P element insertion-dependent gene activation in the Drosophila eye

Insights into the function of a gene can be gained in multiple ways, including loss-of-function phenotype, sequence similarity, expression pattern, and by the consequences of its misexpression. Analysis of the phenotypes produced by expression of a gene at an abnormal time, place, or level may provide clues to a gene’s function when other approaches are not illuminating. Here we report that an eye-specific, enhancer–promoter present in the P element expression vector pGMR is able to drive high level expression in the eye of genes near the site of P element insertion. Cell fate determination, differentiation, proliferation, and death are essential for normal eye development. Thus the ability to carry out eye-specific misexpression of a significant fraction of genes in the genome, given the dispensability of the eye for viability and fertility of the adult, should provide a powerful approach for identifying regulators of these processes. To test this idea we carried out two overexpression screens for genes that function to regulate cell death. We screened for insertion-dependent dominant phenotypes in a wild-type background, and for dominant modifiers of a reaper overexpression-induced small eye phenotype. Multiple chromosomal loci were identified, including an insertion 5′ to hid, a potent inducer of apoptosis, and insertions 5′ to DIAP1, a cell death suppressor. To facilitate the cloning of genes near the P element insertion new misexpression vectors were created. A screen with one of these vectors identified eagle as a suppressor of a rough eye phenotype associated with overexpression of an activated Ras1 gene.

Fragile X mental retardation protein is translated near synapses in response to neurotransmitter activation

Local translation of proteins in distal dendrites is thought to support synaptic structural plasticity. We have previously shown that metabotropic glutamate receptor (mGluR1) stimulation initiates a phosphorylation cascade, triggering rapid association of some mRNAs with translation machinery near synapses, and leading to protein synthesis. To determine the identity of these mRNAs, a cDNA library produced from distal nerve processes was used to screen synaptic polyribosome-associated mRNA. We identified mRNA for the fragile X mental retardation protein (FMRP) in these processes by use of synaptic subcellular fractions, termed synaptoneurosomes. We found that this mRNA associates with translational complexes in synaptoneurosomes within 1–2 min after mGluR1 stimulation of this preparation, and we observed increased expression of FMRP after mGluR1 stimulation. In addition, we found that FMRP is associated with polyribosomal complexes in these fractions. In vivo, we observed FMRP immunoreactivity in spines, dendrites, and somata of the developing rat brain, but not in nuclei or axons. We suggest that rapid production of FMRP near synapses in response to activation may be important for normal maturation of synaptic connections.

Calcium channel activation and self-biting in mice

The L type calcium channel agonist (±)Bay K 8644 has been reported to cause characteristic motor abnormalities in adult mice. The current study shows that administration of this drug can also cause the unusual phenomenon of self-injurious biting, particularly when given to young mice. Self-biting is provoked by injecting small quantities of (±)Bay K 8644 directly into the lateral ventricle of the brain, suggesting a central effect of the drug. Similar behaviors can be provoked by administration of another L type calcium channel agonist, FPL 64176. The self-biting provoked by (±)Bay K 8644 can be inhibited by pretreating the mice with dihydropyridine L type calcium channel antagonists such as nifedipine, nimodipine, or nitrendipine. However, self-biting is not inhibited by nondihydropyridine antagonists including diltiazem, flunarizine, or verapamil. The known actions of (±)Bay K 8644 as an L type calcium channel agonist, the reproduction of similar behavior with another L type calcium channel agonist, and the protection afforded by certain L type calcium channel antagonists implicate calcium channels in the mediation of the self-biting behavior. This phenomenon provides a model for studying the neurobiology of this unusual behavior.

Dynamic regulation of c-Jun N-terminal kinase activity in mouse brain by environmental stimuli

Activation of the recently identified c-Jun N-terminal kinases (JNKs) typically results in programmed cell death (apoptosis) in neurons and other cell types grown in culture. However, the effects of JNK activation in the central nervous system in vivo are unknown. At baseline, JNK activity in mice was on average 17-fold higher in brain than in peripheral organs, whereas JNK protein levels were similar. In brain, JNK was expressed primarily in neurons. Restraining mice or allowing them to explore a novel environment rapidly increased JNK activity 3- to 15-fold in various brain regions, but these manipulations did not increase brain activity of the extracellular signal-regulated kinase. Because noninvasive environmental stimuli that do not induce neurodegeneration elicited prominent increases in JNK activity in the brain, we conclude that acute activation of the JNK cascade in central nervous system neurons does not induce neuronal apoptosis in vivo. In contrast, the high baseline activity of JNK in the brain and the activation of the JNK cascade by environmental stimuli suggest that this kinase may play an important physiological role in neuronal function.

CD4-independent, CCR5-dependent infection of brain capillary endothelial cells by a neurovirulent simian immunodeficiency virus strain

Brain capillary endothelial cells (BCECs) are targets of CD4-independent infection by HIV-1 and simian immunodeficiency virus (SIV) strains in vitro and in vivo. Infection of BCECs may provide a portal of entry for the virus into the central nervous system and could disrupt blood–brain barrier function, contributing to the development of AIDS dementia. We found that rhesus macaque BCECs express chemokine receptors involved in HIV and SIV entry including CCR5, CCR3, CXCR4, and STRL33, but not CCR2b, GPR1, or GPR15. Infection of BCECs by the neurovirulent strain SIV/17E-Fr was completely inhibited by aminooxypentane regulation upon activation, normal T cell expression and secretion in the presence or absence of ligands, but not by eotaxin or antibodies to CD4. We found that the envelope (env) proteins from SIV/17E-Fr and several additional SIV strains mediated cell–cell fusion and virus infection with CD4-negative, CCR5-positive cells. In contrast, fusion with cells expressing the coreceptors STRL33, GPR1, and GPR15 was CD4-dependent. These results show that CCR5 can serve as a primary receptor for SIV in BCECs and suggest a possible CD4-independent mechanism for blood–brain barrier disruption and viral entry into the central nervous system.

Characterization of Mayven, a Novel Actin-binding Protein Predominantly Expressed in Brain

The cytoskeleton plays an important role in neuronal morphogenesis. We have identified and characterized a novel actin-binding protein, termed Mayven, predominantly expressed in brain. Mayven contains a BTB (broad complex, tramtrack, bric-a-brac)/POZ (poxvirus, zinc finger) domain-like structure in the predicted N terminus and “kelch repeats” in the predicted C-terminal domain. Mayven shares 63% identity (77% similarity) with the Drosophila ring canal (“kelch”) protein. Somatic cell-hybrid analysis indicated that the human Mayven gene is located on chromosome 4q21.2, whereas the murine homolog gene is located on chromosome 8. The BTB/POZ domain of Mayven can self-dimerize in vitro, which might be important for its interaction with other BTB/POZ-containing proteins. Confocal microscopic studies of endogenous Mayven protein revealed a highly dynamic localization pattern of the protein. In U373-MG astrocytoma/glioblastoma cells, Mayven colocalized with actin filaments in stress fibers and in patchy cortical actin-rich regions of the cell margins. In primary rat hippocampal neurons, Mayven is highly expressed in the cell body and in neurite processes. Binding assays and far Western blotting analysis demonstrated association of Mayven with actin. This association is mediated through the “kelch repeats” within the C terminus of Mayven. Depolarization of primary hippocampal neurons with KCl enhanced the association of Mayven with actin. This increased association resulted in dynamic changes in Mayven distribution from uniform to punctate localization along neuronal processes. These results suggest that Mayven functions as an actin-binding protein that may be translocated along axonal processes and might be involved in the dynamic organization of the actin cytoskeleton in brain cells.

Detection of cortical activation during averaged single trials of a cognitive task using functional magnetic resonance imaging

Functional neuroimaging studies in human subjects using positron emission tomography or functional magnetic resonance imaging (fMRI) are typically conducted by collecting data over extended time periods that contain many similar trials of a task. Here methods for acquiring fMRI data from single trials of a cognitive task are reported. In experiment one, whole brain fMRI was used to reliably detect single-trial responses in a prefrontal region within single subjects. In experiment two, higher temporal sampling of a more limited spatial field was used to measure temporal offsets between regions. Activation maps produced solely from the single-trial data were comparable to those produced from blocked runs. These findings suggest that single-trial paradigms will be able to exploit the high temporal resolution of fMRI. Such paradigms will provide experimental flexibility and time-resolved data for individual brain regions on a trial-by-trial basis.

When the brain changes its mind: Interocular grouping during binocular rivalry

The prevalent view of binocular rivalry holds that it is a competition between the two eyes mediated by reciprocal inhibition among monocular neurons. This view is largely due to the nature of conventional rivalry-inducing stimuli, which are pairs of dissimilar images with coherent patterns within each eye’s image. Is it the eye of origin or the coherency of patterns that determines perceptual alternations between coherent percepts in binocular rivalry? We break the coherency of conventional stimuli and replace them by complementary patchworks of intermingled rivalrous images. Can the brain unscramble the pieces of the patchwork arriving from different eyes to obtain coherent percepts? We find that pattern coherency in itself can drive perceptual alternations, and the patchworks are reassembled into coherent forms by most observers. This result is in agreement with recent neurophysiological and psychophysical evidence demonstrating that there is more to binocular rivalry than mere eye competition.

Dopamine receptor subtypes modulate olfactory bulb γ-aminobutyric acid type A receptors

The γ-aminobutyric acid type A (GABAA) receptor is the predominant Cl− channel protein mediating inhibition in the olfactory bulb and elsewhere in the mammalian brain. The olfactory bulb is rich in neurons containing both GABA and dopamine. Dopamine D1 and D2 receptors are also highly expressed in this brain region with a distinct and complementary distribution pattern. This distribution suggests that dopamine may control the GABAergic inhibitory processing of odor signals, possibly via different signal-transduction mechanisms. We have observed that GABAA receptors in the rat olfactory bulb are differentially modulated by dopamine in a cell-specific manner. Dopamine reduced the currents through GABA-gated Cl- channels in the interneurons, presumably granule cells. This action was mediated via D1 receptors and involved phosphorylation of GABAA receptors by protein kinase A. Enhancement of GABA responses via activation of D2 dopamine receptors and phosphorylation of GABAA receptors by protein kinase C was observed in mitral/tufted cells. Decreasing or increasing the binding affinity for GABA appears to underlie the modulatory effects of dopamine via distinct receptor subtypes. This dual action of dopamine on inhibitory GABAA receptor function in the rat olfactory bulb could be instrumental in odor detection and discrimination, olfactory learning, and ultimately odotopic memory formation.

Autoinhibition of serotonin cells: An intrinsic regulatory mechanism sensitive to the pattern of usage of the cells

After periods of high-frequency firing, the normal rhythmically active serotonin (5HT)-containing neurosecretory neurons of the lobster ventral nerve cord display a period of suppressed spike generation and reduced synaptic input that we refer to as “autoinhibition.” The duration of this autoinhibition is directly related to the magnitude and duration of the current injection triggering the high-frequency firing. More interesting, however, is that the autoinhibition is inversely related to the initial firing frequency of these cells within their normal range of firing (0.5–3 Hz). This allows more active 5HT neurons to resume firing after shorter durations of inhibition than cells that initially fired at slower rates. Although superfused 5HT inhibits the spontaneous firing of these cells, the persistence of autoinhibition in saline with no added calcium, in cadmium-containing saline, and in lobsters depleted of serotonin suggests that intrinsic membrane properties account for the autoinhibition. A similar autoinhibition is seen in spontaneously active octopamine neurons but is absent from spontaneously active γ-aminobutyric acid cells. Thus, this might be a characteristic feature of amine-containing neurosecretory neurons. The 5HT cells of vertebrate brain nuclei share similarities in firing frequencies, spike shapes, and inhibition by 5HT with the lobster cells that were the focus of this study. However, the mechanism suggested to underlie autoinhibition in vertebrate neurons is that 5HT released from activated or neighboring cells acts back on inhibitory autoreceptors that are found on the dendrites and cell bodies of these neurons.

The roles of prefrontal brain regions in components of working memory: Effects of memory load and individual differences

Using an event-related functional MRI design, we explored the relative roles of dorsal and ventral prefrontal cortex (PFC) regions during specific components (Encoding, Delay, Response) of a working memory task under different memory-load conditions. In a group analysis, effects of increased memory load were observed only in dorsal PFC in the encoding period. Activity was lateralized to the right hemisphere in the high but not the low memory-load condition. Individual analyses revealed variability in activation patterns across subjects. Regression analyses indicated that one source of variability was subjects’ memory retrieval rate. It was observed that dorsal PFC plays a differentially greater role in information retrieval for slower subjects, possibly because of inefficient retrieval processes or a reduced quality of mnemonic representations. This study supports the idea that dorsal and ventral PFC play different roles in component processes of working memory.

Microglial activation precedes acute neurodegeneration in Sandhoff disease and is suppressed by bone marrow transplantation

Sandhoff disease is a lysosomal storage disorder characterized by the absence of β-hexosaminidase and storage of GM2 ganglioside and related glycolipids in the central nervous system. The glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. In symptomatic Sandhoff disease mice, apoptotic neuronal cell death was prominent in the caudal regions of the brain. cDNA microarray analysis to monitor gene expression during neuronal cell death revealed an upregulation of genes related to an inflammatory process dominated by activated microglia. Activated microglial expansion, based on gene expression and histologic analysis, was found to precede massive neuronal death. Extensive microglia activation also was detected in a human case of Sandhoff disease. Bone marrow transplantation of Sandhoff disease mice suppressed both the explosive expansion of activated microglia and the neuronal cell death without detectable decreases in neuronal GM2 ganglioside storage. These results suggest a mechanism of neurodegeneration that includes a vigorous inflammatory response as an important component. Thus, this lysosomal storage disease has parallels to other neurodegenerative disorders, such as Alzheimer's and prion diseases, where inflammatory processes are believed to participate directly in neuronal cell death.

Reactivation of encoding-related brain activity during memory retrieval

Neuronal models predict that retrieval of specific event information reactivates brain regions that were active during encoding of this information. Consistent with this prediction, this positron-emission tomography study showed that remembering that visual words had been paired with sounds at encoding activated some of the auditory brain regions that were engaged during encoding. After word-sound encoding, activation of auditory brain regions was also observed during visual word recognition when there was no demand to retrieve auditory information. Collectively, these observations suggest that information about the auditory components of multisensory event information is stored in auditory responsive cortex and reactivated at retrieval, in keeping with classical ideas about “redintegration,” that is, the power of part of an encoded stimulus complex to evoke the whole experience.

Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs

Heterotrimeric G proteins mediate the earliest step in cell responses to external events by linking cell surface receptors to intracellular signaling pathways. Gz is a member of the Gi family of G proteins that is prominently expressed in platelets and brain. Here, we show that deletion of the α subunit of Gz in mice: (i) impairs platelet aggregation by preventing the inhibition of cAMP formation normally seen at physiologic concentrations of epinephrine, and (ii) causes the mice to be more resistant to fatal thromboembolism. Loss of Gzα also results in greatly exaggerated responses to cocaine, reduces the analgesic effects of morphine, and abolishes the effects of widely used antidepressant drugs that act as catecholamine reuptake inhibitors. These changes occur despite the presence of other Giα family members in the same cells and are not accompanied by detectable compensatory changes in the level of expression of other G protein subunits. Therefore, these results provide insights into receptor selectivity among G proteins and a model for understanding platelet function and the effects of psychoactive drugs.

Connexin expression in electrically coupled postnatal rat brain neurons

Electrical coupling by gap junctions is an important form of cell-to-cell communication in early brain development. Whereas glial cells remain electrically coupled at postnatal stages, adult vertebrate neurons were thought to communicate mainly via chemical synapses. There is now accumulating evidence that in certain neuronal cell populations the capacity for electrical signaling by gap junction channels is still present in the adult. Here we identified electrically coupled pairs of neurons between postnatal days 12 and 18 in rat visual cortex, somatosensory cortex, and hippocampus. Notably, coupling was found both between pairs of inhibitory neurons and between inhibitory and excitatory neurons. Molecular analysis by single-cell reverse transcription–PCR revealed a differential expression pattern of connexins in these identified neurons.