Separate Groups Of Cells Research Articles

Lentiviral vector gene therapy and CFTR modulators show comparable effectiveness in cystic fibrosis rat airway models.

Mutation-agnostic treatments such as airway gene therapy have the potential to treat any individual with cystic fibrosis (CF), irrespective of their CF transmembrane conductance regulator (CFTR) gene variants. The aim of this study was to employ two CF rat models, Phe508del and CFTR knockout (KO), to assess the comparative effectiveness of CFTR modulators and lentiviral (LV) vector-mediated gene therapy. Cells were isolated from the tracheas of rats and used to establish air-liquid interface (ALI) cultures. Phe508del rat ALIs were treated with the modulator combination, elexacaftor-tezacaftor-ivacaftor (ETI), and separate groups of Phe508del and KO tracheal epithelial cells were treated with LV-CFTR followed by differentiation at ALI. Ussing chamber measurements were performed to assess CFTR function. ETI-treated Phe508del ALI cultures demonstrated CFTR function that was 59% of wild-type level, while gene-addition therapy restored Phe508del to 68% and KO to 47% of wild-type level, respectively. Our findings show that rat Phe508del-CFTR protein can be successfully rescued with ETI treatment, and that CFTR gene-addition therapy provides significant CFTR correction in Phe508del and KO ALI cultures to levels that were comparable to ETI. These findings highlight the potential of an LV vector-based gene therapy for the treatment of CF lung disease.

Variable organization of symbiont-containing tissue across planthoppers hosting different heritable endosymbionts.

Sap-feeding hemipteran insects live in associations with diverse heritable symbiotic microorganisms (bacteria and fungi) that provide essential nutrients deficient in their hosts' diets. These symbionts typically reside in highly specialized organs called bacteriomes (with bacterial symbionts) or mycetomes (with fungal symbionts). The organization of these organs varies between insect clades that are ancestrally associated with different microbes. As these symbioses evolve and additional microorganisms complement or replace the ancient associates, the organization of the symbiont-containing tissue becomes even more variable. Planthoppers (Hemiptera: Fulgoromorpha) are ancestrally associated with bacterial symbionts Sulcia and Vidania, but in many of the planthopper lineages, these symbionts are now accompanied or have been replaced by other heritable bacteria (e.g., Sodalis, Arsenophonus, Purcelliella) or fungi. We know the identity of many of these microbes, but the symbiont distribution within the host tissues and the bacteriome organization have not been systematically studied using modern microscopy techniques. Here, we combine light, fluorescence, and transmission electron microscopy with phylogenomic data to compare symbiont tissue distributions and the bacteriome organization across planthoppers representing 15 families. We identify and describe seven primary types of symbiont localization and seven types of the organization of the bacteriome. We show that Sulcia and Vidania, when present, usually occupy distinct bacteriomes distributed within the body cavity. The more recently acquired gammaproteobacterial and fungal symbionts generally occupy separate groups of cells organized into distinct bacteriomes or mycetomes, distinct from those with Sulcia and Vidania. They can also be localized in the cytoplasm of fat body cells. Alphaproteobacterial symbionts colonize a wider range of host body habitats: Asaia-like symbionts often colonize the host gut lumen, whereas Wolbachia and Rickettsia are usually scattered across insect tissues and cell types, including cells containing other symbionts, bacteriome sheath, fat body cells, gut epithelium, as well as hemolymph. However, there are exceptions, including Gammaproteobacteria that share bacteriome with Vidania, or Alphaproteobacteria that colonize Sulcia cells. We discuss how planthopper symbiont localization correlates with their acquisition and replacement patterns and the symbionts' likely functions. We also discuss the evolutionary consequences, constraints, and significance of these findings.

A Compartmentalized Neuronal Cell-Culture Platform Compatible With Cryo-Fixation by High-Pressure Freezing for Ultrastructural Imaging.

The human brain contains a wide array of billions of neurons and interconnections, which are often simplified for analysis in vitro using compartmentalized microfluidic devices for neuronal cell culturing, to better understand neuronal development and disease. However, such devices are traditionally incompatible for high-pressure freezing and high-resolution nanoscale imaging and analysis of their sub-cellular processes by methods including electron microscopy. Here we develop a novel compartmentalized neuronal co-culture platform allowing reconstruction of neuronal networks with high variable spatial control, which is uniquely compatible for high-pressure freezing. This cryo-fixation method is well-established to enable high-fidelity preservation of the reconstructed neuronal networks and their sub-cellular processes in a near-native vitreous state without requiring chemical fixatives. To direct the outgrowth of neurites originating from two distinct groups of neurons growing in the two different compartments, polymer microstructures akin to microchannels are fabricated atop of sapphire disks. Two populations of neurons expressing either enhanced green fluorescent protein (EGFP) or mCherry were grown in either compartment, facilitating the analysis of the specific interactions between the two separate groups of cells. Neuronally differentiated PC12 cells, murine hippocampal and striatal neurons were successfully used in this context. The design of this device permits direct observation of entire neuritic processes within microchannels by optical microscopy with high spatial and temporal resolution, prior to processing for high-pressure freezing and electron microscopy. Following freeze substitution, we demonstrate that it is possible to process the neuronal networks for ultrastructural imaging by electron microscopy. Several key features of the embedded neuronal networks, including mitochondria, synaptic vesicles, axonal terminals, microtubules, with well-preserved ultrastructures were observed at high resolution using focused ion beam – scanning electron microscopy (FIB-SEM) and serial sectioning – transmission electron microscopy (TEM). These results demonstrate the compatibility of the platform with optical microscopy, high-pressure freezing and electron microscopy. The platform can be extended to neuronal models of brain disease or development in future studies, enabling the investigation of subcellular processes at the nanoscale within two distinct groups of neurons in a functional neuronal pathway, as well as pharmacological testing and drug screening.

Novel miRNA-based biomarker panel for detection β2-agonists in goats

miRNA sequencing was applied in this work to screen miRNA biomarkers related to β2-agonists from the test and control goat samples. A total of 10 selected miRNAs were proven by qRT-PCR to be able to separate treatment cell groups from the control. With previously reported differentially expressed genes (DEGs), we used target gene prediction to build a miRNA-mRNA regulatory network related to β2-agonists, which validated the miRNA biomarkers and provided a reference for identifying the mechanism of β2-agonists. Our subsequent in vivo experiments revealed that the regulation trends of the miRNAs were the same as in vitro experiments. DD-SIMCA and heatmap analysis also indicated concordant separation effects with the 10 miRNAs, which could therefore be used as biomarkers to monitor illegal use of β2-agonists in goats.

Distribution and regulation of gonadotropin-releasing hormone, kisspeptin, RF-amide related peptide-3, and dynorphin in the bovine hypothalamus.

Recent work has led to the hypothesis that kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the arcuate nucleus (ARC) play a key role in gonadotropin-releasing hormone (GnRH) pulse generation and gonadal steroid feedback, with kisspeptin driving GnRH release and neurokinin B and dynorphin acting as pulse start and stop signals, respectively. A separate cell group, expressing RFamide-related peptide-3 (RFRP-3) has been shown to be a primary inhibitor of GnRH release. Very little is known regarding these cell groups in the bovine. In this study, we examined the relative immunoreactivity of kisspeptin, dynorphin, and RFRP-3 and their possible connectivity to GnRH neurons in the hypothalami of periestrus and diestrus bovine. While GnRH and RFRP-3 immunoreactivity were unchanged, kisspeptin and dynorphin immunoreactivity levels varied in relation to plasma progesterone concentrations and estrous status. Animals with higher plasma progesterone concentrations in diestrus had lower kisspeptin and increased dynorphin immunoreactivity in the ARC. The percentage of GnRH cells with kisspeptin or RFRP-3 fibers in close apposition did not differ between estrous stages. However, the proportions of GnRH cells with kisspeptin or RFRP-3 contacts (∼49.8% and ∼31.3%, respectively) suggest direct communication between kisspeptin and RFRP-3 cells to GnRH cells in the bovine. The data produced in this work support roles for kisspeptin and dynorphin, within the KNDy neural network, in controlling GnRH release over the ovarian cycle and conveying progesterone-negative feedback onto GnRH neurons in the bovine.

Abstract 12625: Mechanical Shear Stress Restores Mitochondrial Cytoarchitecture in Human Endothelial Cells Exposed to Angiotensin II by Modulating Activity of the Fission-Inducing Protein Dynamin Related Protein 1

Background: The mitochondria exist in a state of balance between fission (fragmentation) and fusion (joining of mitochondrial segments). Disease is characterized by excessive fission, leading to an overproduction of mitochondrial reactive oxygen species (ROS) which mediate pathological changes in vascular endothelium. Dynamin-related protein-1 (DRP-1) is the primary mediator of mitochondrial fission. The therapeutic benefit of exercise is largely due to increased endothelial shear; however the effects of shear stress on DRP-1 expression and mitochondrial structure are unknown. Hypothesis: Shear stress reduces mitochondrial fission in cultured human umbilical vein endothelial cells (HUVECs) exposed to the fission-inducing stimulus angiotensin II (ANG II) by reducing DRP-1 expression and activity. Methods: HUVECs were treated with MitoRFP for 24 hours to fluorescently label the mitochondria, then were exposed to ANG II (10 pM; 4 hours), fixed, and 25 random cells/group were imaged with a confocal microscope. Separate groups of cells were sheared (15 dyn/cm2; 24 hours) immediately following ANG II exposure using the Ibidi Pump System. To quantify mitochondrial fission, images were converted to binary color and ImageJ was used to count non-contiguous mitochondrial fragments. This value was divided by the number of pixels in the mitochondrial network and multiplied by 1000 to yield a Mitochondrial Fragmentation Index (MFI). Total DRP-1 expression and phosphorylation of the S616 activator and S637 suppressor sites were analyzed by Western blot. Results: ANG II increased MFI (1.13±0.25 vs. 0.75±0.06), DRP-1 expression (+48%) and phosphorylation of the S616 activator site (+141%; p<0.05 for all) compared to static controls. Conversely, ANG II-treated cells sheared for 24 hours showed reduced mitochondrial fission (MFI = 0.50 vs. 0.75±0.06), DRP-1 expression (-24%) and phosphorylation at S616 (-11%) compared to control. ANG II treated cells exposed to shear also showed a robust increase (+528%) in phosphorylation of the S637 suppressor site of DRP-1. Conclusions: High shear stress restores mitochondrial network integrity in HUVECs exposed to ANG II, suggesting exercise may promote vascular health by modulating mitochondrial fission.

Effects of Prefrontal Cortical Inactivation on Neural Activity in the Ventral Tegmental Area

Dopamine (DA) cells have been suggested to signal discrepancies between expected and actual rewards in reinforcement learning. DA cells in the ventral tegmental area (VTA) receive direct projections from the medial prefrontal cortex (mPFC), a structure known to be one of the brain areas that represents expected future rewards. To investigate whether the mPFC contributes to generating reward prediction error signals of DA cells, we recorded VTA cells from rats foraging for different amounts of reward in a spatial working memory task. Our results showed that DA cells initially responded after the acquisition of rewards, but over training, they exhibited phasic responses when rats detected sensory cues originating from the rewards before obtaining them. We also observed two separate groups of non-DA cells activated in expectation of upcoming rewards or during reward consumption. Bilateral injections of muscimol, a GABAA agonist, into the mPFC significantly decreased the non-DA activity that encoded reward expectation. By contrast, the same manipulation of the mPFC elevated DA responses to reward-predicting cues. However, neither DA nor non-DA responses elicited after reward acquisition were affected by mPFC inactivation. These results suggest that the mPFC provides information about expected rewards to the VTA, and its functional loss elevates DA responses to reward-predicting cues by altering expectations about forthcoming rewards.

Electrogram Fractionation

Background— Fractionated electrograms are used by some as targets for ablation in atrial and ventricular arrhythmias. Fractionation has been demonstrated to result when there is repetitive or asynchronous activation of separate groups of cells within the recording region of a mapping electrode(s). Methods and Results— Using a computer model, we generated tissue activation patterns with increasing spatiotemporal variation and calculated virtual electrograms from electrodes with decreasing resolution. We then quantified electrogram fractionation. In addition, we recorded unipolar electrograms during atrial fibrillation in 20 patients undergoing atrial fibrillation ablation. From these we constructed bipolar electrograms with increasing interelectrode spacing and quantified fractionation. During modeling of spatiotemporal variation, fractionation varied directly with electrode length, diameter, height, and interelectrode spacing. When resolution was held constant, fractionation increased with increasing spatiotemporal variation. In the absence of spatial variation, fractionation was independent of resolution and proportional to excitation frequency. In patients with atrial fibrillation, fractionation increased as interelectrode spacing increased. Conclusions— We created a model for distinguishing the roles of spatial and temporal electric variation and electrode resolution in producing electrogram fractionation. Spatial resolution affects fractionation attributable to spatiotemporal variation but not temporal variation alone. Electrogram fractionation was directly proportional to spatiotemporal variation and inversely proportional to spatial resolution. Spatial resolution limits the ability to distinguish high-frequency excitation from overcounting. In patients with atrial fibrillation, complex fractionated atrial electrogram detection varies with spatial resolution. Electrode resolution must therefore be considered when interpreting and comparing studies of fractionation.

Anti-glioma response of autologous T cells stimulated by autologous dendritic cells electrofused with CD133+ or CD133− glioma cells

Glioma, the most common tumor of the central nervous system (CNS), currently results in a high rate of morbidity and mortality. The expression of CD133, a stem-like cell marker expressed in the glioma cells, is believed to lead to tumorigenesis in the human brain. Thus, it is necessary to find a proper method to specifically kill the CD133+ glioma cells. Dendritic cell (DC)/tumor hybrids are proven to be able to induce an effective immune response, leading to killing of glioma cells in vitro. We isolated CD133+ cells from a population of primary glioma cells, and cultured autologous DCs and T cells at the same time. Next, we electrofused the DCs with the CD133+ glioma cells and with CD133− ones, in order to explore a new strategy for glioma therapy. We then exposed the T cells to five separate groups of cells: DC/CD133+ hybrids, DC/CD133− hybrids, DCs alone, unsorted glioma cells alone and mixed DCs-glioma cells. A cytotoxicity assay showed that T cells stimulated by either type of hybrid were able to kill cultured autologous glioma cells significantly more effectively than those stimulated by the other three cell types (P<0.05). The amounts of IFN-γ secreted by T cells stimulated by the two types of fused cells were obviously increased compared to those stimulated by the other three cell types (P<0.05). However, no significant differences were noted between the effects of the two hybrids, neither in the cytotoxicity assay nor in the IFN-γ release assay (P>0.05). Therefore, both DC/CD133+ and DC/CD133− hybrids can cause significant T cell immune responses in vitro. There were no significant differences between the immune responses caused by the two types of hybrids.

Fish reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts reveals high accuracy of diagnosis and no sign of mosaicism or preferential allocation

To study the blastocyst genetic constitution (possible chromosomal mosaicism between ICM and TE and within TE), after isolating ICM by a novel method.and to evaluate array-CGH accuracy against FISH testing Cross-sectional study. 32 blastocysts diagnosed as genetically abnormal by array-CGH were donated to research. ICM was isolated from the trophectoderm (TE) using a novel mechanical separation which was confirmed by KRT18 immunostaining. 24 of the 32 embryo had single or double aneuploidy and 8 aneusomic for unbalanced product of translocation or for long single arm deletions/duplications but with normal ploidy (by aCGH). After biopsy, individual samples were fixed by Carnoy method and analyzed with conventional 9-chromosomes FISH and locus specific probes. We reanalyzed 32 ICM and 96 TE samples (3 separate groups of cells from each TE of the same embryo). Looking at the single chromosome level, 28 out of 28 aneuploidies diagnosed by a-CGH were confirmed by FISH reanalysis both in ICM and TE samples resulting in a analytical sensitivity of 100%(95%CI 90.2-100). For almost all embryos, the reported aneuploidies were constitutive. As regard to chromosomes diagnosed as disomic, only 2 out of 248 (specificity 99.1 95%CI 97.4-99.8) were misdiagnosed by a-CGH, a trisomy 13 in an unbalanced embryo from reciprocal translocation carrier observed in a constitutive state and a mosaic nullisomy/monosomy 16 in a double aneuploid embryo. Consequently, only one out of 32 embryos was misdiagnosed as false negative. Only in one case we observed a confined trisomy 17 in all nuclei of a TE sample from a trisomic embryo for chromosome 13. In all other embryos ICM and TE samples were concordant and confirmed the original a-CGH diagnosis. Our data show a very high analytical accuracy of a-CGH to diagnose embryo chromosomal constitution. Concordance between ICM and TE was reported for all embryos analyzed, showing no sign of mosaicism or preferential allocation at blastocyst stage.

Taste-Specific Cell Assemblies in a Biologically Informed Model of the Nucleus of the Solitary Tract

Although the cellular organization of many primary sensory nuclei has been well characterized, questions remain about the functional architecture of the first central relay for gustation, the rostral nucleus of the solitary tract (NTS). Here we used electrophysiological data recorded from single cells in the NTS to inform a network model of taste processing. Previous studies showed that electrical stimulation of the chorda tympani (CT) nerve initiates two types of inhibitory influences with different time courses in separate groups of NTS cells. Each type of inhibition targeted cells with distinct taste response properties. Further analyses of these data identified three NTS cell types differentiated by their latency of evoked response, time course of CT evoked inhibition, and degree of selectivity across taste qualities. Based on these results, we designed a model of the NTS consisting of discrete, reciprocally connected, stimulus-specific "cell" assemblies. Input to the network of integrate-and-fire model neurons was based on electrophysiological recordings from the CT nerve. Following successful simulation of paired-pulse CT stimulation, the network was tested for its ability to discriminate between two "taste" stimuli. Network dynamics of the model produced biologically plausible responses from each unit type and enhanced discrimination between taste qualities. We propose that an interactive network of taste quality specific cell assemblies, similar to our model, may account for the coherence in across-neuron patterns of NTS responses between similar tastants.

Neuron-specific enolase-like immunoreactivity in human Leydig cells

Using the peroxidase anti-peroxidase immunocytochemical technique, neuron-specific enolase (NSE)-like immunoreactivity (NSE-LI) was revealed in Leydig cells of adult human testes at the light microscopic level. Differences in the NSE staining intensity were observed between the individual Leydig cells, separate cell groups within a testis and between the testes of individual patients. Together with the already established substance P-like immunoreactivity (SP-LI), the results obtained provided further evidence for the possible neuroectodermal origin of human Leydig cells and their presumable relation to the APUD- or the Diffuse Neuroendocrine System (DNES).

Two types of inhibitory influences target different groups of taste-responsive cells in the nucleus of the solitary tract of the rat

Electrical stimulation of the chorda tympani nerve (CT; innervating taste buds on the rostral tongue) is known to initiate recurrent inhibition in cells in the nucleus of the solitary tract (NTS, the first central relay in the gustatory system). Here, we explored the relationship between inhibitory circuits and the breadth of tuning of taste-responsive NTS neurons. Initially, NTS cells with evoked responses to electrical stimulation of the CT (0.1 ms pulses; 1 Hz) were tested with each of four tastants (0.1 M NaCl, 0.01 M HCl, 0.01 M quinine and 0.5 M sucrose) in separate trials. Next, the CT was electrically stimulated using a paired-pulse (10–2000 ms interpulse interval; blocks of 100 trials) paradigm. Forty-five (30 taste-responsive) of 51 cells with CT-evoked responses (36 taste-responsive) were tested with paired pulses. The majority (34; 75.6%) showed paired-pulse attenuation, defined as fewer evoked spikes in response to the second (test) pulse compared with the first (conditioning) pulse. A bimodal distribution of the peak of paired-pulse attenuation was found with modes at 10 ms and 50 ms in separate groups of cells. Cells with early peak attenuation showed short CT-evoked response latencies and large responses to relatively few taste stimuli. Conversely, cells with late peak attenuation showed long CT-evoked response latencies and small taste responses with less selectivity. Results suggest that the breadth of tuning of an NTS cell may result from the combination of the sensitivities of peripheral nerve inputs and the recurrent influences generated by the circuitry of the NTS.

Localization of Amiloride-Sensitive Sodium Current and Voltage-Gated Calcium Currents in Rat Fungiform Taste Cells

Recent studies have shown that taste cells transducing bitter, sweet, and umami stimuli do not possess high-threshold voltage-gated calcium channels required for synaptic transmission at conventional synapses, suggesting some sort of signal processing inside taste buds prior to the activation of nerve endings. To evaluate whether this is a general paradigm for the physiology of taste reception, we studied the transduction pathway underlying the detection of sodium ions (salty stimulus). In laboratory rodents, Na(+) is thought to be transduced, at least in part, through amiloride-sensitive sodium channels (ASSCs). Therefore we used the patch-clamp techniques to analyze the occurrence pattern of amiloride-sensitive sodium currents and voltage-gated calcium currents (both low-voltage-activated T-type current and high-voltage-activated L-type current) among taste cells in rat fungiform papillae. Because taste cells turnover, we focused our attention on cells possessing large voltage-gated sodium currents, a sign of "mature" cells. We found that cells expressing functional ASSCs either did not possess any calcium currents or exhibited only T-type calcium currents, which is believed to play a role in repetitive firing. On the contrary, cells lacking functional ASSCs were endowed with L-type calcium currents, which are thought to mediate calcium influx required for neurotransmitter exocytosis. Therefore our data suggest that sodium-detecting cells are unlikely to use conventional synaptic communication to transfer taste information to nerve endings. Our findings on sodium taste detection support the recent model of taste transduction, involving separate groups of taste cells: chemosensitive cells and cells forming conventional synapses.

Structural and functional classes of multipolar cells in the ventral cochlear nucleus

Multipolar cells in the ventral cochlear nucleus (VCN) are a structurally and functionally diverse group of projection neurons. Understanding their role in the ascending pathway involves partitioning multipolar cells into distinct populations and determining where in the brain each sends its coded messages. In this study, we used retrograde labeling techniques in rats to identify multipolar neurons that project their axons to the ipsilateral dorsal cochlear nucleus (DCN), the contralateral CN, or both structures. Three rats received injections of biotinylated dextran amine in the ipsilateral DCN and diamidino yellow in the contralateral CN. Several radiate multipolar neurons (defined by their axonal projections to the ipsilateral DCN and their dendrites that traverse VCN isofrequency sheets) were double-labeled but over 70% were not. This result suggests two distinct populations: (1) radiate-commissural (RC) multipolar cells that project to the ipsilateral DCN and the contralateral CN, and (2) radiate multipolar cells that project exclusively (in this context) to the ipsilateral DCN. In a different group of animals, we retrogradely labeled multipolar neurons that project their axons to the contralateral CN and measured the size of their cell bodies. The mean size of this population (266 +/- 156 microm2) was significantly smaller than those of RC-multipolar cells (418 +/- 140 microm2). We conclude that the CN commissural pathway is composed of at least two components: (1) RC multipolar cells and (2) commissural multipolar cells that are small- and medium-sized neurons that project exclusively (in this context) to the contralateral CN. These results identify separate structural groups of multipolar cells that may correspond to physiological unit types described in the literature. They also provide protocols for isolating and studying different populations of multipolar cells to determine the neural mechanisms that govern their responses to sound.

Afferent projections to pharynx and soft palate motoneurons: A light and electron microscopical tracing study in the cat

Pharynx and soft palate are muscles for respiration, vocalization, swallowing, and vomiting. In cat, motoneurons innervating pharynx/soft palate are located in the dorsal group of the nucleus ambiguus (dgNA) in the medulla oblongata. In cat, dgNA is the only part of nucleus ambiguus that can be distinguished as a separate cell group, which makes it possible to study its afferent input. In two cats, WGA-HRP injections in dgNA and surrounding tegmentum resulted in retrogradely labeled cells at several levels of the neuraxis. In 170 cases anterograde tracers were injected in areas in which the cells of origin were identified. Results demonstrate that dgNA afferents originate from the tegmentum dorsolateral to the superior olivary complex, medullary ventromedial tegmentum, caudal raphe nuclei, medullary lateral tegmental field, nucleus retroambiguus (NRA), and adjoining tegmentum, extending into the first cervical segment of the spinal cord. In order to determine whether periaqueductal gray (PAG) and parabrachial nuclei (PB) make synaptic contacts with dgNA, ultrastructural studies combined anterograde tracing from PAG, PB, and NRA with retrograde tracing of pharyngeal and soft palate motoneurons. The results showed that PB, but not PAG, projects to the dgNA and that NRA afferent synapses are three times as numerous as those from PB. The morphology of PB and NRA synapses is consistent with excitatory input. In conclusion, pharyngeal and soft palate motoneurons receive their afferents almost exclusively from the pontine and medullary tegmentum and first cervical spinal segment.

Immunophenotyping of acute leukemias and myelodysplastic syndromes.

Alberto Orfao,* Francisco Ortuno, Maria de Santiago, Antonio Lopez, and Jesus San Miguel Servicio General de Citometria, Universidad de Salamanca, Salamanca, Spain Centro de Investigacion del Cancer y Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain Servicio de Hematologia y Oncologia Medica, Hospital General Universitario J.M. Morales Meseguer, Murcia, Spain Servicio de Hematologia, Hospital Universitario de Salamanca, Salamanca, Spain

Pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in melatonin release via the specific receptor PACAP-r1, but not in the circadian oscillator, in chick pineal cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates melatonin release from pineal cells and modulates glutamatergic regulation of the suprachiasmatic circadian clock in rodents. We investigated whether PACAP is involved in melatonin release and the circadian oscillation system in chick pineal cells, and if so, whether its effects are mediated by the PACAP-specific receptor (PACAP-r1) or the vasoactive intestinal polypeptide (VIP) receptor. Chick pineal cells were maintained for 4 days under a 12-h light/dark cycle, and thereafter in constant darkness. In the dose-range 10 −10 to 10 −6 M, PACAP increased melatonin release dose-dependently during the 12-h light period on day 3 of culture, and the degree of stimulation was greater than that produced by VIP. VIP receptor antagonists only slightly inhibited PACAP-stimulated melatonin release. Simultaneous addition of VIP and PACAP produced almost additive melatonin release. Under constant dark conditions, 6-h pulses of PACAP started at zeitogaber times (ZT) 15, 21, 3 and 9 h in separate groups of pineal cells did not cause any phase shift in their melatonin rhythm. In addition, PACAP did not affect the light-induced phase advance (ZT 15 h) and delay (ZT 9 h) in melatonin rhythms. The expression of mRNA for the PACAP-r1 (including its splicing variant with a hop cassette) was observed in chick pineal cells. These results suggest that PACAP participates in melatonin release, but not in the circadian oscillator system, via the specific receptor PACAP-r1 in chick pineal cells.

Expression of Foxb1 Reveals Two Strategies for the Formation of Nuclei in the Developing Ventral Diencephalon

Fork head b1 (Foxb1; also called Fkh5, HFH-e5.1, Mf3) is a winged helix transcription factor gene whose widespread early expression in the developing neural tube is soon restricted to the ventral and caudal diencephalon. During diencephalic neurogenesis, Foxb1 is expressed in one patch of neuroepithelium comprising a large mammillary portion and a smaller tuberal portion. The labeled cells coming from this patch contribute to nuclear formation by means of two different strategies: (1) caudally, the young neurons aggregate and settle immediately, giving rise to the nuclei of the mammillary body; (2) rostrally, the young neurons separate from the neuroepithelium forming a trail of cells which spans the mantle layer mediolaterally and which will give rise to two separate cell groups (the dorsal premammillary and part of the lateral hypothalamic area). Our results show the elaborate, regionalized histogenetic mechanisms necessary for the differentiation of the caudal diencephalon; moreover, they suggest that specifically labeled populations, arising from specifically labeled neuroepithelial patches and giving place to specific brain nuclei could be a common mechanism to build complex, nonlaminar regions of the forebrain.

In ovo time-lapse analysis of chick hindbrain neural crest cell migration shows cell interactions during migration to the branchial arches.

Hindbrain neural crest cells were labeled with DiI and followed in ovo using a new approach for long-term time-lapse confocal microscopy. In ovo imaging allowed us to visualize neural crest cell migration 2-3 times longer than in whole embryo explant cultures, providing a more complete picture of the dynamics of cell migration from emergence at the dorsal midline to entry into the branchial arches. There were aspects of the in ovo neural crest cell migration patterning which were new and different. Surprisingly, there was contact between neural crest cell migration streams bound for different branchial arches. This cell-cell contact occurred in the region lateral to the otic vesicle, where neural crest cells within the distinct streams diverted from their migration pathways into the branchial arches and instead migrated around the otic vesicle to establish a contact between streams. Some individual neural crest cells did appear to cross between the streams, but there was no widespread mixing. Analysis of individual cell trajectories showed that neural crest cells emerge from all rhombomeres (r) and sort into distinct exiting streams adjacent to the even-numbered rhombomeres. Neural crest cell migration behaviors resembled the wide diversity seen in whole embryo chick explants, including chain-like cell arrangements; however, average in ovo cell speeds are as much as 70% faster. To test to what extent neural crest cells from adjoining rhombomeres mix along migration routes and within the branchial arches, separate groups of premigratory neural crest cells were labeled with DiI or DiD. Results showed that r6 and r7 neural crest cells migrated to the same spatial location within the fourth branchial arch. The diversity of migration behaviors suggests that no single mechanism guides in ovo hindbrain neural crest cell migration into the branchial arches. The cell-cell contact between migration streams and the co-localization of neural crest cells from adjoining rhombomeres within a single branchial arch support the notion that the pattern of hindbrain neural crest cell migration emerges dynamically with cell-cell communication playing an important guidance role.