Behavioral and neurochemical phenotyping of Homer1 mutant mice: possible relevance to schizophrenia.

Homer proteins are involved in the functional assembly of postsynaptic density proteins at glutamatergic synapses and are implicated in learning, memory and drug addiction. Here, we report that Homer1-knockout (Homer1-KO) mice exhibit behavioral and neurochemical abnormalities that are consistent with the animal models of schizophrenia. Relative to wild-type mice, Homer1-KO mice exhibited deficits in radial arm maze performance, impaired prepulse inhibition, enhanced 'behavioral despair', increased anxiety in a novel objects test, enhanced reactivity to novel environments, decreased instrumental responding for sucrose and enhanced MK-801- and methamphetamine-stimulated motor behavior. No-net-flux in vivo microdialysis revealed a decrease in extracellular glutamate content in the nucleus accumbens and an increase in the prefrontal cortex. Moreover, in Homer1-KO mice, cocaine did not stimulate a rise in frontal cortex extracellular glutamate levels, suggesting hypofrontality. These behavioral and neurochemical data derived from Homer1 mutant mice are consistent with the recent association of schizophrenia with a single-nucleotide polymorphism in the Homer1 gene and suggest that the regulation of extracellular levels of glutamate within limbo-corticostriatal structures by Homer1 gene products may be involved in the pathogenesis of this neuropsychiatric disorder.

Using reporter genes to label selected neuronal populations in transgenic mice for gene promoter, anatomical, and physiological studies.

This review summarizes recent work on the use of reporter genes to label selected neuronal populations in transgenic mice, with particular emphasis on gonadotropin-releasing hormone (GnRH) neurons. Reporter genes discussed are the lacZ, green fluorescent protein (GFP), luc, and bla genes, which encode the reporter proteins beta-galactosidase, GFP, luciferase, and beta-lactamase, respectively. Targeted transgenic expression of these reporter proteins is obtained by fusing the corresponding reporter gene, with or without a subcellular localization signal, to a cell type- or brain region-specific gene promoter. Mice carrying GnRH promoter-driven reporter genes have proven useful for revealing the promoter elements required for cell type-specific expression of GnRH, the full anatomical profile of the GnRH neuronal network, and its electrophysiological activity, suggesting that similar approaches will assist in elucidating the properties of other neuronal populations as well.

The distribution of splice variants of the NMDAR1 subunit mRNA in adult rat brain.

Regional variation in the alternative splice forms of the NMDAR1 subunit mRNA was investigated by in situ hybridization in the adult rat brain, using radiolabelled splice-specific oligonucleotide probes. Each splice variant was detected in an individual distribution. The NMDAR1-a and NMDAR1-2 forms were widely and abundantly distributed throughout the brain, except for the inferior colliculus. The NMDAR1-b and NMDAR1-4 variants were located in similar patterns in fewer areas (e.g. parietal cortex, hippocampus CA3, thalamus, inferior colliculus, cerebellar granule cells). In contrast, the NMDAR1-1 forms were distributed in a pattern approximately complementary in the forebrain to that of NMDAR1-4 (weakly expressed in thalamus and inferior colliculus). The NMDAR1-3 variants were not abundant in any structure. Considerable overlap of the in situ hybridization images was noted, so all eight splice combinations are possible in heterogenous distributions. Correlation of the distribution of NMDAR1 mRNA splice forms with functional analyses of heteromeric recombinant receptors will be necessary to ascertain if alternative splicing of the NMDAR1 subunit can account for some of the known heterogeneity of endogenous NMDA receptors.

Small N-terminal deletion by splicing in cerebellar alpha 6 subunit abolishes GABAA receptor function.

Sequence variation was found in cDNA coding for the extracellular domain of the rat gamma-aminobutyric acid type A (GABAA) receptor alpha 6 subunit. About 20% of polymerase chain reaction (PCR)-amplified alpha 6 cDNA prepared from rat cerebellar mRNA lacked nucleotides 226-255 as estimated by counting single-stranded phage plaques hybridized specifically to the short (alpha 6S) and long (wild-type) forms of the alpha 6 mRNA. Genomic PCR revealed an intron located upstream of the 30-nucleotide sequence. Both splice forms were detected in the cerebellum by in situ hybridization. Recombinant receptors, resulting from coexpression of the alpha 6S subunit with the GABAA receptor beta 2 and gamma 2 subunits in human embryonic kidney 293 cells, were inactive at binding [3H]muscimol and [3H]Ro 15-4513. In agreement, injection of complementary RNAs encoding the same subunits into Xenopus oocytes produced only weak GABA-induced currents, indistinguishable from those produced by beta 2 gamma 2 receptors. Therefore, the 10 amino acids encoded by the 30-nucleotide fragment may be essential for the correct assembly or folding of the alpha 6 subunit-containing receptors.

A complex mosaic of high-affinity kainate receptors in rat brain.

The significance for CNS function of glutamate-gated cation channels that exhibit high-affinity kainate sites is not understood. Such receptors, which on dorsal root ganglia and in recombinant systems exhibit currents that rapidly desensitize to kainate application, have not been detected electrophysiologically in the brain. However, a comparison of the distribution of mRNAs encoding five glutamate receptor subunits exhibiting high-affinity kainate sites (GluR-5-GluR-7, KA-1, and KA-2) indicates that high-affinity kainate receptors are most likely involved in all central neuronal circuits of the rat brain. The KA-1 mRNA occurs mainly in the CA3 field of the hippocampus and dentate gyrus, with much lower amounts being found in inner cortical layers, cerebellar Purkinje cells, and white matter (e.g., corpus callosum and anterior commissure). The KA-2 gene is widely expressed in many neuronal nuclei including layers II-VI of neocortex, hippocampal pyramidal (CA1-CA3) and dentate granule cells, septal nuclei such as the bed nucleus of the stria terminalis, medial preoptic, suprachiasmatic, and ventral medial hypothalamic nuclei, dorsal raphe, locus coeruleus, and cerebellar granule cells. KA-2 mRNA is also found in the pineal gland. GluR-5 transcripts are in the cingulate and piriform cortex, the subiculum, lateral septal nuclei, anteroventral thalamus, suprachiasmatic nucleus, the tegmental nuclei, pontine nuclei, and Purkinje cells. GluR-6 mRNA is most abundant in cerebellar granule cells, with lower levels in caudate-putamen and the pyramidal cell layers and dentate granule cells of hippocampus. The GluR-7 gene is prominently expressed in the inner neocortical layers and some cells in layer II, subiculum, caudate-putamen, reticular thalamus, ventral medial hypothalamic nucleus, pontine nuclei, and in putative stellate/basket cells in the cerebellum. These findings suggest that a complex mosaic of receptor variants underlies the high-affinity kainate receptor in the vertebrate brain.

Heteromeric NMDA receptors: molecular and functional distinction of subtypes.

The N-methyl D-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent sensitivity to magnesium and is modulated by glycine. Molecular cloning identified three complementary DNA species of rat brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% identical in sequence. These are structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the NMDA receptor subunit NMDAR1 (NR1). Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate- and NMDA-activated currents only when they were in heteromeric configurations with NR1. NR1-NR2A and NR1-NR2C channels differed in gating behavior and magnesium sensitivity. Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution.

The gonadotropin-releasing hormone associated peptide reduces calcium entry in prolactin-secreting cells.

The precursor molecule to the GnRH contains a peptide named GnRH-associated peptide (GAP) with PRL-inhibiting properties. In this work, we have studied the electrophysiological properties and responses to GAP of three different types of PRL-secreting cells: 1) the rat tumor cell line GH3, 2) normal rat pituitary cells in primary culture, and 3) human PRL-secreting adenoma cells. Using different but complementary techniques we show that GAP reduces intracellular Ca++ levels, [Ca++]i, and inhibits Ca++ transients in these cells. This reduction of [Ca++]i results from coordinate actions of GAP on K+ and Ca++ conductances and may explain the inhibitory effect of GAP on hormonal secretion by PRL-secreting cells.

The rat gonadotropin-releasing hormone: SH locus: structure and hypothalamic expression.

The rat GnRH gene as expressed in the central nervous system is comprised of four exons and three introns and spans 4.5 kilobases of genomic DNA. Recently it has been shown that the DNA strand opposite that which is transcribed to produce GnRH mRNA is transcribed in heart to produce a set of transcripts, SH RNAs, which share significant exonic sequences with the GnRH gene. The nucleotide sequence of this locus and approximately 3 kilobases on either side has been determined. Northern analysis of hypothalamic RNA probed with GnRH and SH strand specific probes demonstrate that both GnRH and SH RNAs are present within the preoptic hypothalamus. The cap sites for GnRH and SH transcripts have been localized using polymerase chain reaction technology. Results from these experiments indicate that in the preoptic hypothalamus GnRH transcription initiates from three sites. The majority of GnRH transcripts is spliced efficiently and gives rise to the major class of GnRH mRNA. A second spliced population is present in lower abundance, while a third population is not spliced. The SH gene contains at least two distinct promoters, from which two populations of transcripts are derived containing unique 5'-sequences spliced to a common 3'-region.

Structure and functional expression of cloned rat serotonin 5HT-2 receptor.

A complementary DNA (cDNA) encoding a serotonin receptor with 51% sequence identity to the 5HT-1C subtype was isolated from a rat brain cDNA library by homology screening. Transient expression of the cloned cDNA in mammalian cells was used to establish the pharmacological profile of the encoded receptor polypeptide. Membranes from transfected cells showed high-affinity binding of the serotonin antagonists spiperone, ketanserin and mianserin, low affinity for haloperidol (a dopamine D2 receptor antagonist), 8-OH-DPAT as well as MDL-72222 and no detectable binding of [3H]serotonin. This profile is consonant with the 5HT-2 subtype of serotonin receptors. In agreement with this assignment, serotonin increased the intracellular Ca2+ concentration and activated phosphoinositide hydrolysis in transfected mammalian cells. The agonist also elicited a current flow, blocked by spiperone, in Xenopus oocytes injected with in vitro synthesized RNA containing the cloned nucleotide sequences.

Molecular forms of gonadotropin-releasing hormone associated peptide (GAP): changes within the rat hypothalamus and release from hypothalamic cells in vitro.

We have developed RIAs using antisera directed against the cryptic peptide of the GnRH precursor (termed GnRH-associated peptide, GAP) and have used these together with a GnRH assay to characterize proGnRH-derived peptides in rat hypothalamic extracts. On Sephadex chromatography we have identified three molecular forms of GAP-like immunoreactivity (GAP-LI), in addition to the GnRH decapeptide. The largest of these forms is an 8.0-kilodalton (kD) GAP-LI which appears to be the complete proGnRH peptide. The second is a 6.5-kD GAP-LI, and is similar to the complete cryptic peptide (i.e. proGnRH14-69 or GAP1.56). The third peptide is a 2.5 kD C-terminal fragment of the cryptic peptide, representing a processed form of GAP. In whole hypothalamic extracts from normal rats the 8.0-kD form was the major form, comprising 60-70% of the total GAP-LI. All three forms were present in three distinct areas of the rat hypothalamus, namely median eminence (ME), anterior and mid-hypothalamus. However in the ME the proportion of 8.0-kD GAP-LI was significantly reduced and the proportion of 6.5-kD GAP-LI significantly increased compared to anterior and mid-hypothalamic samples (p less than 0.05). In whole hypothalamic extracts from pregnant and lactating rats the total content of proGnRH-derived peptides was reduced but the relative proportions of these peptides were not significantly changed from normal female rats. However, in postlactating rats, 2 weeks after removal of pups, the total levels of GAP-LI were unchanged compared to normals, but the percentage of 8.0-kD GAP-LI was significantly decreased and the percentage of 2.5-kD GAP-LI significantly increased compared to normals (p less than 0.05), suggesting that proGnRH may undergo additional processing dependent on physiological condition. In fetal and neonatal rats the proportion of the 6.5-kD peptide was increased and that of the 8.0-kD peptide decreased compared to adults, and this change became less significant with increasing age. In ovariectomized rats the proportion of 6.5-kD GAP-LI was increased and that of 8.0-kD GAP-LI decreased; this change was partially reversed with steroid treatment. Both the 6.5 and 2.5-kD forms were released by high K+ stimulation of neonatal hypothalamic cells in culture. These results indicate that there is differential processing of the proGnRH precursor within the hypothalamus and in altered physiological states.

In situ hybridization histochemistry for messenger ribonucleic acid (mRNA) encoding gonadotropin-releasing hormone (GnRH): effect of estrogen on cellular levels of GnRH mRNA in female rat brain.

Using in situ hybridization histochemistry, we have detected perikarya containing mRNA encoding GnRH and GnRH-associated peptide (GAP) in rat brains. Synthetic DNA oligomers with sequences complementary to the rat cDNA encoding the GnRH structural region and the GAP structural region were hybridized to formaldehyde-fixed coronal sections. The distribution and number of cells containing GnRH/GAP mRNA were similar to those shown by immunocytochemical studies. The areas in which GnRH mRNA perikarya were shown included the medial septal area, the diagonal band of Broca, the preoptic area, and the anterior hypothalamus. Up to 55 cells were detected in a single 12-micron section containing the diagonal band and organum vasculosum lamina terminalis (OVLT) whereas cell numbers diminished in more caudal regions. In addition, both probes labeled the same cells contained within adjacent sections. We used this technique to examine the effect of estrogen on GnRH mRNA levels in the area of the OVLT of normal and androgen-sterilized female rats, using an estrogen treatment paradigm previously characterized in studies investigating the hypothalamic regulation of negative and positive estrogen feedback. We found that 7 days after ovariectomy, 2 days of estrogen treatment resulted in a significant reduction in the average cellular level of GnRH mRNA in both normal and androgen-sterilized females. Analysis of histograms relating the intensity of labeling to the abundance of cells suggested that a small population of GnRH cells responded to the estrogen treatment. However, we found no evidence for a discrete neuroanatomical segregation of such a subpopulation of GnRH-responsive cells within the area of the OVLT.

Processing of luteinizing hormone-releasing hormone precursor in rat neurons.

Results of previous immunocytochemical studies indicate that in the rat brain proteolytic cleaving of LHRH precursors to generate the physiologically active decapeptide takes place within neuronal fibers and terminals and not within perikarya. A 69-amino acid (aa) LHRH precursor comprised of the decapeptide, a 3-aa cleavage and amidation site, and a 56-aa C-terminal extension has recently been characterized. Two antisera generated to specific aa sequences of the C-terminal extension (RM 8/5, anti aa 14-26; PS 39A, anti aa 40-53) and two directed to specific regions of the LHRH decapeptide (RM 1076, anti aa 4-8; A 422 generated to the N-terminal pGlu and C-terminal amidated Gly) were used to further examine intraneuronal sites of precursor processing. Patterns of immunoreactivity revealed with antisera directed to non-LHRH sequences of LHRH precursor paralleled those observed with antisera to the decapeptide. Immunopositive perikarya, processes, and neurovascular terminals were observed with PS 39A. Antiserum PS 39A binds to an internal aa sequence of the C-terminal extension and would, therefore, be expected to detect intact precursor LHRH as well as products of proteolytic cleavage. In contrast, only immunopositive processes and neurovascular terminals were observed with RM 8/5, an antiserum directed to an initial aa sequence of the C-terminal extension. The pattern of immunoreactivity revealed with RM 8/5 resembled that observed with an antiserum that binds the fully processed decapeptide (A 422), indicating that proteolytic cleavage of the decapeptide from the C-terminal extension is required for binding by this antiserum. Furthermore, the restricted distribution of reaction product observed with RM 8/5 relative to A 422 suggests that additional processing of the C-terminal extension may be required for binding. Such additional processing appears to occur in neurovascular terminals of the median eminence.

GnRH-associated peptide (GAP) is cosecreted with GnRH into the hypophyseal portal blood of ovariectomized sheep.

The secretion of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) into sheep hypothalamo-hypophyseal portal blood was investigated in ovariectomized ewes. GAP and GnRH were cosecreted into portal blood as determined in pooled 'peak' and 'trough' samples. The temporal pattern of GAP secretion into portal blood was coincidental with that of luteinizing hormone (LH) secretion into peripheral blood in three individual animals. The data provide the first evidence that GAP is a secretory product from mammalian hypothalamus and establish the temporal coexistence of the two peptides which appears to be of physiological significance in the regulation of pituitary function.

The hypogonadal mouse: reproductive functions restored by gene therapy.

The hypogonadal (hpg) mouse lacks a complete gonadotropin-releasing hormone (GnRH) gene and consequently cannot reproduce. Introduction of an intact GnRH gene into the genome of these mutant mice resulted in complete reversal of the hypogonadal phenotype. Transgenic hpg/hpg homozygotes of both sexes were capable of mating and producing offspring. Pituitary and serum concentrations of luteinizing hormone, follicle-stimulating hormone, and prolactin were restored to those of normal animals. Immunocytochemistry and in situ hybridization showed that GnRH expression was restored in the appropriate hypothalamic neurons of the transgenic hpg animals, an indication of neural-specific expression of the introduced gene.

Isolation of the gene and hypothalamic cDNA for the common precursor of gonadotropin-releasing hormone and prolactin release-inhibiting factor in human and rat.

Cloned cDNAs encoding the precursor protein for gonadotropin-releasing hormone (Gn-RH) and prolactin release-inhibiting factor (PIF) were isolated from libraries derived from human and rat hypothalamic mRNA. Nucleotide sequence analyses predict precursor proteins of 92 amino acids for both species and show identity between the human placental and human hypothalamic precursor proteins. Whereas the Gn-RH peptide structure is completely conserved in human and rat, the PIF domain of the precursor displays 70% interspecies homology. Genomic analyses revealed the presence of a single Gn-RH-PIF gene in human and rat containing sequences corresponding to the cDNA distributed across four exons.