Circadian dysregulation of clock genes: clues to rapid treatments in major depressive disorder.

Conventional antidepressants require 2-8 weeks for a full clinical response. In contrast, two rapidly acting antidepressant interventions, low-dose ketamine and sleep deprivation (SD) therapy, act within hours to robustly decrease depressive symptoms in a subgroup of major depressive disorder (MDD) patients. Evidence that MDD may be a circadian-related illness is based, in part, on a large set of clinical data showing that diurnal rhythmicity (sleep, temperature, mood and hormone secretion) is altered during depressive episodes. In a microarray study, we observed widespread changes in cyclic gene expression in six regions of postmortem brain tissue of depressed patients matched with controls for time-of-death (TOD). We screened 12 000 transcripts and observed that the core clock genes, essential for controlling virtually all rhythms in the body, showed robust 24-h sinusoidal expression patterns in six brain regions in control subjects. In MDD patients matched for TOD with controls, the expression patterns of the clock genes in brain were significantly dysregulated. Some of the most robust changes were seen in anterior cingulate (ACC). These findings suggest that in addition to structural abnormalities, lesion studies, and the large body of functional brain imaging studies reporting increased activation in the ACC of depressed patients who respond to a wide range of therapies, there may be a circadian dysregulation in clock gene expression in a subgroup of MDDs. Here, we review human, animal and neuronal cell culture data suggesting that both low-dose ketamine and SD can modulate circadian rhythms. We hypothesize that the rapid antidepressant actions of ketamine and SD may act, in part, to reset abnormal clock genes in MDD to restore and stabilize circadian rhythmicity. Conversely, clinical relapse may reflect a desynchronization of the clock, indicative of a reactivation of abnormal clock gene function. Future work could involve identifying specific small molecules capable of resetting and stabilizing clock genes to evaluate if they can rapidly relieve symptoms and sustain improvement.

Altered expression of glutamate signaling, growth factor, and glia genes in the locus coeruleus of patients with major depression.

Several studies have proposed that brain glutamate signaling abnormalities and glial pathology have a role in the etiology of major depressive disorder (MDD). These conclusions were primarily drawn from post-mortem studies in which forebrain brain regions were examined. The locus coeruleus (LC) is the primary source of extensive noradrenergic innervation of the forebrain and as such exerts a powerful regulatory role over cognitive and affective functions, which are dysregulated in MDD. Furthermore, altered noradrenergic neurotransmission is associated with depressive symptoms and is thought to have a role in the pathophysiology of MDD. In the present study we used laser-capture microdissection (LCM) to selectively harvest LC tissue from post-mortem brains of MDD patients, patients with bipolar disorder (BPD) and from psychiatrically normal subjects. Using microarray technology we examined global patterns of gene expression. Differential mRNA expression of select candidate genes was then interrogated using quantitative real-time PCR (qPCR) and in situ hybridization (ISH). Our findings reveal multiple signaling pathway alterations in the LC of MDD but not BPD subjects. These include glutamate signaling genes, SLC1A2, SLC1A3 and GLUL, growth factor genes FGFR3 and TrkB, and several genes exclusively expressed in astroglia. Our data extend previous findings of altered glutamate, astroglial and growth factor functions in MDD for the first time to the brainstem. These findings indicate that such alterations: (1) are unique to MDD and distinguishable from BPD, and (2) affect multiple brain regions, suggesting a whole-brain dysregulation of such functions.

In situ transcription: specific synthesis of complementary DNA in fixed tissue sections.

A technique, in situ transcription, is described, in which reverse transcription of mRNAs is achieved within fixed tissue sections. An oligonucleotide complementary to proopiomelanocortin (POMC) mRNA was used as a primer for the specific synthesis of radiolabeled POMC cDNA in fixed sections of rat pituitary, thus permitting the rapid anatomical localization of POMC mRNA by autoradiography. Intermediate lobe signal intensities were sensitive to dopaminergic drugs, demonstrating that the method can be used for studies of mRNA regulation. The transcripts may also be eluted from tissue sections for a variety of uses, including the identification and cloning of autoradiographically localized cDNAs from small amounts of tissue.

Enkephalin-like material elevated in ventricular cerebrospinal fluid of pain patients after analgetic focal stimulation.

Enkephalin-like activity has been measured in the ventricular cerebrospinal fluid of patients with intractable pain. Electrical stimulation of periventricular brain sites resulted in significant decrease in persistent pain in these subjects. This analgesia, which was blocked by naloxone in 80% of the cases, was accompanied by a significant rise in ventricular enkephalin-like activity, as measured by two different methods. The results present evidence of in vivo release of enkephalin-like material in humans and suggest that stimulation analgesia may be partially due to this release.

Adrenocorticotropin in rat brain: immunocytochemical localization in cells and axons.

By means of antiserum (purified by affinity chromatography) directed against adrenocorticotropin (ACTH) 11-24, cell bodies and beaded axons were visualized in rat brain. The ACTH-like immunoreactivity (ACTH-LI) was primarily located in the hypothalamus (cells and axons). Fibers were scattered throughout thalamus, amygdala, periaqueductal gray area, and reticular formation. There was no change in the distribution of ACTH-LI in rats that had been subjected to hypophysectomy. This distribution of ACTH-LI parallels that of beta-lipotropin and is altered by specific lesions in a similar fashion. The presence of ACTH-LI in cells and beaded axons in brain raises the possibility that it is a neuroregulator functioning as a neurotransmitter, neuromodulator, or neurohormone.

Identification of pro-opiomelanocortin-derived peptides in the human adrenal medulla.

Extracts from adult human adrenals contained high concentrations of immunoreactive beta-endorphin and alpha-melanotropin. Lower quantities of immunoreactive adrenocorticotropic hormone could also be detected. Distribution studies showed the presence of pro-opiomelanocortin fragments in the adrenal medulla. No alpha-melanotropin, beta-endorphin, or adrenocorticotropic hormone could be found in adrenal extracts from several other mammalian species. Analysis of the beta-endorphin-like immunoreactivity using region specific radioimmunoassays interfacing with gel filtration and reverse-phase high-performance liquid chromatography showed the majority of the beta-endorphin-like material to exist as nonacetylated beta-endorphin-(1-31) with a small percentage of lipotropin-sized molecules. The alpha-melanotropin-like immunoreactivity cochromatographed on gel filtration and reverse-phase high-performance liquid chromatography with desacetyl alpha-melanotropin. The data suggest that pro-opiomelanocortin is expressed in the adrenal medulla of humans but is not detectable in the adrenal glands of many other mammalian species.

Epinephrine metabolism in mammalian brain after intravenous and intraventricular administration.

Epinephrine given intravenously or intraventricularly has a half-life in the brain of the rat of 2 to 2.5 hours. After intravenous administration of the drug the principal route of metabolism is O-methylation, whereas after intraventricular administration the principal route is conjugation.

Rapid increase of phenylethanolamine N-methyltransferase by environmental stress in an inbred mouse strains.

The activity of-phenylethanolamine N-methyltransferase in mice of the C57Bl/Ka strain was determined after a 4 degrees C stress. The enzyme activity increased 1.2-fold at the end of 3 hours and by 1.4-fold by the end of 6 hours of the stress. The results are in contrast to those from other species with intact animals in which the enzyme changes only after several days of chronic stress. Cycloheximide prevents the rise in enzyme activity, suggesting the increase may be due to protein synthesis. The increase may provide a model system for studying regulation of catecholamine biosynthetic enzymes.

Effects of naloxone on schizophrenia: reduction in hallucinations in a subpopulation of subjects.

Endogenous opiate-like peptides (endorphins) are putative neuroregulators located throughout the mammalian brainstem. There is some evidence for their role in pain, stress, and affect. We report that the opiate antagonist, naloxone, alters some schizophrenic symptoms. In a double-blind, cross-over study, naloxone produced decreases in auditory hallucinations in some schizophrenic patients. This finding supports the hypothesis that the endorphins may play a roll in modulating hallucinations in a highly selected subgroup of chronically hallucinating schizophrenic patients.

Dopamine beta-hydroxylase activity in brains of chronic schizophrenic patients.

Postmorten brain specimens from nine chronic schizophrenic patients and nine control were assayed for activity of dopamine beta-hydroxylase, the enzyme responsible for the conversion of dopamine to norepinephrine. Unlike the results of previous reports, there was no statistically significant difference in enzyme activity between the patient and control groups. There were, however, significant negative correlations between dopamin beta-hydroxylase activity and the tim spent in the morgue before autopsy, and between enzyme activity of schizophrenics and dosage of chlorpromazine or its equivalent.

Immunoreactive dynorphin-(1-8) and corticotropin- releasing factor in subpopulation of hypothalamic neurons.

Immunoreactive corticotropin-releasing factor (CRF) and dynorphin-(I-8) were visualized in rat hypothalamus by immunohistofluorescence with specific antibodies. In brains from colchicine-treated, adrenalectomized rats, neuronal perikarya with immunoreactive CRF were observed in the paraventricular nucleus of the hypothalamus. The CRF occurred together with the dynorphin-(1-8). However, the CRF immunoreactivity occurred only in a subpopulation of the dynorphin-(1-8) immunoreactive cells. These findings suggest that there may be a functional interrelationship of CRF with dynorphin-related opioid peptides and provide further evidence that neurons may contain more than one bioactive substance.

Novel peptide neuronal system in rat brain and pituitary.

Immunohistofluorescence studies of the rat central nervous system with antibodies to Phe-Met-Arg-Phe-NH2 (molluskan cardioexcitatory peptide) revealed a widespread neuronal system in the brain, spinal cord, and posterior pituitary. Immunoreactive axons and cell bodies were mainly located in cortical, limbic, and hypothalamic areas. Immunostaining of serial sections of the brain and pituitary showed that the Phe-Met-Arg-Phe-NH2 immunoreactive neurons were different from neurons labeled by antibodies to either Met-enkephalin or the putative Met-enkephalin precursor Tyr-Gly-Gly-Phe-Met-Arg-Phe, which is structurally related to Phe-Met-Arg-Phe-NH2. Control staining by antiserum absorption and radioimmunoassay indicated that the antibodies that caused the specific immunofluorescence recognized peptides with an amidated Arg-Phe sequence at the carboxyl terminus.

Brain norepinephrine: enhanced turnover after rubidium treatment.

After biosynthesis of norepinephrine was inhibited, treatment of rats for 10 days with rubidium chloride (0.6 milliequivalent per kilogram of body weight) caused an increase in the rate of disappearance of norepinephrine in the brainstem but not in the telencephalon. Also the utilization of intracisternally injected tritiated norepinephrine was increased and was accompanied by a shift in the pattern of norepinephrine metabolism to normetanephrine. These data suggest that greater amounts of neuronally stored norepinephrine were released to central adrenergic receptors.

Sexual behavior of male cats after administration of parachlorophenylalanine.

The behavior of 12 male cats was observed before and after six or eight daily injections of parachlorophenylalanine. Sexual performance was either unchanged or diminished; aggressive behavior was not seen. Serotonin concentrations in the brains were uniformly lowered.

Behavioral neurochemistry: neuroregulators and behavioral states.

There is compelling evidence that behavioral events after neurochemical function and that altered neurochemical function can change behavior. Such processes have been related both to neurotransmitters and to neuromodulators, together termed neuroregulators. Available research tools and theoretical constructs have begun to permit studies of certain types of behavior, primarily those related to emotional states and drives. This work is changing long-held concepts about severe mental disorders and the treatment of them.

Tryptoline formation by a preparation from brain with 5-methyltetrahydrofolic acid and tryptamine.

An enzymatic preparation from human brain converts tryptamine to tryptoline (9H-1,2,3,4-tetrahydropyrido[3,4-b]indole) in the presence of 5-methyltetrahydrofolic acid. Similarly, N-methyltryptamine and 5-hydroxytryptamine yield 1-methyltryptoline and 5-hydroxytryptoline, respectively. Neither in vitro nor in vivo formation of these compounds by human tissues has been described.

Narcolepsy: biogenic amine deficits in an animal model.

Concentrations of biogenic amine metabolites in discrete brain areas differed significantly between dogs with genetically transmitted narcolepsy and age- and breed-matched controls. Dopamine and 3,4-dihydroxyphenylacetic acid were consistently elevated in the brains of narcoleptic animals, while homovanillic acid was not. Narcoleptic animals consistently exhibited lower utilization of dopamine and higher intraneuronal degradation of dopamine but no uniform decrease in serotonin utilization. Hence neuropathology appears to be associated with genetically transmitted canine narcolepsy. The data indicate a nonglobal depression of dopamine utilization or turnover or both.

Corticosterone: a critical factor in an opioid form of stress-induced analgesia.

The finding that some opioid-mediated forms of stress-induced analgesia are antagonized by hypophysectomy and dexamethasone has led to the suggestion that beta-endorphin, released from the pituitary, may mediate these analgesic reactions. "Long-term analgesia" (an opioid-mediated form of stress-induced analgesia), which is blocked by dexamethasone and hypophysectomy, was also blocked by adrenalectomy and reinstated with corticosterone therapy. Corticosterone is proposed to play a permissive role in long-term analgesia and to be a critical hormone mediating this phenomenon.

Electrophysiological evaluation of extracellular spermine and alkaline pH on synaptic human GABAA receptors.

Polyamines have fundamental roles in brain homeostasis as key modulators of cellular excitability. Several studies have suggested alterations in polyamine metabolism in stress related disorders, suicide, depression, and neurodegeneration, making the pharmacological modulation of polyamines a highly appealing therapeutic strategy. Polyamines are small aliphatic molecules that can modulate cationic channels involved in neuronal excitability. Previous indirect evidence has suggested that polyamines can modulate anionic GABAA receptors (GABAARs), which mediate inhibitory signaling and provide a direct route to reduce hyperexcitability. Here, we attempted to characterize the effect that spermine, the polyamine with the strongest reported effect on GABAARs, has on human postmortem native GABAARs. We microtransplanted human synaptic membranes from the dorsolateral prefrontal cortex of four cases with no history of mental or neurological disorders, and directly recorded spermine effects on ionic GABAARs responses on microtransplanted oocytes. We show that in human synapses, inhibition of GABAARs by spermine was better explained by alkalization of the extracellular solution. Additionally, spermine had no effect on the potentiation of GABA-currents by diazepam, indicating that even if diazepam binding is enhanced by spermine, it does not translate to changes in functional activity. Our results clearly demonstrate that while extracellular spermine does not have direct effects on human native synaptic GABAARs, spermine-mediated shifts of pH inhibit GABAARs. Potential spermine-mediated increase of pH in synapses in vivo may therefore participate in increased neuronal activity observed during physiological and pathological states, and during metabolic alterations that increase the release of spermine to the extracellular milieu.

Quantitative validation of immunofluorescence and lectin staining using reduced CLARITY acrylamide formulations.

The CLARITY technique enables three-dimensional visualization of fluorescent-labeled biomolecules in clarified intact brain samples, affording a unique view of molecular neuroanatomy and neurocircuitry. It is therefore, essential to find the ideal combination for clearing tissue and detecting the fluorescent-labeled signal. This method requires the formation of a formaldehyde-acrylamide fixative-generated hydrogel mesh through which cellular lipid is removed with sodium dodecyl sulfate. Several laboratories have used differential acrylamide and detergent concentrations to achieve better tissue clearing and antibody penetration, but the potential effects upon fluorescent signal retention is largely unknown. In an effort to optimize CLARITY processing procedures we performed quantitative parvalbumin immunofluorescence and lectin-based vasculature staining using either 4 or 8% sodium dodecyl sulfate detergent in combination with different acrylamide formulas in mouse brain slices. Using both confocal and CLARITY-optimized lightsheet microscope-acquired images, we demonstrate that 2% acrylamide monomer combined with 0.0125% bis-acrylamide and cleared with 4% sodium dodecyl sulfate generally provides the most optimal signal visualization amongst various hydrogel monomer concentrations, lipid removal times, and detergent concentrations.