Mihran J. Bakalian

Neuronal tryptophan hydroxylase mRNA expression in the human dorsal and median raphe nuclei: major depression and suicide.

Major depressive disorder (MDD) and suicide are associated with deficient serotonergic neurotransmission. Tryptophan hydroxylase (TPH) is the rate-limiting biosynthetic enzyme for serotonin. Previously, we reported elevated levels of TPH protein in the dorsal raphe nucleus (DRN) of depressed suicides and now examine expression of neuronal TPH2 mRNA in a cohort of matched controls and depressed suicides (n=11 pairs). DRN TPH2 mRNA was measured by densitometric analysis of autoradiograms from in situ hybridization histochemistry experiments. TPH2 mRNA is confirmed as the raphe-specific isoform of TPH in human brain, and is expressed in neurons throughout the anteroposterior extent of the DRN and median raphe nucleus (MRN). TPH2 mRNA expression correlates with TPH protein distribution in the DRN, and has a negative correlation with age. In drug-free suicides, TPH2 expression is 33% higher in the DRN and 17% higher in the MRN as compared to matched nonpsychiatric controls. Higher levels of TPH2 mRNA were found throughout the entire extent of the rostrocaudal axis of the DRN, and were not specific to any single subnucleus. Higher TPH2 mRNA expression may explain more TPH protein observed in depressed suicides and reflect a homeostatic response to deficient brain serotonergic transmission.

Neuron Density and Serotonin Receptor Binding in Prefrontal Cortex in Suicide

Although serotonin receptor and cytoarchitectonic alterations are reported in prefrontal cortex (PFC) in suicide and depression, no study has considered binding relative to neuron density. Therefore, we measured neuron density and serotonin transporter (SERT), 5-HT1A and 5-HT2A binding in matched suicides and controls. Suicides and normal controls (n=15 matched pairs) were psychiatrically characterized. Neuron density and binding were determined in dorsal [Brodmann area (BA) 9] and ventral (BA 47) PFC by stereology and quantitative autoradiography in near-adjacent sections. Binding index was defined as the ratio of receptor binding to neuron density. Suicides had lower neuron density in the gyrus of both areas. The binding index was lower for SERT in BA 47 but not in BA9; the 5-HT1A binding index was higher in BA 9 but not in BA 47, while the 5-HT2A binding index was not different between groups. SERT binding was lower in suicides in BA 47 but not BA 9, while 5-HT1A binding was higher in BA 9 but not BA 47. SERT binding negatively correlated with 5-HT1A binding in BA 47 in suicides. Neuron density decreased with age. The 5-HT1A binding index was higher in females than males. We found lower neuron density and lower SERT binding index in both PFC regions in suicides. More 5-HT1A binding with less SERT binding and the negative correlation in depressed suicides suggests post-synaptic receptor up-regulation, and it is independent of the difference in neuron density. Thus, abnormalities in both cortical neurons and in their serotonergic innervation are present in suicides and future studies will need to determine whether cortical changes reflect the trophic effect of altered serotonin innervation.

Regulation of cortical blood flow by the dorsal raphe nucleus : topographic organization of cerebrovascular regulatory regions

We examined in rat: (1) the time-course and magnitude of change in cortical blood flow (CoBF) following electrical stimulation of the dorsal raphe nucleus (DRN) and (2) whether DRN lesions affect resting CoBF or the cerebrovascular response to CO2. Animals were anesthetized (chloralose), paralyzed, and artificially ventilated. The effect of stimulus frequency (1–200 Hz) and intensity (10–100 μA) on arterial pressure, heart rate, and CoBF was examined; lesions were made electrolytically. CoBF was measured using a laser-Doppler flowmeter with the probe placed extradurally over the parietal sensorimotor cortex. The DRN was computer reconstructed in three dimensions from Nissl stained coronal sections for localization of electrode placements. Brief stimuli (8 s; n = 6) elicited frequency and intensity-dependent increases in arterial pressure, heart rate, and CoBF. Sustained intermittent trains of stimuli of rostral DRN (200 Hz; 1 s on/1 s off; 70 μA) elicited a decrease (85 ± 12% of baseline; n = 9) in CoBF (p < 0.05) while stimulation in caudal DRN resulted in increased CBF (126 ± 13% of baseline; n = 9). Phenylephrine infusion (0.1–1 μg; i.v.; n = 8) increased arterial pressure and CoBF less than that elicited by brief DRN stimulation (p < 0.05). DRN lesions did not affect resting CoBF (140 ± 25 perfusion units (PU) before; 127 ± 16 PU after DRN lesion; p > 0.05, n = 5) or mean arterial pressure (127 ± 13 before; 120 ± 11 after); nor did it affect the cerebrovascular response to change in arterial Pco2. Sustained intermittent stimulation of the DRN can evoke either increases or decreases in CoBF depending on the anatomical sublocalization. The DRN does not tonically maintain resting CoBF, nor participate in the cerebrovascular response to change in Pco2.

Effects of l-tryptophan and other amino acids on electroencephalographic sleep in the rat

Electroencephalographic sleep was quantitated in adult male Sprague-Dawley rats following single injections of the methylesters of tryptophan, valine or alanine. The amino acids were administered at the onset of the daily light period (09.00 h); electrographic data were collected for the succeeding 6-h period. Saline served as the injection control, and fluoxetine, a serotonin-reuptake blocker, as a positive control. The injection of tryptophan methylester (125 mg/kg) caused a delay in rapid eye movement (REM) sleep onset, and significantly reduced the amount of REM sleep during the first 2 h postinjection. Tryptophan produced no effect on sleep onset, nor did it influence total sleep time. Fluoxetine (2.5 mg/kg) produced similar effects, as previously observed. The methylesters of valine and alanine were without effect on REM sleep, when injected at a molar dose equivalent to that for tryptophan. No consistent effects of any of the test substances were noted on non-REM (NREM) sleep or waking time, or on any of the other sleep indices quantitated. Together, the data indicate that tryptophan selectively reduces REM sleep; the effect is not due to a non-specific action of amino acids or their methylesters. The effect on REM sleep may be the consequence of a tryptophan-induced stimulation of 5-HT synthesis and release, since it is like that produced by fluoxetine, a drug that enhances transmission across serotonin synapses.

Synthesis and in vitro evaluation of [18F]BMS-754807: a potential PET ligand for IGF-1R.

Radiosynthesis and in vitro evaluation of [(18)F](S)-1-(4-((5-cyclopropyl-1H-pyrazol-3-yl)amino)pyrrolo[2,1-f][1,2,4]triazin-2-yl)-N-(6-fluoropyridin-3-yl)-2-methylpyrrolidine-2-carboxamide ([(18)F]BMS-754807 or [(18)F]1) a specific IGF-1R inhibitor was performed. [(18)F]1 demonstrated specific binding in vitro to human cancer tissues. Synthesis of reference standard 1 and corresponding bromo derivative (1a), the precursor for radiolabeling were achieved from 2,4-dichloropyrrolo[2,1-f][1,2,4]triazine (4) in three steps with 50% overall yield. The radioproduct was obtained in 8% yield by reacting 1a with [(18)F]TBAF in DMSO at 170°C at high radiochemical purity and specific activity (1-2Ci/μmol, N=10). The proof of concept of IGF-IR imaging with [(18)F]1 was demonstrated by in vitro autoradiography studies using pathologically identified surgically removed grade IV glioblastoma, breast cancer and pancreatic tumor tissues. These studies indicate that [(18)F]1 can be a potential PET tracer for monitoring IGF-1R.

Synthesis and in vitro evaluation of [18F]FECIMBI-36: A potential agonist PET ligand for 5-HT2A/2C receptors.

Radiosynthesis and in vitro evaluation of [(18)F]-2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-(2-fluoroethoxy)benzyl)ethanamine, ([(18)F]FECIMBI-36) or ([(18)F]1), a potential agonist PET imaging agent for 5-HT2A/2C receptors is described. Syntheses of reference standard 1 and the corresponding des-fluoroethyl radiolabeling precursor (2) were achieved with 75% and 65% yields, respectively. In vitro pharmacology assay of FECIMBI-36 by [(3)H]-ketanserin competition binding assay obtained from NIMH-PDSP showed high affinities to 5-HT2AR (Ki = 1nM) and 5-HT2CR (Ki=1.7 nM). Radiolabeling of FECIMBI-36 was achieved from the boc-protected precursor 2 using [(18)F]-fluoroethyltosylate in presence of Cs2CO3 in DMSO followed by removal of the protective group. [(18)F]1 was isolated using RP-HPLC in 25 ± 5% yield, purity > 95% and specific activity 1-2Ci/μmol (N = 6). In vitro autoradiography studies demonstrate that [(18)F]1 selectively label 5-HT2A and 5-HT2C receptors in slide-mounted sections of postmortem human brain using phosphor imaging. Our results indicate the potential of [(18)F]1 for imaging 5-HT2A/2C receptors in the high affinity state in vivo using PET imaging.

Association of BDNF Val66Met Polymorphism and Brain BDNF Levels with Major Depression and Suicide

Abstract Background Brain-derived neurotrophic factor is implicated in the pathophysiology of major depressive disorder and suicide. Both are partly caused by early life adversity, which reduces brain-derived neurotrophic factor protein levels. This study examines the association of brain-derived neurotrophic factor Val66Met polymorphism and brain brain-derived neurotrophic factor levels with depression and suicide. We hypothesized that both major depressive disorder and early life adversity would be associated with the Met allele and lower brain brain-derived neurotrophic factor levels. Such an association would be consistent with low brain-derived neurotrophic factor mediating the effect of early life adversity on adulthood suicide and major depressive disorder. Methods Brain-derived neurotrophic factor Val66Met polymorphism was genotyped in postmortem brains of 37 suicide decedents and 53 nonsuicides. Additionally, brain-derived neurotrophic factor protein levels were determined by Western blot in dorsolateral prefrontal cortex (Brodmann area 9), anterior cingulate cortex (Brodmann area 24), caudal brainstem, and rostral brainstem. The relationships between these measures and major depressive disorder, death by suicide, and reported early life adversity were examined. Results Subjects with the Met allele had an increased risk for depression. Depressed patients also have lower brain-derived neurotrophic factor levels in anterior cingulate cortex and caudal brainstem compared with nondepressed subjects. No effect of history of suicide death or early life adversity was observed with genotype, but lower brain-derived neurotrophic factor levels in the anterior cingulate cortex were found in subjects who had been exposed to early life adversity and/or died by suicide compared with nonsuicide decedents and no reported early life adversity. Conclusions This study provides further evidence implicating low brain brain-derived neurotrophic factor and the brain-derived neurotrophic factor Met allele in major depression risk. Future studies should seek to determine how altered brain-derived neurotrophic factor expression contributes to depression and suicide.

Computerized Three-Dimensional Reconstruction Reveals Cerebrovascular Regulatory Subregions in Rat Brain Stem

Three-dimensional wireframe reconstructions were used to examine the relationship between the anatomical localization of electrode sites and the cerebrovascular response which was elicited by electrical stimulation of the dorsal raphe nucleus (DRN). Reconstructions of the rat brain and DRN were done from atlas plates and from Nissl-stained coronal sections (100-micron increments). Data points were entered and three-dimensional reconstructions were performed using commercially available software and a personal computer. Display of the entire brain yielded views which obscured visualization of the DRN. The data file was edited to reduce the number of contours without affecting the display resolution of the DRN. Selective display of the DRN and electronic rotation from the coronal to a sagittal view revealed a functional organization of the cerebral blood flow responses which was not apparent in two-dimensional coronal sections.

A microcomputer-based sleep system: Data acquisition and system calibration programs

A data acquisition program is described for the Apple II series of microcomputers that allows for continuous, direct monitoring of electrographic elements from cortical, hippocampal and muscle leads from rats. The program detects cortical delta waves and sigma activity, hippocampal theta activity and electromyographic activity. The detected elements are counted and stored in memory at 15 second intervals (bins). Every three hours, the data are transferred to disks for permanent storage and off-line analysis.

Dysregulation of Striatal Dopamine Receptor Binding in Suicide.

Inconsistent evidence implicates disruptions of striatal dopaminergic indices in suicide and major depression. To determine whether there are alterations in the striatal dopamine system in suicide, we conducted a quantitative autoradiographic survey of dopamine transporter (DAT; [3H]mazindol), D1 receptor ([3H]SCH23390), and D2 receptor ([3H]sulpiride) binding in the dorsal striatum postmortem from matched suicides and controls. Axis I and axis II psychiatric diagnosis, recent treatment history, and early life adversity (ELA) were determined by psychological autopsy. Mean DAT, D2, and D1 receptor binding did not differ in suicide. However, there was a positive correlation between D1 and D2 receptor binding in the dorsal striatum of control subjects (R2=0.31, p<0.05) that was not present in suicides (R2=0.00, p=0.97). In suicides and controls with reported ELA, there was no correlation between striatal DAT and D1 receptor binding (R2=0.07, p=0.33), although DAT and D1 receptor binding was positively correlated in subjects with no report of ELA (R2=0.32, p<0.05). After controlling for age, there were no significant ELA-related mean differences. Binding of D1 receptors and DAT throughout the striatum correlated negatively with age (D1 receptor: R2=0.12, p<0.05; DAT: R2=0.36, p<0.001). There appears to be an imbalance in dopaminergic receptor and transporter expression related to suicide that differs from that associated with ELA or age.