Localized decrease in serotonin transporter-immunoreactive axons in the prefrontal cortex of depressed subjects committing suicide.

A variety of postmortem brain studies and clinical investigations have provided evidence that reduced serotonin neurotransmission is associated with suicidal behavior and depression, and several serotonergic parameters have been found to be altered in the prefrontal cortex of suicide victims. However, the integrity of the serotonin innervation of the prefrontal cortex in mood disorders has not been directly investigated. The present study used immunocytochemical methods and an antibody against the serotonin transporter to examine the relative density of serotonin axons in the dorsolateral prefrontal cortex of suicide victims with a diagnosis of major depression. The mean total length of serotonin transporter-immunoreactive axons per unit area was unchanged in layers 2 and 4 of area 46 in the depressed suicide subjects compared to controls, but was significantly (P < 0.01) decreased by 24% in layer 6 in the depressed suicide group. The total length of serotonin transporter-positive axons in layer 6 was reduced in eight of the 12 depressed suicide subjects compared to their matched control subjects. These findings reveal that depressed subjects who have committed suicide exhibit a lamina-specific reduction in a marker of serotonin axons in the dorsolateral prefrontal cortex that may reflect an alteration in cortical serotonin neurotransmission.

Dopamine transporter immunoreactivity in monkey cerebral cortex: regional, laminar, and ultrastructural localization.

Dopamine (DA) influences a number of cognitive and motor functions that are mediated by the primate cerebral cortex, and the DA membrane transporter (DAT) is known to be a critical regulator of DA neurotransmission in subcortical structures in rodents. To gain insight into the possible functional role of cortical DAT, we compared the regional, laminar, and ultrastructural distribution of DAT immunoreactivity to that of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA synthesis, in the cerebral cortex of macaque monkeys. DAT-immunoreactive (DAT-IR) axons were present throughout the cortical mantle, with substantial differences in density and laminar distribution across cytoarchitectonic areas. In particular, high densities of DAT-IR axons were present in certain regions (e.g., posterior parietal cortex, dentate gyrus) not previously thought to receive a substantial DA input. The laminar distribution of DAT-IR axons ranged from a restricted localization of labeled axons to layer 1 in lightly innervated regions to the presence of axons in all six cortical layers, with a particularly dense plexus in deep layer 3, in highly innervated regions. These regional and laminar patterns paralleled those of TH-IR axons, but several differences in fiber morphology and ultrastructural localization of DAT were observed. For example, in contrast to TH, DAT immunoreactivity in the cortex was localized predominantly to small-diameter profiles, whereas, in the dorsolateral caudate nucleus, DAT and TH immunoreactivities were present in both large-diameter and small-diameter profiles, which may represent varicose and intervaricose axon segments, respectively. Overall, the distribution of DAT-IR axons confirms and extends the results of previous reports, using other markers of DA axons, that the DA innervation of the primate cerebral cortex is global but specialized on both a regional basis and a laminar basis. In particular, these observations reveal an anatomical substrate for a direct and potent influence of DA over neuronal activity in posterior parietal cortex and in certain regions of the temporal lobe. However, due to its predominant distribution to small-diameter profiles, immunoreactivity for DAT may not be an appropriate ultrastructural marker for larger DA varicosities in the primate cortex. Moreover, this distribution of DAT suggests that cortical DA fibers may permit greater neurotransmitter diffusion than subcortical DA axons.

Reaction of oxidized dopamine with endogenous cysteine residues in the human dopamine transporter.

There is evidence to suggest that dopamine (DA) oxidizes to form dopamine ortho-quinone (DAQ), which binds covalently to nucleophilic sulfhydryl groups on protein cysteinyl residues. This reaction has been shown to inhibit dopamine uptake, as well as other biological processes. We have identified specific cysteine residues in the human dopamine transporter (hDAT) that are modified by this electron-deficient substrate analog. DAQ reactivity was inferred from its effects on the binding of [(3)H]2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (beta-CFT) to hDAT cysteine mutant constructs. One construct, X5C, had four cysteines mutated to alanine and one to phenylalanine (Cys(90)A, Cys(135)A, C306A, C319F and Cys(342)A). In membrane preparations 1 mM DAQ did not affect [(3)H]beta-CFT binding to X5C hDAT, in contrast to its effect in wild-type hDAT in which it reduced the B:(max) value by more than half. Wild-type cysteines were substituted back into X5C, one at a time, and the ability of DAQ to inhibit [(3)H]beta-CFT binding was assessed. Reactivity of DAQ with Cys(90) increased the affinity of [(3)H]beta-CFT for the transporter, whereas reactivity with Cys(135) decreased the affinity of [(3)H]beta-CFT. DAQ did not change the K:(D) for [(3)H]beta-CFT binding to wild-type. The reactivity of DAQ at Cys(342) decreased B:(max) to the same degree as wild-type. The latter result suggests that Cys(342) is the wild-type residue most responsible for DAQ-induced inhibition of [(3)H]beta-CFT binding.

Lamina-specific alterations in the dopamine innervation of the prefrontal cortex in schizophrenic subjects.

OBJECTIVE: Abnormalities in dopamine neurotransmission in the prefrontal cortex have been implicated in the pathophysiology of schizophrenia. However, the integrity of the dopamine projections to the prefrontal cortex in this disorder has not been directly examined. METHOD: The authors employed immunocytochemical methods and antibodies against tyrosine hydroxylase, the rate-limiting enzyme in dopamine biosynthesis, and the dopamine membrane transporter to examine dopamine axons in the dorsomedial prefrontal cortex (area 9) from 16 pairs of schizophrenic and matched control subjects. RESULTS: Compared to the control subjects, the total length of tyrosine hydroxylase-immunoreactive axons was unchanged in the superficial and middle layers of the schizophrenic subjects but was reduced by an average of 33.6% in layer 6. The total length of tyrosine hydroxylase-positive axons in layer 6 was decreased in 13 of the schizophrenic subjects compared to their control subjects. Axons immunoreactive for the dopamine membrane transporter showed a similar pattern of change. In contrast, axons labeled for the serotonin transporter did not differ between schizophrenic and control subjects in any layer examined. In addition, the density of tyrosine hydroxylase-containing axons did not differ between monkeys chronically treated with haloperidol and matched control animals. CONCLUSIONS: These findings reveal that schizophrenia is associated with an altered dopamine innervation of prefrontal cortex area 9 that is lamina- and neurotransmitter-specific and that does not appear to be a consequence of pharmacological treatment. Together, these data provide direct evidence for a disturbance in dopamine neurotransmission in the prefrontal cortex of schizophrenic subjects.

MR imaging of the hippocampus: measurement of T2 with four dual-echo techniques.

PURPOSE: To assess the effect of cerebrospinal fluid (CSF) signal nulling and different magnetic resonance (MR) imaging read-out implementations on the precision of T2 measurements in the hippocampus. MATERIALS AND METHODS: Oblique coronal MR imaging was performed in 12 healthy volunteers by using four dual-echo MR sequences: conventional spin-echo (SE), fast SE, gradient-echo and SE (GRASE), and fast fluid-attenuated inversion-recovery (FLAIR) imaging. T2 measurements were obtained from T2 maps derived from regions of interest within the head of both hippocampi. T2 measurements obtained with the four MR sequences were compared, as were differences in T2 between right and left hippocampi. Interrater and test-retest consistency were determined. RESULTS: Fast FLAIR imaging provided the smallest normal range and SD of T2 measurements. Fast FLAIR and fast SE imaging provided the smallest coefficients of variation. GRASE imaging provided a lower coefficient of variation than that of conventional SE. The mean difference between left and right hippocampi was smallest at fast FLAIR imaging. CONCLUSION: Use of a CSF-nulling inversion prepulse, fast SE read-out, or GRASE read-out MR sequence helps improve the precision of the hippocampal T2 measurements in comparison with use of conventional SE imaging. T2 measurements obtained at dual-echo fast FLAIR imaging may help detect subtle hippocampal abnormalities.

Spontaneous labyrinthine hemorrhage in sickle cell disease.

We report the clinical and MR imaging findings in two African-American patients with manifestations of sickle cell disease affecting the inner ear. Both suffered sudden-onset sensorineural hearing loss and vestibular symptoms, and both had high labyrinthine signal on T1-weighted MR images attributed to labyrinthine hemorrhage. Follow-up studies of the first patient revealed a decrease in abnormal vestibular signal. Careful attention to the labyrinth on T1-weighted MR images can reveal vestibulocochlear clinical findings in sickle cell patients, with important implications for management and prognosis.

MR imaging of hemorrhagic brain lesions: a comparison of dual-echo gradient- and spin-echo and fast spin-echo techniques.

OBJECTIVE: Our objective was to assess the usefulness of the dual-echo gradient- and spin-echo (GRASE) technique in revealing acute hemorrhagic brain lesions and compare GRASE and fast spin-echo techniques for revealing acute hemorrhagic lesions and image artifacts. MATERIALS AND METHODS: Thirty-two consecutive patients with acute intracranial hemorrhage underwent dual-echo GRASE (TEeff1/TEeff2, 35/85) and fast spin-echo (25/110) imaging. The techniques were matched for TR (3032 msec), spatial resolution, and acquisition time. Two neuroradiologists reviewed the images independently, documenting the number, size (<1, >1, or 1 cm in diameter), location, and signal characteristics (hypointense versus hyperintense compared with brain) of detectable lesions. These observers also compared matched T2- and proton density-weighted GRASE and fast spin-echo images for paramagnetic lesion conspicuity, diamagnetic susceptibility artifacts, chemical shift artifacts along the phase- and frequency-encoding directions, and artifactual CSF hyperintensity in the thin curvilinear cortical sulci and the Virchow-Robin spaces on only the proton density-weighted images. RESULTS: The average number and conspicuity of dark (paramagnetic) lesions were significantly greater on GRASE than on fast spin-echo images (p < .05 and p < .001, respectively). We found no significant difference in the average number of bright lesions revealed by either technique (p > .1). Chemical shift artifacts along the phase-encoding directions were more prominent on GRASE than on fast spin-echo imaging. Chemical shift artifacts along the frequency-encoding directions and artifactual CSF hyperintensity were more prominent on fast spin-echo than on GRASE imaging. No visually apparent difference was found in the degree of diamagnetic susceptibility artifacts seen with the two techniques. CONCLUSION: Dual-echo GRASE imaging can be helpful in the examination of patients with suspected acute brain hemorrhage.

A subclass of prefrontal gamma-aminobutyric acid axon terminals are selectively altered in schizophrenia.

In the primate cerebral cortex, morphologically and functionally diverse classes of local circuit neurons containing the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) differentially regulate the activity of pyramidal cells, the principal type of excitatory output neurons. In schizophrenia, GABA neurotransmission in the prefrontal cortex (PFC) appears to be disturbed but whether specific populations of GABA neurons are affected is not known. The chandelier class of GABA neurons are of particular interest because their axon terminals, which form distinctive arrays termed "cartridges," provide inhibitory input exclusively to the axon initial segment of pyramidal cells. Thus, chandelier cells are positioned to powerfully regulate the excitatory output of pyramidal neurons and, consequently, to substantially affect the patterns of neuronal activity within the PFC. In this study, an antibody directed against the GABA membrane transporter GAT-1 was used to label GABA axon terminals in postmortem human brains. The relative density of GAT-1-immunoreactive axon cartridges furnished by chandelier neurons was decreased by 40% in the PFC of schizophrenic subjects compared with matched groups of normal control and nonschizophrenic psychiatric subjects. In contrast, markers of the axon terminals of other populations of GABA neurons were not altered in the schizophrenic subjects. Furthermore, the density of GAT-1-immunoreactive axon cartridges was not altered in psychiatric subjects who had been treated with antipsychotic medications. The changes in GAT-1-immunoreactive axon cartridges of chandelier neurons in schizophrenia are likely to reflect altered information processing within the PFC and in its output connections to other brain regions and could contribute to the cognitive impairments seen in this disorder.

Telangiectatic osteosarcoma of the skull base.

We present a patient with a distinctive lesion of the skull base, which, at imaging, showed expansile destruction of the sphenoid bone, hemorrhage with fluid-fluid levels, and contrast enhancement of a solid portion. These features ordinarily suggest either giant cell tumor or aneurysmal bone cyst; however, pathologic examination confirmed instead a rare variant of osteosarcoma of the telangiectatic type. Although imaging findings can indicate any of these possibilities, biopsy is an essential step in arriving at the diagnosis.

Effect of fasting on insulin-like growth factor (IGF)-IA and IGF-IB messenger ribonucleic acids and prehormones in rat liver.

The insulin-like growth factor I (IGF-I) gene generates by alternative splicing two IGF-I messenger RNAs (mRNAs) coding for IGF-I prehormones with different E domain sequences. In rats, these two mRNAs differ by the presence (IGF-IB) or absence (IGF-IA) of a 52-bp insert in the E domain coding region. The purpose of this study was to investigate the effect of nutritional perturbation on IGF-IA and -IB expression in rat liver. Northern blot analysis of liver mRNA revealed that the 1.5-1.9 kb and 0.9-1.2 kb IGF-I mRNA species were decreased in rats fasted for 48 h compared with either fasted-refed (48 h of each) or control-fed rats (each, P < 0.01), whereas the 7.5 kb IGF-I mRNA was decreased only when compared with the fasted-refed animals. Using semiquantitative RT-PCR, the IGF-IA transcript (114 bp amplicon) was not altered, whereas the IGF-IB transcript (166 bp amplicon) was decreased in fasted rats compared with the other two groups (both P < 0.01). We confirmed the RT-PCR results by RNase protection assay (RPA), observing that the IGF-IA (224 and 100 bases protected) was not decreased and that the IGF-IB transcript (376 bases protected), accounting for only 23% of the total IGF-I transcripts of control fed rats, was decreased by fasting. Because the results from RT-PCR and RPA do not necessarily predict full-length translatable mRNA, we subjected hepatic IGF-I transcripts to in vitro translation, and we immunoprecipitated IGF-IA and -IB prehormones. Both prehormones were translated principally from exon 1-containing mRNAs, with molecular weights of about 17K and 18K, representing 80% and 20% of the total IGF-I prehormones observed in control fed rats, respectively. Both peptides were reduced in fasted rats compared with controls (P < 0.01), and refeeding restored both. By immunoblotting of the protein extract from liver of fasted rats, IGF-IA was decreased by 77% compared with control-fed animals. Refeeding returned IGF-IA to normal. The lack of reduction of IGF-IA transcript at the alternative splice site suggests that posttranscriptional mechanisms are responsible for the reduction in steady-state IGF-I mRNAs that occurs during fasting. Additionally, we present evidence that biosynthesis of IGF-IA and -IB prehormones by liver is impaired at a posttranscriptional level.

Effects of D2 dopamine receptor antagonists on Fos protein expression in the striatal complex and entorhinal cortex of the nonhuman primate.

Recent studies have reported that acute administration of dopamine D2 receptor antagonists increases expression of the immediate early gene c-fos in the rat striatal complex. There have been no corresponding studies of the effects of D2 antagonists in primate species. Since all clinically effective antipsychotic drugs share D2 receptor antagonism, it is important to define the extent to which these drugs may alter expression of c-fos or its protein product, Fos, in primates. We therefore examined the effects of administration of two D2 receptor antagonists, haloperidol and metoclopramide, on Fos expression in the striatum and temporal cortices of the vervet monkey. Metoclopramide does not appear to possess significant antipsychotic efficacy but potently produces extra-pyramidal side effects, while haloperidol is an effective antipsychotic drug that produces extrapyramidal side effects. Both drugs increased the number of Fos-like immunoreactive (Fos-li) neurons in the caudate nucleus and putamen; the numbers of Fos-li neurons in these regions were increased in both the patch and matrix compartments. Haloperidol but not metoclopramide increased the number of Fos-li neurons in the nucleus accumbens shell. Similarly, haloperidol but not metoclopramide increased the number of Fos-li neurons in the entorhinal cortex. Neither drug altered Fos expression in the inferior temporal cortex. These data suggest that the dorsolateral caudate nucleus and putamen may be sites at which D2 receptor antagonists elicit extrapyramidal side effects, and the nucleus accumbens shell and entorhinal cortex may be loci at which the therapeutic actions of antipsychotic drugs are manifested.

Detection of multiple tumor suppressor genes for Syrian hamster fibrosarcomas by somatic cell hybridization.

Identification of tumor suppressor gene loci in rodent cell culture systems has relied upon the use of somatic cell hybridization studies. Although normal rodent fibroblasts are capable of suppressing the tumorigenicity of a variety of tumor cells, the lack of complementation in tumor cell x tumor cell hybrids has left the possibility that a single tumor suppressor gene may be responsible for tumor suppression in a particular rodent cell culture system. Using this same approach, we found no evidence for complementation resulting in suppression of the transformed phenotype when three viral oncogene-transformed Syrian hamster embryo (SHE) cell lines and one spontaneously transformed baby hamster kidney (BHK) cell line were fused to benzo[a]pyrene-transformed SHE cells (BP6T-M3). However, v-src oncogene-transformed cell line (srcT) x BP6T-M3 hybrids did demonstrate limited suppression of the transformed phenotype, suggesting at least two complementing tumor suppressor genes in this system. We were able to confirm and extend this finding using another experimental approach with preneoplastic hamster cell lines that are immortal in culture but nontumorigenic in nude mice. We propose that fusion of these preneoplastic cells to various tumor cells may reveal tumor suppressor genes not evident in the tumor cell x tumor cell complementation studies. Subclones of two nontumorigenic, immortal hamster cell lines, 10W and DES4, displayed differing abilities to suppress BP6T-M3 cells in somatic cell hybrids, as quantitated by the ability of the hybrid cells to form colonies in soft agar. With a panel of preneoplastic hamster cell x BP6T-M3 hybrids, a distinct pattern of suppression or expression of the transformed phenotype was observed. Marked differences in this pattern were seen when the same 10W and DES4 subclones were fused to other hamster fibrosarcoma cell lines, indicating different tumor suppressing activities of multiple tumor suppressor genes. Analysis of this data suggests that as few as three or as many as six different tumor suppressor genes may be active in the Syrian hamster embryo cell culture system. Thus, this system may provide a useful model for identifying and studying the effects and regulation of a number of different tumor suppressor genes for fibrosarcomas.

Tyrosine phosphorylation of the receptor for insulin-like growth factor II is inhibited in plasma membranes from insulin-treated rat adipocytes.

Insulin action in intact adipocytes leads to a rapid increase in the concentration of receptors for insulin-like growth factor (IGF) II on the adipocyte cell surface, and to a decrease in the [32P]phosphate content of these receptors on the plasma membrane [Corvera & Czech (1985) Proc. Natl. Acad. Sci. U.S.A. 82. 7314-7318]. It has been previously shown that the receptor for IGF-II can be phosphorylated on tyrosine residues by a kinase activity which is expressed in isolated adipocyte plasma membranes. It is now shown that IGF-II-receptor phosphorylation in vitro, in plasma membranes derived from insulin-treated cells, is markedly decreased compared with the phosphorylation of the receptor in membranes from control cells. This effect of insulin cannot be attributed to an increase in the activity of phosphotyrosyl phosphatase in the membranes. The tyrosine kinase that catalyses the phosphorylation of IGF-II receptors is associated with a fraction of the plasma membrane which is insoluble in Triton X-100. Removal of the Triton X-100-soluble components of the membrane markedly enhances receptor phosphorylation. Moreover, the expression of the inhibitory effect of insulin requires the presence of one or several Triton X-100-soluble components of the plasma membrane.

The insulin-like growth factor II receptor is phosphorylated by a tyrosine kinase in adipocyte plasma membranes.

Incorporation of 32P from [gamma-32P]ATP into tyrosine residues of the insulin-like growth factor (IGF)-II receptor was observed in a Triton X-100-insoluble fraction of rat adipocyte plasma membranes. IGF-II receptor phosphorylation proceeded to a stoichiometry of approximately 0.5 mol of phosphate/IGF-II binding site after 10 min of incubation at 4 degrees C. A Km for ATP of 6 microM was calculated for this phosphorylation reaction. Addition of IGF-II caused an approximately 2-fold increase in tyrosine phosphorylation of the IGF-II receptor in this preparation. In contrast, phosphorylation of angiotensin II by the Triton X-100 washed membranes was not stimulated by IGF-II. Incubation of purified receptor immobilized on IGF-II agarose or of receptor-enriched low density microsomal membranes with [gamma-32P]ATP did not result in appreciable incorporation of [32P]phosphate into the IGF-II receptor nor into exogenous substrates. These data suggest that the IGF-II receptor is not a tyrosine protein kinase capable of autophosphorylation but that it is a substrate for a tyrosine protein kinase endogenous to the adipocyte plasma membrane. The stimulatory effect of IGF-II on the tyrosine phosphorylation of its receptor may be due to a conformational change which converts the receptor to a better substrate for this tyrosine kinase.