High-resolution magnetic resonance imaging reveals nuclei of the human amygdala: manual segmentation to automatic atlas.

The amygdala is composed of multiple nuclei with unique functions and connections in the limbic system and to the rest of the brain. However, standard in vivo neuroimaging tools to automatically delineate the amygdala into its multiple nuclei are still rare. By scanning postmortem specimens at high resolution (100-150µm) at 7T field strength (n = 10), we were able to visualize and label nine amygdala nuclei (anterior amygdaloid, cortico-amygdaloid transition area; basal, lateral, accessory basal, central, cortical medial, paralaminar nuclei). We created an atlas from these labels using a recently developed atlas building algorithm based on Bayesian inference. This atlas, which will be released as part of FreeSurfer, can be used to automatically segment nine amygdala nuclei from a standard resolution structural MR image. We applied this atlas to two publicly available datasets (ADNI and ABIDE) with standard resolution T1 data, used individual volumetric data of the amygdala nuclei as the measure and found that our atlas i) discriminates between Alzheimer's disease participants and age-matched control participants with 84% accuracy (AUC=0.915), and ii) discriminates between individuals with autism and age-, sex- and IQ-matched neurotypically developed control participants with 59.5% accuracy (AUC=0.59). For both datasets, the new ex vivo atlas significantly outperformed (all p < .05) estimations of the whole amygdala derived from the segmentation in FreeSurfer 5.1 (ADNI: 75%, ABIDE: 54% accuracy), as well as classification based on whole amygdala volume (using the sum of all amygdala nuclei volumes; ADNI: 81%, ABIDE: 55% accuracy). This new atlas and the segmentation tools that utilize it will provide neuroimaging researchers with the ability to explore the function and connectivity of the human amygdala nuclei with unprecedented detail in healthy adults as well as those with neurodevelopmental and neurodegenerative disorders.

Nicotinic antagonists enhance process outgrowth by rat retinal ganglion cells in culture.

Functional nicotinic cholinergic receptors are found on mammalian retinal ganglion cell neurons in culture. The neurotransmitter acetylcholine (ACh) can be detected in the medium of many of these retinal cultures, after release presumably from the choline acetyltransferase-positive amacrine cells. The postsynaptic effect of endogenous or applied ACh on the ganglion cells can be blocked with specific nicotinic antagonists. Here it is shown that within 24 hours of producing such a pharmacologic blockade, the retinal ganglion cells begin to sprout or regenerate neuronal processes. Thus, the growth-enhancing effect of nicotinic antagonists may be due to the removal of inhibition to growth by tonic levels of ACh present in the culture medium. Since there is a spontaneous leak of ACh in the intact retina, the effects of nicotinic cholinergic drugs on process outgrowth in culture may reflect a normal control mechanism for growth or regeneration of retinal ganglion cell processes that is exerted by ACh in vivo.

Ubiquitination of a new form of alpha-synuclein by parkin from human brain: implications for Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive accumulation in selected neurons of protein inclusions containing alpha-synuclein and ubiquitin. Rare inherited forms of PD are caused by autosomal dominant mutations in alpha-synuclein or by autosomal recessive mutations in parkin, an E3 ubiquitin ligase. We hypothesized that these two gene products interact functionally, namely, that parkin ubiquitinates alpha-synuclein normally and that this process is altered in autosomal recessive PD. We have now identified a protein complex in normal human brain that includes parkin as the E3 ubiquitin ligase, UbcH7 as its associated E2 ubiquitin conjugating enzyme, and a new 22-kilodalton glycosylated form of alpha-synuclein (alphaSp22) as its substrate. In contrast to normal parkin, mutant parkin associated with autosomal recessive PD failed to bind alphaSp22. In an in vitro ubiquitination assay, alphaSp22 was modified by normal but not mutant parkin into polyubiquitinated, high molecular weight species. Accordingly, alphaSp22 accumulated in a non-ubiquitinated form in parkin-deficient PD brains. We conclude that alphaSp22 is a substrate for parkin's ubiquitin ligase activity in normal human brain and that loss of parkin function causes pathological alphaSp22 accumulation. These findings demonstrate a critical biochemical reaction between the two PD-linked gene products and suggest that this reaction underlies the accumulation of ubiquitinated alpha-synuclein in conventional PD.

Presenilin-mediated modulation of capacitative calcium entry.

We studied a novel function of the presenilins (PS1 and PS2) in governing capacitative calcium entry (CCE), a refilling mechanism for depleted intracellular calcium stores. Abrogation of functional PS1, by either knocking out PS1 or expressing inactive PS1, markedly potentiated CCE, suggesting a role for PS1 in the modulation of CCE. In contrast, familial Alzheimer's disease (FAD)-linked mutant PS1 or PS2 significantly attenuated CCE and store depletion-activated currents. While inhibition of CCE selectively increased the amyloidogenic amyloid beta peptide (Abeta42), increased accumulation of the peptide had no effect on CCE. Thus, reduced CCE is most likely an early cellular event leading to increased Abeta42 generation associated with FAD mutant presenilins. Our data indicate that the CCE pathway is a novel therapeutic target for Alzheimer's disease.

Analysis of meningiomas by methylation- and transcription-based clonality assays.

The clonal derivation of tumors can be determined by X chromosome inactivation analysis based on differential expression of genes or differential methylation of cytosine residues in CpG islands near polymorphic loci. In this report, we compared a transcription-based RNA analysis with a methylation-based DNA assay to determine clonality of meningiomas. Both clonality assays use PCR-based analysis at the hunan androgen-receptor gene (HUMARA) on the X chromosome. Among 23 meningiomas from female patients, 19 were informative heterozygotes at this locus (83%). The patterns of X chromosome inactivation in four patients were extremely skewed towards one allele in blood (unequal Lyonization), which precluded clonality determination in the tumor samples. Concordant clonality results with methylation- and transcription-based clonality assays were demonstrated in 9 of 13 informative tumors expressing the androgen receptor. Seven meningiomas were monoclonal, but surprisingly, two pathologically documented cases of meningiomas were polyclonal. There was disparity in 4 of 13 tumor specimens that were polyclonal by the methylation-based assay but monoclonal by the transcription assay. Clonality examination of these tumors by the methylation-based phosphoglycerate kinase assay provided identical results to the methylation-based analysis at the HUMARA locus. In addition, loss of heterozygosity (LOH) studies of chromosome 22, which is frequently deleted in meningiomas, showed that four of four informative samples of the six polyclonal tumors had partial LOH in tumor tissues. However, complete LOH was observed in primary cultured cells, which were also monoclonal by the methylation assay. Taken together, these data suggest that the disparity of the two assays in these four cases may be due to differences in the level of expression of the androgen receptor gene in tumors. Therefore, we conclude that: (a) clonal derivation of meningiomas determined by both transcription- and methylation-based clonality assays are in full agreement in many (9 of 13) but not all cases (4 of 13); and (b) most meningiomas (9 of 15) are monoclonal in origin, whereas some meningioma samples (6 of 15) are polyclonal or may contain heterogeneous components.

Presenilin 1 facilitates the constitutive turnover of beta-catenin: differential activity of Alzheimer's disease-linked PS1 mutants in the beta-catenin-signaling pathway.

Although an association between the product of the familial Alzheimer's disease (FAD) gene, presenilin 1 (PS1), and beta-catenin has been reported recently, the cellular consequences of this interaction are unknown. Here, we show that both the full length and the C-terminal fragment of wild-type or FAD mutant PS1 interact with beta-catenin from transfected cells and brains of transgenic mice, whereas E-cadherin and adenomatous polyposis coli (APC) are not detected in this complex. Inducible overexpression of PS1 led to increased association of beta-catenin with glycogen synthase kinase-3beta (GSK-3beta), a negative regulator of beta-catenin, and accelerated the turnover of endogenous beta-catenin. In support of this finding, the beta-catenin half-life was dramatically longer in fibroblasts deficient in PS1, and this phenotype was completely rescued by replacement of PS1, demonstrating that PS1 normally stimulates the degradation of beta-catenin. In contrast, overexpression of FAD-linked PS1 mutants (M146L and DeltaX9) failed to enhance the association between GSK-3beta and beta-catenin and interfered with the constitutive turnover of beta-catenin. In vivo confirmation was demonstrated in the brains of transgenic mice in which the expression of the M146L mutant PS1 was correlated with increased steady-state levels of endogenous beta-catenin. Thus, our results indicate that PS1 normally promotes the turnover of beta-catenin, whereas PS1 mutants partially interfere with this process, possibly by failing to recruit GSK-3beta into the PS1-beta-catenin complex. These findings raise the intriguing possibility that PS1-beta-catenin interactions and subsequent activities may be consequential for the pathogenesis of AD.

A compendium of gene expression in normal human tissues.

This study creates a compendium of gene expression in normal human tissues suitable as a reference for defining basic organ systems biology. Using oligonucleotide microarrays, we analyze 59 samples representing 19 distinct tissue types. Of approximately 7,000 genes analyzed, 451 genes are expressed in all tissue types and designated as housekeeping genes. These genes display significant variation in expression levels among tissues and are sufficient for discerning tissue-specific expression signatures, indicative of fundamental differences in biochemical processes. In addition, subsets of tissue-selective genes are identified that define key biological processes characterizing each organ. This compendium highlights similarities and differences among organ systems and different individuals and also provides a publicly available resource (Human Gene Expression Index, the HuGE Index, http://www.hugeindex.org) for future studies of pathophysiology.

Meningioangiomatosis is associated with neurofibromatosis 2 but not with somatic alterations of the NF2 gene.

Meningioangiomatosis occurs sporadically and in patients with neurofibromatosis. The literature, however, is unclear concerning the type of neurofibromatosis associated with meningioangiomatosis. Because determining which form of neurofibromatosis predisposes to meningioangiomatosis would clarify the genetic alterations of this lesion, we reviewed all reported cases of meningioangiomatosis associated with neurofibromatosis in light of current diagnostic criteria for neurofibromatosis 1 (NF1) and neurofibromatosis 2 (NF2). All well-documented cases of meningioangiomatosis occurred in the setting of NF2, implying that germline alterations of the NF2 gene predispose to meningioangiomatosis. To determine whether sporadic (non-NF) cases of meningioangiomatosis arise from somatic alterations of the same gene, we screened the NF2 gene for mutations in 12 sporadic cases of meningioangiomatosis and in constitutional DNA from 6 of these 12 patients. No mutations were found in either the lesional or constitutional DNA, which suggests that sporadic meningioangiomatosis is not a forme fruste of NF2 and that somatic alterations of the NF2 gene do not play a major role in sporadic meningioangiomatosis. For some tumor suppressor genes, germline mutations may predispose to specific tumors, while similar sporadic lesions only rarely suffer somatic mutations in these genes. The present findings suggest a similar dichotomy for the NF2 gene in meningioangiomatosis.

The gonadotropin-releasing hormone receptor gene promoter directs pituitary-specific oncogene expression in transgenic mice.

Our previous work has shown that 1.2 kb of the 5' flanking region of the mouse GnRH receptor (mGnRH-R) gene is sufficient to direct tissue-specific expression in vitro. In this study, we have used the cell-specific regulatory sequences of the mGnRH-R gene promoter to target the expression of the simian virus 40 virus T antigen (TAg) to the pituitary gland of transgenic mice. A hybrid transgene, GnRH-R/TAg, was prepared using the -1164/+52 region of the mGnRH-R gene and +2533/+5234 sequences encoding the large T antigen of the simian virus 40. Two founders developed tumors of apparent pituitary origin at 44 (M28, female) and 50 (M25, male) days of age. M28 and M25 mice were about 50% underweight, and their gonads were grossly underdeveloped compared with wild-type litter mates. A third male founder, M29, developed a tumor at a later time (109 days). M29 was able to breed successfully and stably transmit the GnRH-R/TAg transgene. Mice of the M29 transgene line developed tumors at 4-5 months of age. Gross examination showed that the tumors extend from the sella and infiltrate into the inferior surface of the brain. In small tumors collected from young transgenic animals, normal pituitary cells as well as transition areas of increasing cellular atypia are evident. Frankly malignant cells are seen in all tumors. The pituitary tumors express the alpha-, FSHbeta-, and LHbeta-subunits and the GnRH-R messenger RNA, all markers of a gonadotrope but not of other anterior pituitary cell lineages. In summary, our studies indicate that 1.2 kb of the 5'-flanking region of the mGnRH-R gene can be used to target expression specifically to the gonadotropes of the pituitary gland in transgenic mice. The GnRH-R gene promoter-directed expression appears to be cell-specific and results in the formation of tumors that are primarily of gonadotropic origin.

August 2000: Two cases with necrosis and hemorrhage in the putamen and white matter.

The August COM: Acute methanol poisoning is an uncommon, but well-recognized, cause of central nervous system injury. We present two autopsy cases showing the classic neuropathologic injuries in acute methanol poisoning: putamen and white matter necrosis and hemorrhage. In Case 1, putamen hemorrhages were striking; white matter pathology predominated in Case 2. The precise mechanism of methanol toxicity is unclear. Direct toxicity of metabolites, particularly formic acid, as well as ischemic injury and acidosis likely play a role. Methanol is readily available in many commercial products, and may be ingested accidentally or intentionally.

Synthetic amyloid beta-protein fails to produce specific neurotoxicity in monkey cerebral cortex.

Because progressive amyloid beta-protein (A beta P) deposition and surrounding neuritic dystrophy occur spontaneously in primates, we evaluated the in vivo effects of synthetic A beta P in monkey cortex. Experimental and control A beta P were stereotactically injected into multiple neocortical sites of adult rhesus monkeys in a vehicle of either artificial cerebrospinal fluid or acetonitrile. After 2 weeks or 3 months, injection sites were identified and characterized histologically and immunocytochemically. A beta P antibodies specifically detected the injected A beta P1-40 peptide. Serial sections stained with silver and antineurofilament protein demonstrated comparable degrees of degenerating neurons, dystrophic neurites, and axonal spheroids associated with both experimental and control peptide injections. Alz 50 staining was sparse or absent in all sites. We conclude that specific cellular changes closely resembling AD pathology were not detected in these experiments, and that control and experimental A beta P peptides produced indistinguishable effects. Methodological concerns regarding the in vivo modeling of A beta P bioactivity are discussed.

Nonvasculitic, steroid-responsive mononeuritis multiplex.

We report two patients with mononeuritis multiplex, both of whom had focal inflammation of the perineurium and endoneurium on sural nerve biopsy without necrosis of blood vessel walls, histologic evidence of lymphoid malignancy, or mycobacterial infection. The predominant early sensory symptoms were asymmetric pain and paresthesias; subsequently, muscle weakness developed. Electrophysiologic studies showed an asymmetric sensorimotor axon loss radiculoneuropathy with denervation of limb and paraspinal muscles. Spinal fluid protein was elevated in one patient. There was no cause or underlying systemic disease. Marked improvement occurred with steroid therapy.

C455R notch3 mutation in a Colombian CADASIL kindred with early onset of stroke.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by mutations in the notch3 epidermal growth factor-like repeats. A Colombian kindred carries a novel C455R mutation located in the predicted ligand-binding domain. Stroke occurred in the patients at an unusually early age (median age: 31 years) in comparison to the more frequent onset in the fourth decade of life in other CADASIL populations, including a second Colombian kindred with an R1031C mutation.

Characterization of GABA- and glycine-induced currents of solitary rodent retinal ganglion cells in culture.

Ganglion cells were fluorescently labeled, dissociated from 7- to 11-day-old rodent retinas, and placed in tissue culture. Whole-cell recordings with patch electrodes were obtained from solitary cells lacking processes, which permitted a high-quality space clamp. Both GABA (1-200 microM) and glycine (10-300 microM) produced large increases in membrane conductance in virtually every ganglion cell tested, including ganglion cells from different size classes in both rats and mice. Taurine evoked responses similar to those of glycine, but considerably greater concentrations of taurine (150-300 microM) were necessary to observe any effect. Since 20 microM GABA produced approximately the same response as 100 microM glycine, the effects of these two concentrations were compared under various conditions. When recording with chloride distributed equally across the membrane, the reversal potential of the agonist-induced currents was approximately 0 mV. When the internal chloride was reduced by substitution with aspartate, the reversal potential shifted in a negative direction by about 42 mV, indicating that the current was carried mainly by chloride ions. Strychnine (1-5 microM) completely and reversibly blocked the actions of glycine (100 microM) but not those of GABA (20 microM); however, higher concentrations of strychnine (20 microM) nearly totally inhibited the current elicited by GABA (20 microM). The responses to glycine (100 microM) were not affected by bicuculline methiodide (20 microM) or picrotoxinin (20 microM). In contrast, bicuculline methiodide (10 microM) and picrotoxinin (10 microM) reversibly blocked the current evoked by GABA (20 microM); d-tubocurarine (100 microM) only slightly decreased the response to GABA (20 microM). The antagonists were effective over a wide range of holding potentials (-90 mV to +30 mV). The responses to a steady application of both GABA and glycine decayed in a few seconds when recorded under conditions of both symmetric and asymmetric chloride across the membrane. During this decay the current and conductance decreased simultaneously, reflecting receptor desensitization rather than a change in the driving force for chloride caused by agonist-induced ionic fluxes. The time-course of desensitization was usually described by a single exponential with time constants for GABA (20 microM) and glycine (100 microM) of 4.0 +/- 1.6 s and 4.4 +/- 1.9 s (mean +/- S.D.), respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

FDG hypermetabolism associated with inflammatory necrotic changes following radiation of meningioma.

PET with 18F-fluoro-2-deoxy-D-glucose (FDG) is currently the noninvasive gold standard for distinguishing brain tumor recurrence from radiation necrosis. We present a case report that appears to contradict this doctrine. The patient had a history of atypical meningioma and was treated with surgical resection and postoperative proton-beam radiation therapy. Approximately 16 mo after completion of therapy, MRI demonstrated two new regions of enhancement, and an FDG-PET study was performed to further characterize these lesions. FDG-PET demonstrated an area of intense hypermetabolism, and wide surgical resection was performed. Histological examination of the surgical specimen revealed reactive changes and areas of necrosis. There was no evidence of either recurrent or radiation-induced tumor.

Physiology and pharmacology of olfactory bulb neurons in dissociated cell culture.

Cells from olfactory bulbs of embryonic rats were grown in dissociated cell culture for up to 5 weeks. Both neurons and non-neuronal cells grew in these cultures, with a variety of neuronal populations appearing. A population of 20-25% of the neurons were GABAergic by the criterion of [3H]GABA uptake. Electrophysiologic measurements were made of the baseline activity of the cultured neurons. Cells showed a mean resting potential of 60.1 +/- 1.2 mV and a mean input resistance of 87.6 +/- 9.5 M omega. All cells were sensitive to microperfusion of GABA with half-maximal effect occurring at about 20 microM. Glutamate was universally excitatory but with variations in degree. Carnosine (beta-Ala-L-His), tested over the concentration range of 10 nM to 100 microM, had no effect on input resistance, resting potential, action potential shape, on-going synaptic activity or the responsiveness to either GABA or glutamate. These results are further evidence against a role for carnosine as the excitatory transmitter of the primary olfactory afferents.

Non-invasive laser microsurgery selectively damages populations of labeled mouse neurons: dependence on incident laser dose and absorption.

Selective photothermolysis (SP) is a novel technique by which brief, unfocused laser pulses are selectively absorbed by, and cause selective thermal damage to, endogenously pigmented structures. This report describes the use of an exogenous non-fluorescent chromophore (Procion blue) to effect cellular damage by SP. Cultured dorsal root ganglia neurons were selectively labeled with Procion blue and subjected to varying doses of laser illumination. Progressive cellular damage was assessed by leakage of propidium iodide through damaged membranes. The neurons targeted via an exogenous chromophore sustained damage which was proportionate to laser dose and chromophore concentration. The results of these experiments demonstrated that the rate and extent of neuronal damage can be controlled by adjusting either the incident dose of laser energy or the amount of target chromophore within cells. Selective photothermolysis will provide an experimental tool for neurobiologists in particular and will find general use within the biomedical field.

Non-uniform distribution of GABA activated chloride channels in cultured cortical neurons.

Several different neurotransmitter receptors and ion channels exist in non-random distributions in the membrane of nerve and muscle cells, a pattern which appears to have functional implications. The data presented here, based on the distribution of single channels in outside-out patches, directly demonstrate that receptors for gamma-aminobutyric acid (GABA) and their associated chloride channels (GABAA receptors) exist almost exclusively in clusters in the membranes of cultured rat cortical neurons. This non-uniform distribution is present both in innervated and uninnervated neurons.

Adult medulloblastoma: prognostic factors and patterns of relapse.

OBJECTIVE: To determine the patterns of relapse and the prognostic factors for adult medulloblastomas treated in the magnetic resonance imaging era. METHODS: Between 1986 and 1996, 32 adult patients (age, > or =16 yr) with medulloblastomas confined to the craniospinal axis were treated in our institutions. Twenty cases involved classic histological features and 12 involved the desmoplastic variant. The Chang staging distribution was as follows: T1, 2; T2, 17; T3, 10; T4, 3; M0, 24; M1, 1; M2, 4; M3, 3. Brainstem invasion was present in nine patients. Lesions were midline in 13 cases and lateral in 19. Resection was complete in 17 cases, subtotal in 6, and partial in 5, with biopsy only in 4 cases. All patients received postoperative radiotherapy, with median doses of 36 Gy to the entire craniospinal axis and 55 Gy to the posterior fossa. Twenty-four patients received chemotherapy (20 before radiotherapy, 3 after radiotherapy, and 1 before and after radiotherapy). RESULTS: With a median follow-up period of 5.4 years, 17 patients experienced recurrences. At 5 and 8 years, overall survival rates were 83 and 45% and disease-free survival rates were 57 and 40%, respectively. The 5- and 8-year posterior fossa control rates were 67 and 59%, respectively. Twenty-nine percent of all relapses occurred more than 5 years after treatment. The posterior fossa was the most common site of relapses. In univariate analyses, factors adversely affecting posterior fossa control were less than complete resection (P<0.001), the presence of brainstem invasion (P = 0.02), and the use of chemotherapy (P = 0.03). The overall radiotherapy duration was marginally significant in predicting posterior fossa control, with 5-year posterior fossa control rates of 81 and 49% for durations of less than 48 days and 48 days or more, respectively (P = 0.06). In a multivariate analysis, complete resection was predictive of improved posterior fossa control (P = 0.02) and disease-free survival (P = 0.02) rates. Of the eight low-risk patients who received radiotherapy alone, three experienced recurrences in the bone as the only site of relapse. CONCLUSION: Late relapse is common among adult patients with medulloblastomas, and long-term follow-up monitoring is important. Because of the high risk of systemic failure among the low-risk patients treated with radiotherapy alone, the role of chemotherapy for this group of patients needs to be further investigated. Complete resection, the absence of brainstem invasion, and an overall radiotherapy duration of less than 48 days are important prognostic factors.