimulates POMC gene expression within the primate brain and that this regulation occurs within a specific subset of POMC neurons residing in the arcuate nucleus of the hypothalamus. We used in situ hybridization to compare cellular levels of POMC messenger RNA in intact (n = 4), castrated (n = 4), and castrated/testosterone-treated (n = 4) monkeys. We report that after castration of the male macaque (Macaca fascicularis), cellular POMC messenger RNA levels decline significantly (P less than 0.05) in neurons within the arcuate nucleus and that this decline is prevented by replacement with physiological doses of testosterone. Moreover, we found that this testosterone-dependent modulation of POMC gene expression is restricted to a small fraction of the numerous POMC neurons located within the most anterior region of the arcuate nucleus in the brain of this primate species. These observations provide evidence that sex steroids regulate expression of the POMC gene in the primate brain.

Pubertal changes in pro-opiomelanocortin and gonadotropin-releasing hormone gene expression in the brain of the male monkey.

Puberty in the primate is initiated by an amplification of the pulsatile secretion of gonadotropin-releasing hormone (GnRH); however, the factors responsible for this a pubertal activation of GnRH neurons are unknown. We examined whether the biosynthetic capacity of GnRH neurons could be limiting for GnRH secretion in the juvenile monkey by determining whether the prepubertal macaque (Maraca fascicularis) expresses the GnRH gene. Using in situ hybridization for GnRH mRNA, we found no evidence for differences between prepubertal and adult animals in either the cellular level of GnRH mRNAs or the number of GnRH mRNA-containing cells in any area of the brain. These observations suggest that expression of the GnRH gene, at least to the point of messenger mRNA production, is not limiting for puberty onset. beta-Endorphin, derived from its precursor proopiomelanocortin (POMC), has been implicated as an inhibitory neurotransmitter in the control of GnRH secretion. We examined whether expression of the POMC gene changes as a function of sexual maturation by comparing the cellular content of POMC mRNA in the arcuate nucleus between prepubertal and adult male macaques. We report that cellular POMC mRNA levels are significantly higher in the adult than in the juvenile macaque (P < 0.02). We suggest that an increase in POMC biosynthesis may be part of the mechanism whereby a continuous mode of GnRH secretion is shaped into discrete pulses, which in turn initiate puberty in the primate.

Pro-opiomelanocortin messenger RNA in hypothalamic neurons is increased by testosterone through aromatization to estradiol.

We have previously demonstrated that neurons in the rostral arcuate nucleus expressing the messenger RNA (mRNA) for pro-opiomelanocortin (POMC) are responsive to modulation by physiological levels of testosterone. It is uncertain, however, whether testosterone's action is mediated through direct activation of androgen receptors or through aromatization to estradiol and subsequent binding to estrogen receptors. We examined this question by evaluating the effectiveness of estradiol and dihydrotestosterone (DHT), a nonaromatizable androgen, in reversing the castration-induced diminution of POMC mRNA in the arcuate nucleus. Using in situ hybridization, we measured POMC mRNA content within arcuate neurons of intact, castrated, castrated testosterone-replaced, castrated estradiol-replaced, and castrated DHT-replaced male rats. Adult male rats were castrated and implanted (s.c.) with a Silastic capsule filled to one of the following specifications: crystalline testosterone (30 mm; n = 4); 17 beta-estradiol (E2) diluted 1:1 with cholesterol (5 mm; n = 4); DHT (40 mm; n = 4); or empty (30 mm; n = 4). Control, sham-operated animals (n = 4) were left intact. Analysis of the results showed that following castration, POMC mRNA content was significantly reduced in cells of the arcuate nucleus (intact: 152 +/- 3 grains/cell vs. castrate: 110 +/- 3 grains/cell). Replacement with physiological levels of testosterone prevented the decline of POMC mRNA levels (castrated testosterone-replaced: 143 +/- 6 grains/cell), as did replacement with physiological levels of estrogen (castrated estrogen-replaced: 149 +/- 8 grains/cell). Treatment with DHT failed to prevent the postcastration decline in POMC mRNA content (castrated DHT-treated: 118 +/- 4 grains/cell).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of somatostatin and growth hormone-releasing hormone gene expression in the rat brain.

We have studied the regulation of somatostatin (SS) and growth hormone-releasing hormone (GHRH) gene expression in the brain of the laboratory rat. We report that hypophysectomy in the adult male reduces SS mRNA in cells of the periventricular nucleus (PeN), while GH reverses this effect. We demonstrate that cellular levels of SS mRNA in the PeN are higher in male compared to female animals. We report that castration reduces cellular levels of GHRH mRNA and SS mRNA in the arcuate nucleus and PeN, respectively, and that testosterone reverses this effect through an androgen receptor-dependent mechanism. Finally, we present a theoretical model to explain the generation of the ultradian rhythm in GH secretion, which implicates the reciprocal interaction between GH feedback and the transcriptional regulation of the SS and GHRH genes and the kinetics of these relationships.

Sex differences in vasopressin neurons in the bed nucleus of the stria terminalis by in situ hybridization.

To determine whether a sex difference exists in the biosynthetic capacity of vasopressingergic (AVP) neurons in the bed nucleus of the stria terminalis (BNST), we have used in situ hybridization and quantitative autoradiography to measure propressophysin messenger RNA levels in these cells from adult male and female rats. We have found that significantly more (p less than 0.01) neurons are labeled in male rats than in female rats and that these labeled cells averaged more grains/cell (p less than 0.05) in males than in females. Therefore, the sexual dimorphism of AVP pathways in the BNST and lateral septum recently shown by immunohistochemistry results from a sex difference in the biosynthetic capacity of these AVP neurons.

Testosterone regulation of proopiomelanocortin messenger ribonucleic acid in the arcuate nucleus of the male rat.

GnRH regulates the secretion of LH and FSH, which stimulate the secretion of testicular hormones. Acting in a reciprocal fashion, these hormones, including testosterone and inhibin, exert a negative feedback effect on GnRH and gonadotropin secretion. Endogenous opioid peptides (EOPs) have been implicated to play a role in steroid-mediated regulation of gonadotropin secretion. In this context, certain steroid hormones (e.g. testosterone) increase EOP activity and ultimately inhibit GnRH secretion; however, the cellular mechanism by which this occurs is unknown. beta-Endorphin is one of these EOPs, and it is derived from a larger precursor molecule, POMC. We tested the hypothesis that testicular hormones and testosterone, in particular, stimulate POMC gene expression in the arcuate nucleus of the male rat brain. First, we compared POMC mRNA levels between intact and castrated male rats. Adult male rats were killed 4 days (n = 4) and 21 days (n = 5) after castration. Intact animals (sham-operated; n = 6) were used as controls. Using in situ hybridization and a computerized image analysis system, we measured the POMC mRNA content in individual cells of the arcuate nucleus. POMC mRNA signal was significantly lower (P less than 0.0003) in both 4-day (126 +/- 2 grains/cell) and 21-day (117 +/- 5 grains/cell) castrates than in controls (142 +/- 2 grains/cell). In a second experiment we tested whether testosterone would reverse the castration-induced loss of POMC message. Again, we castrated animals and immediately implanted them with either empty (sham; n = 6) or testosterone-containing Silastic implants (n = 5) of a size that would deliver physiological levels of testosterone (3.6 +/- 1.5 ng/ml). We observed that testosterone-treated animals had significantly higher levels of POMC mRNA signal (121.8 +/- 3.8 grains/cell) than sham-treated castrates (111.4 +/- 3.6 grains/cell; P less than 0.03) and that the testosterone-treated castrates had POMC mRNA signal levels indistinguishable from those of intact controls (122.0 +/- 1.1 grains/cell). These observations lend credence to the theory that one mechanism by which testosterone may regulate GnRH secretion is by increasing the synthesis of POMC in the arcuate nucleus.