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Both gene expression profiling in postmortem human brain and studies using animal models have implicated the fibroblast growth factor (FGF) family in affect regulation and suggest a potential role in the pathophysiology of major depressive disorder (MDD). FGF2, the most widely characterized family member, is down-regulated in the depressed brain and plays a protective role in rodent models of affective disorders. By contrast, using three microarray analyses followed by quantitative RT-PCR confirmation, we show that FGF9 expression is up-regulated in the hippocampus of individuals with MDD, and that FGF9 expression is inversely related to the expression of FGF2. Because little is known about FGF9's function in emotion regulation, we used animal models to shed light on its potential role in affective function. We found that chronic social defeat stress, an animal model recapitulating some aspects of MDD, leads to a significant increase in hippocampal FGF9 expression, paralleling the elevations seen in postmortem human brain tissue. Chronic intracerebroventricular administration of FGF9 increased both anxiety- and depression-like behaviors. In contrast, knocking down FGF9 expression in the dentate gyrus of the hippocampus using a lentiviral vector produced a decrease in FGF9 expression and ameliorated anxiety-like behavior. Collectively, these results suggest that high levels of hippocampal FGF9 play an important role in the development or expression of mood and anxiety disorders. We propose that the relative levels of FGF9 in relation to other members of the FGF family may prove key to understanding vulnerability or resilience in affective disorders.

A cardinal symptom of major depressive disorder (MDD) is the disruption of circadian patterns. However, to date, there is no direct evidence of circadian clock dysregulation in the brains of patients who have MDD. Circadian rhythmicity of gene expression has been observed in animals and peripheral human tissues, but its presence and variability in the human brain were difficult to characterize. Here, we applied time-of-death analysis to gene expression data from high-quality postmortem brains, examining 24-h cyclic patterns in six cortical and limbic regions of 55 subjects with no history of psychiatric or neurological illnesses ("controls") and 34 patients with MDD. Our dataset covered ~12,000 transcripts in the dorsolateral prefrontal cortex, anterior cingulate cortex, hippocampus, amygdala, nucleus accumbens, and cerebellum. Several hundred transcripts in each region showed 24-h cyclic patterns in controls, and >100 transcripts exhibited consistent rhythmicity and phase synchrony across regions. Among the top-ranked rhythmic genes were the canonical clock genes BMAL1(ARNTL), PER1-2-3, NR1D1(REV-ERBa), DBP, BHLHE40 (DEC1), and BHLHE41(DEC2). The phasing of known circadian genes was consistent with data derived from other diurnal mammals. Cyclic patterns were much weaker in the brains of patients with MDD due to shifted peak timing and potentially disrupted phase relationships between individual circadian genes. This transcriptome-wide analysis of the human brain demonstrates a rhythmic rise and fall of gene expression in regions outside of the suprachiasmatic nucleus in control subjects. The description of its breakdown in MDD suggests potentially important molecular targets for treatment of mood disorders.

Bipolar disorder (BP) is a disabling and often life-threatening disorder that affects approximately 1% of the population worldwide. To identify genetic variants that increase the risk of BP, we genotyped on the Illumina HumanHap550 Beadchip 2,076 bipolar cases and 1,676 controls of European ancestry from the National Institute of Mental Health Human Genetics Initiative Repository, and the Prechter Repository and samples collected in London, Toronto, and Dundee. We imputed SNP genotypes and tested for SNP-BP association in each sample and then performed meta-analysis across samples. The strongest association P value for this 2-study meta-analysis was 2.4 x 10(-6). We next imputed SNP genotypes and tested for SNP-BP association based on the publicly available Affymetrix 500K genotype data from the Wellcome Trust Case Control Consortium for 1,868 BP cases and a reference set of 12,831 individuals. A 3-study meta-analysis of 3,683 nonoverlapping cases and 14,507 extended controls on >2.3 M genotyped and imputed SNPs resulted in 3 chromosomal regions with association P approximately 10(-7): 1p31.1 (no known genes), 3p21 (>25 known genes), and 5q15 (MCTP1). The most strongly associated nonsynonymous SNP rs1042779 (OR = 1.19, P = 1.8 x 10(-7)) is in the ITIH1 gene on chromosome 3, with other strongly associated nonsynonymous SNPs in GNL3, NEK4, and ITIH3. Thus, these chromosomal regions harbor genes implicated in cell cycle, neurogenesis, neuroplasticity, and neurosignaling. In addition, we replicated the reported ANK3 association results for SNP rs10994336 in the nonoverlapping GSK sample (OR = 1.37, P = 0.042). Although these results are promising, analysis of additional samples will be required to confirm that variant(s) in these regions influence BP risk.

The heterogeneity of neural gene expression and the spatially limited expression of many low-abundance messenger RNAs in the brain has made cloning and analysis of such messages difficult. To generate amounts of nucleic acids sufficient for use in standard cloning strategies, we have devised a method for producing amplified heterogeneous populations of RNA from limited quantities of cDNA. Whole cerebellar RNA was primed with a synthetic oligonucleotide containing the T7 RNA polymerase promoter sequence 5' to a polythymidylate region. After second-strand cDNA synthesis, T7 RNA polymerase was used to generate amplified antisense RNA (aRNA). Up to 80-fold molar amplification has been achieved from nanogram quantities of cDNA. The amplified material is similar in size distribution to the parent cDNA and shows sequence heterogeneity as assessed by Southern and Northern blot analysis. Specific messages for moderate-abundance mRNAs for actin and guanine nucleotide-binding protein (G-protein) alpha subunits have been detected in the amplified material. By using in situ transcription to generate cDNA, sequences for cyclophilin have been detected in aRNA derived from single cerebellar tissue sections. cDNA derived from a single cerebellar Purkinje cell also has been amplified and yields material that hybridizes to cognate whole RNA and mRNA but not to Escherichia coli RNA.

beta-Endorphin-like immunoreactivity in human ventricular cerebrospinal fluid was measured with a specific radioimmunoassay. The subjects were undergoing a surgical procedure for relief of chronic intractable pain. This procedure involved the focal stimulation of a medial thalamic site adjacent to the wall of the third ventricle. Samples were collected before and during the analgesic stimulation. No beta-endorphin-like immunoreactivity could be detected prior to stimulation, suggesting that baseline levels are below 25 fmol/ml of cerebrospinal fluid. Electrical stimulation led to substantial increases (13- to 20-fold) in immunoreactive material in every subject. These results suggest that beta-endorphin-like material can be released into the ventricular system and may contribute to the pain blockade that results from periventricular stimulation.

alpha-Neo-endorphin and dynorphin immunoreactivities in rat brain were visualized by double antibody immunofluorescence of frozen sections. Antibodies were used that were specific for their respective antigens. The pattern of neuronal immunostaining produced by alpha-neo-endorphin and dynorphin antisera in adjacent serial sections was completely superimposible. No areas were found in which alpha-neo-endorphin or dynorphin immunoreactivities existed alone. The following brain regions contained alpha-neo-endorphin/dynorphin-immunoreactive fibers and terminals: the median forebrain bundle, the internal capsule, the substantia nigra, the hypothalamus, the nucleus accumbens, the hippocampus, and the medulla oblongata. A few fibers were seen in the cerebral cortex and in the corpus striatum. In many regions, this neuronal fiber system seems to overlap the neuronal system previously described to contain [Met]-/[Leu]enkephalin-immunoreactive material. In brains from colchicine-treated animals, numerous alpha-neo-endorphin/dynorphin-immunoreactive neuronal cell bodies were seen in the supraoptic, retrochiasmatic supraoptic, paraventricular, and magnocellular accessory nuclei of the hypothalamus. It is concluded that alpha-neo-endorphin-like and dynorphin-like immunoreactivities are part of the same neuronal system.

Acid acetone extracts of caudate nucleus from bovine brain were found to contain an amidated opioid octapeptide with the following structure: Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-NH2. The peptide has been named metorphamide. Bovine metorphamide appears to be derived by proteolytic cleavage from proenkephalin, the common precursor to [Met5]enkephalin and [Leu5]enkephalin. The cleavage within the precursor giving rise to the carboxyl terminus of metorphamide occurs at a single arginine residue and is followed by transformation of a carboxyl-terminal glycine into an amide group. Metorphamide was detected in bovine caudate nucleus extracts by radioimmunoassay, and it was purified to homogeneity by gel filtration and reversed-phase high performance liquid chromatography. Amino acid composition analysis and automated Edman degradation in the gas-phase sequencer confirmed the postulated amino acid sequence. Carboxyl-terminal amidation of bovine metorphamide was shown by stability to carboxypeptidase A digestion and full crossreactivity in a radioimmunoassay that required the carboxyl-terminal amide as part of the recognition site. A synthetic replicate of metorphamide as well as several synthetic analogs were tested for opioid activity in several bioassays and binding assays, and metorphamide was found to have a high mu-binding activity. Metorphamide is the only known naturally occurring opioid peptide that has a high mu-binding activity. The kappa-binding activity is approximately equal to 50% that of the mu-binding activity, but delta-binding activity is negligible.

mRNA for dopamine beta-hydroxylase [3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1] has been partially purified from poly(A)+ mRNA isolated from a rat pheochromocytoma cell line. Shared antigenic determinants between tyrosine hydroxylase and dopamine beta-hydroxylase allowed us to obtain enriched fractions of dopamine beta-hydroxylase mRNA by immunoprecipitating translated mRNA products with tyrosine hydroxylase antisera. The enriched dopamine beta-hydroxylase mRNA was used to synthesize the corresponding cDNAs, which were then cloned in the Pst I site of pBR322. Recombinant colonies were characterized by an in situ colony immunoassay and hybrid-selected translation. In vitro translation of the mRNA selected from one recombinant clone produced a protein of 75,000 daltons that comigrated with authentic dopamine beta-hydroxylase. Partial proteolysis of both authentic dopamine beta-hydroxylase and the protein encoded by the recombinant clone produced identical peptide patterns.

A specific antiserum was prepared against dynorphin B, an endogenous opioid peptide contained in a recently isolated 4,000-dalton dynorphin. The antiserum did not crossreact with dynorphin A, alpha-neo-endorphin, beta-neo-endorphin, dynorphin-(1-8), or [Leu]enkephalin. In immunohistochemical staining experiments on frozen sections through rat brains from normal and colchicine-treated animals, the antiserum labeled the same neuronal fiber systems previously described as containing both dynorphin A and alpha-neo-endorphin immunoreactive material. The alpha-neo-endorphin/dynorphin A immunoreactive perikarya in the hypothalamic magnocellular nuclei also were labeled by the dynorphin B antiserum. In addition, the dynorphin B antiserum revealed groups of immunoreactive neuronal cell bodies in several other hypothalamic and extrahypothalamic areas, including brain-stem, midbrain, central nucleus of amygdala, and in the dorsomedial, lateral, and anterior nuclei of hypothalamus. These perikarya had not been detected in previous studies that used dynorphin A and alpha-neo-endorphin antisera. The findings are in agreement with recent studies demonstrating a common precursor for dynorphin A, dynorphin B, and alpha-neo-endorphin. The apparently wider distribution of dynorphin B immunoreactive cell bodies compared to alpha-neo-endorphin/dynorphin A immunoreactive perikarya may be a reflection of differential processing of the precursor in different brain regions.

We report the results of the dynamics of a model of the central dopaminergic neuronal system. In particular, for certain values of a parameter k, which monitors the efficacy of dopamine at the postsynaptic receptor, chaotic solutions of the dynamical equations appear--a prediction that correlates with the observed increased variability in behavior among schizophrenics, the rapid fluctuations in motor activity among Parkinsonian patients treated chronically with L-dopa, and the lability of mood in some patients with an affective disorder. Moreover our hypothesis offers specific results concerning the appearance or disappearance of erratic solutions as a function of k and the external input to the dopamine neuronal system.

It has previously been shown that opiates produce selective abolition of aversively motivated classically conditioned responses in the rabbit. The experiments reported here show that these effects are mediated by specific activation of opiate receptors within the central nervous system in that this central activation is both necessary and sufficient to produce opiate-induced abolition of conditioned responding. Further characterization suggests that selective activation through opiate-mu-receptor interactions within the periaqueductal gray/periventricular region of the fourth ventricle may be critical in mediating this abolition.

The biological activity of many proteins and peptides can be profoundly affected by enzyme-catalyzed covalent modifications such as acetylation, sulfation, glycosylation, or amidation. This article describes the cloning of such an enzyme, a peptide acetyltransferase from rat brain that catalyzes the amino-terminal acetylation of endorphins and perhaps other substrates in vivo. Blot-hybridization analysis suggests that the mRNA encoding the acetyltransferase is approximately 2.0 kilobases, is present in whole rat brain and rat hypothalamus, and is slightly larger in mouse AtT20 tumor cells. The acetyltransferase was cloned by using a strategy whereby a cDNA expression library was screened with a solid-phase enzyme-activity assay; this technique combines the use of the substrate coupled to a solid support and subsequent recognition of the product by using a specific antiserum. We have called this method the enzyme immunodetection assay (EIDA). The EIDA should prove useful in the isolation of other clones for proteins that possess enzymatic activity upon expression in bacterial hosts.

Using specific antisera to human beta-lipotropin, we have visualized cells and axons with beta-lipotropin-like immunoreactivity in rat brain and pituitary. The beta-lipotropin so localized is well delineated and contained in the cytoplasm of cells and in beaded axons. Areas of greatest beta-lipotropin content are hypothalamus (with cell bodies in the medial basal hypothalamus and arcuate regions), periventricular nucleus of the thalamus, ansa lenticularis, zona compacta of the substantia nigra, medial amygdaloid nucleus, zona incerta, periaqueductal central gray area, locus ceruleus, and a few fibers in the reticular formation. The question of the exact relationship of beta-lipotropin and methionine-enkephalin remains open, because some brain areas contain both substances and some areas contain only one or the other.

A genetic analysis of differences in adrenal phenylethanolamine N-methyltransferase (EC 2.1.1.X) and tyrosine hydroxylase (EC 1.99.1.X.) and brain tyrosine hydroxylase was performed in three inbred mouse strains. In adrenal glands, the pattern of inheritance of both enzymes was similar; genes carried by the CBA/J strain appeared to be dominant to those of the other strains. The pattern of inheritance of tyrosine hydroxylase activity in adrenals differed from that in the brain. In both tissues, dominant genes appeared to determine intermediate, rather than extreme, levels of enzyme activities.