The zinc finger transcription factor PLAGL2 enhances stem cell fate and activates expression of ASCL2 in intestinal epithelial cells

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Plasmodium falciparum translational machinery condones polyadenosine repeats

Plasmodium falciparum is a causative agent of human malaria. Sixty percent of mRNAs from its extremely AT-rich (81%) genome harbor long polyadenosine (polyA) runs within their ORFs, distinguishing the parasite from its hosts and other sequenced organisms. Recent studies indicate polyA runs cause ribosome stalling and frameshifting, triggering mRNA surveillance pathways and attenuating protein synthesis. Here, we show that P. falciparum is an exception to this rule. We demonstrate that both endogenous genes and reporter sequences containing long polyA runs are efficiently and accurately translated in P. falciparum cells. We show that polyA runs do not elicit any response from No Go Decay (NGD) or result in the production of frameshifted proteins. This is in stark contrast to what we observe in human cells or T. thermophila, an organism with similar AT-content. Finally, using stalling reporters we show that Plasmodium cells evolved not to have a fully functional NGD pathway

Gold nanoparticle-enhanced X-ray microtomography of the rodent reveals region-specific cerebrospinal fluid circulation in the brain

Cerebrospinal fluid (CSF) is essential for the development and function of the central nervous system (CNS). However, the brain and its interstitium have largely been thought of as a single entity through which CSF circulates, and it is not known whether specific cell populations within the CNS preferentially interact with the CSF. Here, we develop a technique for CSF tracking, gold nanoparticle-enhanced X-ray microtomography, to achieve micrometer-scale resolution visualization of CSF circulation patterns during development. Using this method and subsequent histological analysis in rodents, we identify previously uncharacterized CSF pathways from the subarachnoid space (particularly the basal cisterns) that mediate CSF-parenchymal interactions involving 24 functional-anatomic cell groupings in the brain and spinal cord. CSF distribution to these areas is largely restricted to early development and is altered in posthemorrhagic hydrocephalus. Our study also presents particle size-dependent CSF circulation patterns through the CNS including interaction between neurons and small CSF tracers, but not large CSF tracers. These findings have implications for understanding the biological basis of normal brain development and the pathogenesis of a broad range of disease states, including hydrocephalus

Integrative Neural Behavior in a Mammalian Nervous System Revealed Using Ca2+ Imaging

Historically, research on nervous systems has been hindered by the complexity of such systems, and the inability to record activity in multiple neurons at the same time. As such, our current understanding of the way a nervous system functions has been the result of electrophysiological studies performed on single, or small groups of neurons, as well as research on relatively simple invertebrate models. In the work presented in this dissertation, we have used imaging techniques to explore the functions of several classes of neurons and auxiliary cells in the enteric nervous system of the murine large bowel during a compound motor behavior, the colonic migrating motor complex (CMMC).Using low-powered Ca2+ imaging in whole tissue preparations, we explored the relationship between the longitudinal and circular muscle layers of the colon, showing a high degree of synchronization between the two layers during the CMMC. This synchronization is further shown to be a function of enteric innervation, as addition of nicotinic and muscarinic antagonists disrupted coordinated Ca2+ waves in the two muscle layers. Using a mouse model for Hirschsprung's disease (aganglionosis), we have shown that CMMCs do not occur in mice that lack enteric innervation, and Ca2+ waves that persist are unsynchronized, and do not propagate sufficient distances to generate pellet propulsion.The myenteric plexus of the colon contains 13 different classes of enteric neurons, including sensory neurons, ascending and descending interneurons, and both excitatory and inhibitory motor neurons. The use of Ca2+ imaging as a technique to study the activity of enteric neurons provided little information regarding the class of neurons being observed. This dissertation details a number of techniques which we have developed to facilitate the identification of these enteric neurons using post hoc staining, and have subsequently used to identify inhibitory motor neurons, sensory neurons, types of interneurons and different classes of interstitial cells in the murine colon.Our work has identified AH/Dogiel Type II neurons, which stained intensely with the mitochondrial marker, Mitotracker, as the first responders in the generation of the CMMC in response to mucosal stimulations. Responses in these cells were abolished in the presence of 5- HT3 receptor antagonists, and when the mucosa was removed, suggesting that 5-HT release from enterochromaffin cells in the mucosa activates AH/Dogiel Type II neurons to generate a CMMC. Furthermore, we have shown that during the CMMC, there is an increase in Ca2+ transient frequency in neurons that tested negative for NOS, and were presumably excitatory motor neurons, as well as a marked decrease in the activity of NOS+ve neurons, which were likely inhibitory motor neurons.Recent studies have identified a colonic occult reflex, which is activated by colonic elongation, leading to the activation of mechanosensitive descending inhibitory interneurons, which inhibit the activation of the peristaltic reflex. This occult reflex is thought to underlie slow transit constipation, and as the CMMC in the murine large bowel is responsible for fecal pellet propulsion, we sought to establish the effects of colonic elongation on the CMMC. Our findings show that colonic elongation led to a nitric oxide-mediated reduction in the amplitude of migrating complexes. During such elongation, enteric neurons, including AH/Dogiel Type II neurons exhibited reduced amplitude and frequency of spontaneous Ca2+ transients.Ca2+ imaging experiments of the myenteric plexus region during the CMMC revealed an increase in the Ca2+ transient frequency of ICC-MY, which, in the colon, are believed to underlie synchronization of the longitudinal and circular muscle layers. Using high-power imaging, we observed both excitatory and inhibitory nerve varicosities in close apposition to ICC-MY, which activated or inactivated the cells, respectively. Pharmacological studies revealed that ICC-MY have both muscarinic and NK1 receptors, which are likely the targets of ACh and TK release from excitatory motor neurons.Collectively these findings offer insight into the neuronal mechanism that underlies the generation and propagation of the colonic migrating motor complex in the murine large intestine. From a larger perspective, studies described in this dissertation represent an important step in our understanding of how neural networks in a mammalian nervous system functions to generate a complex rhythmic motor behavior

A sodium afterdepolarization in rat superior colliculus neurons and its contribution to population activity.

The mammalian superior colliculus (SC) is a midbrain structure that integrates multimodal sensory inputs and computes commands to initiate rapid eye movements. SC neurons burst with the sudden onset of a visual stimulus, followed by persistent activity that may underlie shifts of attention and decision making. Experiments in vitro suggest that circuit reverberations play a role in the burst activity in the SC, but the origin of persistent activity is unclear. In the present study we characterized an afterdepolarization (ADP) that follows action potentials in slices of rat SC. Population responses seen with voltage-sensitive dye imaging consisted of rapid spikes followed immediately by a second distinct depolarization of lower amplitude and longer duration. Patch-clamp recordings showed qualitatively similar behavior: in nearly all neurons throughout the SC, rapid spikes were followed by an ADP. Ionic and pharmacological manipulations along with experiments with current and voltage steps indicated that the ADP of SC neurons arises from Na(+) current that either persists or resurges following Na(+) channel inactivation at the end of an action potential. Comparisons of pharmacological properties and frequency dependence revealed a clear parallel between patch-clamp recordings and voltage imaging experiments, indicating a common underlying membrane mechanism for the ADP in both single neurons and populations. The ADP can initiate repetitive spiking at intervals consistent with the frequency of persistent activity in the SC. These results indicate that SC neurons have intrinsic membrane properties that can contribute to electrical activity that underlies shifts of attention and decision making

A hard-wired priority map in the superior colliculus shaped by asymmetric inhibitory circuitry.

The mammalian superior colliculus (SC) is a laminar midbrain structure that translates visual signals into commands to shift the focus of attention and gaze. The SC plays an integral role in selecting targets and ultimately generating rapid eye movements to those targets. In all mammals studied to date, neurons in the SC are arranged topographically such that the location of visual stimuli and the endpoints of orienting movements form organized maps in superficial and deeper layers, respectively. The organization of these maps is thought to underlie attentional priority by assessing which regions of the visual field contain behaviorally relevant information. Using voltage imaging and patch-clamp recordings in parasagittal SC slices from the rat, we found the synaptic circuitry of the visuosensory map in the SC imposes a strong bias. Voltage imaging of responses to electrical stimulation revealed more spread in the caudal direction than the rostral direction. Pharmacological experiments demonstrated that this asymmetry arises from GABAA receptor activation rostral to the site of stimulation. Patch-clamp recordings confirmed this rostrally directed inhibitory circuit and showed that it is contained within the visuosensory layers of the SC. Stimulation of two sites showed that initial stimulation of a caudal site can take priority over subsequent stimulation of a rostral site. Taken together, our data indicate that the circuitry of the visuosensory SC is hard-wired to give higher priority to more peripheral targets, and this property is conferred by a uniquely structured, dedicated inhibitory circuit

A sodium afterdepolarization in rat superior colliculus neurons and its contribution to population activity.

The mammalian superior colliculus (SC) is a midbrain structure that integrates multimodal sensory inputs and computes commands to initiate rapid eye movements. SC neurons burst with the sudden onset of a visual stimulus, followed by persistent activity that may underlie shifts of attention and decision making. Experiments in vitro suggest that circuit reverberations play a role in the burst activity in the SC, but the origin of persistent activity is unclear. In the present study we characterized an afterdepolarization (ADP) that follows action potentials in slices of rat SC. Population responses seen with voltage-sensitive dye imaging consisted of rapid spikes followed immediately by a second distinct depolarization of lower amplitude and longer duration. Patch-clamp recordings showed qualitatively similar behavior: in nearly all neurons throughout the SC, rapid spikes were followed by an ADP. Ionic and pharmacological manipulations along with experiments with current and voltage steps indicated that the ADP of SC neurons arises from Na(+) current that either persists or resurges following Na(+) channel inactivation at the end of an action potential. Comparisons of pharmacological properties and frequency dependence revealed a clear parallel between patch-clamp recordings and voltage imaging experiments, indicating a common underlying membrane mechanism for the ADP in both single neurons and populations. The ADP can initiate repetitive spiking at intervals consistent with the frequency of persistent activity in the SC. These results indicate that SC neurons have intrinsic membrane properties that can contribute to electrical activity that underlies shifts of attention and decision making

A hard-wired priority map in the superior colliculus shaped by asymmetric inhibitory circuitry.

The mammalian superior colliculus (SC) is a laminar midbrain structure that translates visual signals into commands to shift the focus of attention and gaze. The SC plays an integral role in selecting targets and ultimately generating rapid eye movements to those targets. In all mammals studied to date, neurons in the SC are arranged topographically such that the location of visual stimuli and the endpoints of orienting movements form organized maps in superficial and deeper layers, respectively. The organization of these maps is thought to underlie attentional priority by assessing which regions of the visual field contain behaviorally relevant information. Using voltage imaging and patch-clamp recordings in parasagittal SC slices from the rat, we found the synaptic circuitry of the visuosensory map in the SC imposes a strong bias. Voltage imaging of responses to electrical stimulation revealed more spread in the caudal direction than the rostral direction. Pharmacological experiments demonstrated that this asymmetry arises from GABAA receptor activation rostral to the site of stimulation. Patch-clamp recordings confirmed this rostrally directed inhibitory circuit and showed that it is contained within the visuosensory layers of the SC. Stimulation of two sites showed that initial stimulation of a caudal site can take priority over subsequent stimulation of a rostral site. Taken together, our data indicate that the circuitry of the visuosensory SC is hard-wired to give higher priority to more peripheral targets, and this property is conferred by a uniquely structured, dedicated inhibitory circuit