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PubMed2006

Phylogeny of the Procyonidae (Mammalia: Carnivora): molecules, morphology and the Great American Interchange.

The Procyonidae (Mammalia: Carnivora) have played a central role in resolving the controversial systematics of the giant and red pandas, but phylogenetic relationships of species within the family itself have received much less attention. Cladistic analyses of morphological characters conducted during the last two decades have resulted in topologies that group ecologically and morphologically similar taxa together. Specifically, the highly arboreal and frugivorous kinkajou (Potos flavus) and olingos (Bassaricyon) define one clade, whereas the more terrestrial and omnivorous coatis (Nasua), raccoons (Procyon), and ringtails (Bassariscus) define another clade, with the similar-sized Nasua and Procyon joined as sister taxa in this latter group. These relationships, however, have not been tested with molecular sequence data. We examined procyonid phylogenetics based on combined data from nine nuclear and two mitochondrial gene segments totaling 6534bp. We were able to fully resolve relationships within the family with strongly supported and congruent results from maximum parsimony, maximum likelihood, minimum evolution, and Bayesian analyses. We identified three distinct lineages within the family: a (Nasua, Bassaricyon) clade, a (Bassariscus, Procyon) clade, and a Potos lineage, the last of which is sister to the other two clades. These findings, which are in strong disagreement with prior fossil and morphology-based assessments of procyonid relationships, reemphasize the morphological and ecological flexibility of these taxa. In particular, morphological similarities between unrelated genera possibly reflect convergence associated with similar lifestyles and diets rather than ancestry. Furthermore, incongruence between the molecular supermatrix and a morphological character matrix comprised mostly of dental characters [Baskin, J.A., 2004. Bassariscus and Probassariscus (Mammalia, Carnivora, Procyonidae) from the early Barstovian (Middle Miocene). J. Vert. Paleo. 24, 709-720] may be due to non-independence among atomized dental characters that does not take into account the high developmental genetic correlation of these characters. Finally, molecular divergence dating analyses using a relaxed molecular clock approach suggest that intergeneric and intrageneric splits in the Procyonidae mostly occurred in the Miocene. The inferred divergence times for intrageneric splits for several genera whose ranges are bisected by the Panamanian Isthmus is significant because they suggest diversification well precedes the Great American Interchange, which has long been considered a primary underlying mechanism for procyonid evolution.

Animals↗

PubMed2006

Motility disorder in experimentally obstructed intestine: relationship between muscularis inflammation and disruption of the ICC network.

We designed a model of small intestinal obstruction in rats to investigate changes in intestinal contractility associated with the immunologically activated components in the tunica muscularis. Although histochemical study did not reveal any typical inflammatory signs such as leucocyte infiltration in the distended intestinal regions of model rats 2-3 weeks after surgical induction of intestinal obstruction, the number of ED2-positive macrophages appeared to be increased in the tunica muscularis. Expression of tumour necrosis factor (TNF)-alpha mRNA was also significantly increased, and the level of CD14 was also increased significantly in the tunica muscularis. Functional studies of distended intestinal muscle segments showed a marked decrease in absolute force stimulated by a cholinergic agent. In addition, the number of spontaneous rhythmic contractions was also reduced in the distended intestinal regions of the obstructed intestine, and this decrease was associated with a reduction in the number of interstitial cells of Cajal (ICC), as revealed by Kit-like immunoreactivity. These results suggest that, under the pro-inflammatory conditions of the tunica muscularis associated with intestinal obstruction, the release of bioactive substances, possibly from activated resident macrophages, may affect smooth muscle contractility. Furthermore, under these conditions, both the number and the function of neighbouring ICC may also be affected.

Animals↗

PubMed2006

Genetic variation, population structure, and phylogenetic relationships of Triatoma rubida and T. recurva (Hemiptera: Reduviidae: Triatominae) from the Sonoran Desert, insect vectors of the Chagas' disease parasite Trypanosoma cruzi.

Nucleotide and amino acid sequence data from the mitochondrial cytochrome b (Cytb) and cytochrome c oxidase subunit I (COI) gene segments were used to gain insights into the population biology and phylogenetic relationships of two species of hematophagous kissing bugs (Hemiptera: Reduviidae: Triatominae) from the Sonoran Desert of northwestern Mexico and southern Arizona, USA, Triatoma rubida (Uhler, 1894) and T. recurva (Stål, 1868), both of which are vectors of the protozoan parasite Trypanosoma cruzi responsible for Chagas' disease. Analysis of molecular variance of gene sequences indicated significant structure among populations of both species from widely separated geographic localities. Phylogenetic analyses of gene and amino acid sequences employing both Bayesian and parsimony methods showed that T. recurva clustered within the phyllosoma complex of Triatoma species from central and southern Mexico with high statistical support, and that it was closely related to T. longipennis. Triatoma dimidiata also was shown to be closely related to the phyllosoma complex, as was T. sanguisuga which has historically been assigned to the lecticularia complex. Analyses of gene sequences were unable to confidently resolve relationships of T. rubida, although weak support for a T. nitida+T. rubida clade was seen under certain conditions. A provisional calibration of a mitochondrial DNA molecular clock for T. rubida, based on geological dates for the vicariant separation of the Baja California peninsula from mainland Mexico, suggested that pairwise sequence divergences for the Cytb and COI genes were 1.1-1.8% and 0.6-1.0% per million years, respectively. Two highly supported sympatric lineages of T. rubida uhleri from southern Arizona, which are hypothesized to have diverged approximately 550,000-900,000 years ago, were detected in the Cytb gene trees.

Amino Acid Sequence↗

PubMed1994

Phylogeny of all major groups of cetaceans based on DNA sequences from three mitochondrial genes.

Traditionally, living cetaceans (order Cetacea) are classified into two highly distinct suborders: the echolocating toothed whales, Odontoceti, and the filter-feeding baleen whales, Mysticeti. A molecular phylogeny based on 1,352 base pairs of two mitochondrial ribosomal gene segments and the mitochondrial cytochrome b gene for all major groups of cetaceans contradicts this long-accepted taxonomic subdivision. One group of toothed whales, the sperm whales, is more closely related to the morphologically highly divergent baleen whales than to other odontocetes. This finding suggests that the suborder Odontoceti constitutes an unnatural grouping and challenges the conventional scenario of a long, independent evolutionary history of odontocetes and mysticetes. The superfamily Delphinoidea (dolphins, porpoises, and white whales) appears to be monophyletic; the Amazon River dolphin, Inia geoffrensis, is its sister species. This river dolphin is genetically more divergent from the morphologically similar marine dolphins than the sperm whales are from the morphologically dissimilar baleen whales. The phylogenetic relationships among the three families of Delphinoidea remain uncertain, and we suggest that the two cladogenetic events that generated these three clades occurred within a very short period of time. Among the baleen whales, the bowhead is basal, and the gray whale is the sister species to the rorquals (family Balaenopteridae). The phylogenetic position of beaked whales (Ziphioidea) remains weakly supported by molecular data. Based on molecular clock assumptions, the mitochondrial-DNA data suggest a more recent origin of baleen whales (approximately 25 mya) than has been previously assumed (> 40 mya). This revised phylogeny has important implications for the rate and mode of evolution of morphological and physiological innovations in cetaceans.

Animals↗

PubMedDate not supplied

A pulsed neural network model of bursting in the basal ganglia.

We present new techniques for extending the functionality of spiking neurons which allow the incorporation of several aspects of neuron function previously confined to the domain of low level ion-channel based models. These aspects include spontaneous (or endogenous) firing, the complex interaction of multiple ion-species and the spatial distribution of synaptic contacts over the cell membrane. These ideas are applied to a neural circuit consisting of the cortex and a subset of the nuclei in the basal ganglia-the subthalamic nucleus (STN) and the external segment of the globus pallidus (GPe). This circuit has been studied extensively in vitro by Plenz and Kitai [Plenz, D., & Kitai, S. T. (1999). A basal ganglia pacemaker formed by the subthalamic nucleus and external globus pallidus. Nature, 400 677-682] whose data we use to constrain our model. With respect to this circuit, we have obtained three main results. First, that its characteristic burst firing is due to a Ca2+ current mediated mechanism. Second, that noise can assist in the generation of bursting and, paradoxically, stabilise the network behaviour under synaptic weight variations. Third, that a variety of dendritic processing is necessary in order to obtain the full range of bursting behaviour.

Action Potentials↗

PubMed2002

Combined nuclear and mitochondrial DNA sequences resolve generic relationships within the Cracidae (Galliformes, Aves).

The Cracidae is one of the most endangered and distinctive bird families in the Neotropics, yet the higher relationships among taxa remain uncertain. The molecular phylogeny of its 11 genera was inferred using 10,678 analyzable sites (5,412 from seven different mitochondrial segments and 5,266 sites from four nuclear genes). We performed combinability tests to check conflicts in phylogenetic signals of separate genes and genomes. Phylogenetic analysis showed that the unrooted tree of ((curassows, horned guan) (guans, chachalacas)) was favored by most data partitions and that different data partitions provided support for different parts of the tree. In particular, the concatenated mitochondrial DNA (mtDNA) genes resolved shallower nodes, whereas the combined nuclear sequences resolved the basal connections among the major clades of curassows, horned guan, chachalacas, and guans. Therefore, we decided that for the Cracidae all data should be combined for phylogenetic analysis. Maximum parsimony (MP), maximum likelihood (ML), and Bayesian analyses of this large data set produced similar trees. The MP tree indicated that guans are the sister group to (horned guan, (curassows, chachalacas)), whereas the ML and Bayesian analysis recovered a tree where the horned guan is a sister clade to curassows, and these two clades had the chachalacas as a sister group. Parametric bootstrapping showed that alternative trees previously proposed for the cracid genera are significantly less likely than our estimate of their relationships. A likelihood ratio test of the hypothesis of a molecular clock for cracid mtDNA sequences using the optimal ML topology did not reject rate constancy of substitutions through time. We estimated cracids to have originated between 64 and 90 million years ago (MYA), with a mean estimate of 76 MYA. Diversification of the genera occurred approximately 41-3 MYA, corresponding with periods of global climate change and other Earth history events that likely promoted divergences of higher level taxa.

Animals↗

PubMed2000

Pattern segmentation in a binary/analog world: unsupervised learning versus memory storing.

We discuss the problem of segmentation in pattern recognition. We adopt the model and the general approach in the landmark paper by Wang, Buhmann and von der Malsburg (Neural Computation, (1990), 2, 94-106), and expand their model in a number of ways. We review their solution to the segmentation problem in associative memory, which consists in feature binding being expressed by synchrony relations between oscillators or populations of neurons. We extend the model by introducing a law of synaptic change, which allows the network to learn by structuring itself in response to stimuli with relevant features. We discuss the problem of interference between pattern completion and the learning of new memories. We also propose a form of multiplexing of input information taking advantage of the time-structure of the neurons' response. It is based on the assessment of analog as well as of binary properties of the stimuli and provides for an enhancement of the network's processing capacity. The relevance of the results for biological systems is pointed out.

Acetylcholine↗

PubMed2002

Wavelet entropy analysis of event-related potentials indicates modality-independent theta dominance.

Sensory/cognitive stimulation elicits multiple electroencephalogram (EEG)-oscillations that may be partly or fully overlapping over the time axis. To evaluate co-existent multi-frequency oscillations, EEG responses to unimodal (auditory or visual) and bimodal (combined auditory and visual) stimuli were analyzed by applying a new method called wavelet entropy (WE). The method is based on the wavelet transform (WT) and quantifies entropy of short segments of the event-related brain potentials (ERPs). For each modality, a significant transient decrease of WE emerged in the post-stimulus EEG epoch indicating a highly-ordered state in the ERP. WE minimum was always determined by a prominent dominance of theta (4-8 Hz) ERP components over other frequency bands. Event-related 'transition to order' was most pronounced and stable at anterior electrodes, and after bimodal stimulation. Being consistently observed across different modalities, a transient theta-dominated state may reflect a processing stage that is obligatory for stimulus evaluation, during which interfering activations from other frequency networks are minimized.

Acoustic Stimulation↗

PubMed2003

Hox genes as synchronized temporal regulators: implications for morphological innovation.

In vertebrates, clusters of Hox genes express in a nested and hierarchical fashion to endow the embryo's segments with discrete identities. Later in development, members of the same gene family are employed again to pattern the limb, intestinal, and reproductive systems. A careful analysis of the morphologies of Hox mutant mice suggests that the genes provide qualitatively different cues during the specification of segments than they do during the development of more recently derived structures. In addition to the regulatory differences noted by others, the activity of Hox genes during specification of the vertebrate metameres in some recent deletion experiments is inconsistent with a role for them as strictly spatial determinants. On the contrary, the phenotypes observed are suggestive of a role for them as elements of a generic time-keeping mechanism. By contrast, the specification of more recent evolutionary structures appears to be more spatial and gene-specific. These differences in role and effect may suggest some simple mechanisms by which the Hox clusters operate, and rules by which gene networks can be diverted to create new structures over the course of evolution. Specific predictions and experiments are proposed.

Animals↗

PubMed2005

Asymmetric pigmentary glaucoma in a patient with Marfan's syndrome.

BACKGROUND: No relationship between Marfan's syndrome and pigmentary glaucoma has previously been described in the ophthalmic literature. We describe the case of a patient with Marfan's syndrome who had bilateral pigment dispersion syndrome and asymmetrical glaucoma. METHODS: A 34-year-old man with Marfan's syndrome developed pigment dispersion bilaterally. In the right eye, elevated intra-ocular pressure was associated with marked glaucomatous excavation of the right disc and corresponding visual field loss. A localised zonular dehiscence was present at the 6 o'clock position in the right eye. RESULTS: Ultrasound biomicroscopy showed marked iris concavity in the right eye, increased iridolenticular contact and superior subluxation of the right lens, resulting in increased dispersion of pigment unilaterally. This explains the asymmetric pattern of the glaucomatous damage. CONCLUSIONS: Asymmetrical progression of pigmentary glaucoma is uncommon and should prompt a search for some other mechanical factor, which might increase the iridozonular contact on one side or reduce it on the other. The association between the two syndromes in this patient was most likely mechanical due to reduced fibrillin expression throughout the eye and particularly in the iris and the lens zonules, resulting in loss of iris tensile strength and marked iris concavity as well as zonular weakness and partial lens subluxation.

Adult↗

PubMed1999

Two pacemaker channels from human heart with profoundly different activation kinetics.

Cardiac pacemaking is produced by the slow diastolic depolarization phase of the action potential. The hyperpolarization-activated cation current (If) forms an important part of the pacemaker depolarization and consists of two kinetic components (fast and slow). Recently, three full-length cDNAs encoding hyperpolarization-activated and cyclic nucleotide-gated cation channels (HCN1-3) have been cloned from mouse brain. To elucidate the molecular identity of cardiac pacemaker channels, we screened a human heart cDNA library using a highly conserved neuronal HCN channel segment and identified two cDNAs encoding HCN channels. The hHCN2 cDNA codes for a protein of 889 amino acids. The HCN2 gene is localized on human chromosome 19p13.3 and contains eight exons spanning approximately 27 kb. The second cDNA, designated hHCN4, codes for a protein of 1203 amino acids. Northern blot and PCR analyses showed that both hHCN2 and hHCN4 are expressed in heart ventricle and atrium. When expressed in HEK 293 cells, either cDNA gives rise to hyperpolarization-activated cation currents with the hallmark features of native If. hHCN2 and hHCN4 currents differ profoundly from each other in their activation kinetics, being fast and slow, respectively. We thus conclude that hHCN2 and hHCN4 may underlie the fast and slow component of cardiac If, respectively.

Biological Clocks↗

PubMed2002

Synchronization of motor neurons during locomotion in the neonatal rat: predictors and mechanisms.

We describe here the robust synchronization of motor neurons at a millisecond time scale during locomotor activity in the neonatal rat. Action potential activity of motor neuron pairs was recorded extracellularly using tetrodes during locomotor activity in the in vitro neonatal rat spinal cord. Approximately 40% of motor neuron pairs recorded in the same spinal segment showed significant synchronization, with the duration of the central peak in cross-correlograms between motor neurons typically ranging between approximately 30 and 100 msec. The percentage of synchronized motor neuron pairs was considerably higher for pairs with similar locomotor-related activity and strong rhythmic modulation. We also found synchronization between the activities of different motor pools, even if located several segments apart. Such distant synchronization was abolished in the absence of chemical synapses, although local coupling between motor neurons persisted. On the other hand, both local and distant coupling between motor neurons were preserved after antagonism of gap junction coupling between motor neurons. These results demonstrate that motor neuron activity is strongly synchronized at a millisecond time scale during the production of locomotor activity in the neonatal rat. These results also demonstrate that chemical synaptic inputs, in addition to electrical synapses, contribute to this synchronization, suggesting the existence of multiple mechanisms underlying motor neuron synchronization in the neonatal rat. The fast synchronization described here might be involved in activity-dependent processes during development or in the coordination of individual motor neurons into a functional population underlying behavior.

Action Potentials↗

PubMed1986

Dopaminergic regulation of cone retinomotor movement in isolated teleost retinas: I. Induction of cone contraction is mediated by D2 receptors.

In the retinas of lower vertebrates, retinal photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) undergo characteristic movements in response to changes in light intensity and to signals from an endogenous circadian clock. To identify agents responsible for mediating light and/or circadian regulation of these retinomotor movements, we investigated the effects of hormones and neurotransmitters on cone, rod, and RPE movements in the green sunfish, Lepomis cyanellus. We report here that 3,4-dihydroxyphenylethylamine (dopamine) mimics the effect of light by inducing light-adaptive retinomotor movements in all three cell types. In isolated dark-cultured retinas, dopamine induced light-adaptive cone contraction with a half-maximal effect at 10(-8) M. This effect of dopamine was inhibited by antagonists with a potency order characteristic of D2 receptor mediation. The dopamine uptake blocker benztropine also induced light-adaptive cone contraction in isolated dark-cultured retinas, suggesting that there is continuous dopamine release in the dark but that concomitant uptake normally prevents activation of cone contraction. That dopamine plays a role in light regulation of cone movement is further suggested by the observation that light-induced cone contraction was partially inhibited by sulpiride, a selective D2 dopamine antagonist, or by Co2+, a blocker of synaptic transmission. Sulpiride also promoted dark-adaptive cone elongation in isolated light-adapted retinas, suggesting that continuous dopamine action is required in the light to maintain the light-adapted cone position. Dopamine can act directly on D2 receptors located on rod and cone inner/outer segments: dopamine induced light-adaptive retinomotor movements in isolated distal fragments of dark-adapted photoreceptors cultured in the dark. Together our results indicate that dopamine induces light-adaptive retinomotor movements in cones, rods, and RPE cells by activating D2 receptors. We suggest that, in vivo, dopamine plays a role in both light and circadian regulation of retinomotor movements.

Adaptation, Physiological↗

PubMed1997

Phase relations of rhythmic neuronal firing in the supramammillary nucleus and mammillary body to the hippocampal theta activity in urethane anesthetized rats.

Structures in the caudal diencephalon including the posterior hypothalamic nucleus, the supramammillary nucleus (SUM) and the nuclei of the mammillary body (MB) occupy a strategic position in the crossroads of ascending and descending traffic between the brainstem and the limbic forebrain (septum/hippocampus). In this study we analyzed the phase relations of rhythmically discharging SUM/MB cells to hippocampal theta rhythm in urethane anesthetized rats with a dual aim of separating different functional types of SUM and MB neurons and characterizing their coupling to septohippocampal theta oscillators. We found that rhythmically firing neurons in the SUM/MB represent a functionally heterogenous population of cells that are coupled with forebrain theta oscillators at different preferred phases. Based on their phase relations to hippocampal theta four groups of rhythmic SUM/MB cells were identified. Neurons of the first and second groups fired out-of-phase relative to each other and synchronously with the positive (8 degrees +/- 7) or negative peaks (-177 degrees +/- 7) of theta field activity in the hippocampus, recorded above the CA1 pyramidal layer. Cells of the other two groups, also forming out-of-phase counter-parts, fired on the rising (97 degrees +/- 9) or falling segments (-97 degrees +/- 6) of CA1 theta waves. The peaks in the phase distribution histogram were well separated, and the empty zones between them were wider (40-70 degrees) than those comprising the phase data for different groups. The variations of phase values for individual neurons, when tested during several theta epochs, did not exceed the range of a single group. Theta field potentials were also recorded in the SUM/MB and were advanced by one quarter of the cycle (79 degrees +/- 9, range 56-99 degrees) relative to CA1 theta oscillations. The present results indicate that, similar to other theta-generating structures, rhythmically firing neurons can be classified on the basis of their phase relations in the SUM/MB as well. Different classes of SUM/MB neurons might play different roles in generating and/or transmitting theta rhythmic activity of the limbic system.

Anesthetics, Intravenous↗

PubMed1995

Circadian expression of the dnaK gene in the cyanobacterium Synechocystis sp. strain PCC 6803.

The expression of the dnaK gene in the cyanobacterium Synechocystis sp. strain PCC 6803 was continuously monitored as bioluminescence by an automated monitoring system, using the bacterial luciferase genes (luxAB) of Vibrio harveyi as a reporter of promoter activity. A dnaK-reporting bioluminescent Synechocystis strain was constructed by fusing a promoterless segment of the luxAB gene set downstream of the promoter region of the Synechocystis dnaK gene and introduction of this gene fusion into a BglII site downstream of the ndhB gene in the Synechocystis chromosome. Bioluminescence from this strain was continuously monitored and oscillated with a period of about 22 h for at least 5 days in continuous light. The phase of the rhythm was reset by the timing of the 12-h dark period administered prior to the continuous light. The period of the rhythm was temperature compensated between 25 and 35 degrees C. Thus, the bioluminescence rhythm satisfied the three criteria of circadian rhythms. Furthermore, the abundance of dnaK mRNA also oscillated with a period of about 1 day for at least 2 days in continuous light conditions, indicating circadian control of dnaK gene expression in Synechocystis sp. strain PCC 6803.

Base Sequence↗

PubMedDate not supplied

Modelling inter-segmental coordination of neuronal oscillators: synaptic mechanisms for uni-directional coupling during swimming in Xenopus tadpoles.

Locomotion requires longitudinal co-ordination. We have examined uni-directional synaptic coupling processes between two classes of neuronal network oscillators: autonomously active "intrinsic" oscillators, and "potential" oscillators that lack sufficient excitatory drive for autonomous activity. We model such oscillator networks in the bilaterally-symmetrical, Xenopus tadpole spinal cord circuits that co-ordinate swimming. "Glutamate" coupling EPSPs can entrain a second oscillator of lower frequency provided their strength is sufficient. Fast (AMPA) EPSPs advance spiking on each cycle, while slow (NMDA) EPSPs increase frequency over many cycles. EPSPs can also enable rhythmicity in "potential" oscillators and entrain them. IPSPs operate primarily on a cycle-by-cycle basis. They can advance or delay spiking to entrain a second "intrinsic" oscillator with higher, equal or lower frequency. Bilaterally symmetrical coupling connections operate twice per cycle: once in each half-cycle, on each side of the receiving oscillator. Excitatory and inhibitory coupling allow entrainment in complimentary areas of parameter space.

Action Potentials↗

PubMed2006

Vasomotion in human umbilical and placental veins: role of gap junctions and intracellular calcium reservoirs in their synchronous propagation.

Vasomotion was characterized using human placentae vessel rings; force displacement transducers recorded isometric contractions. Umbilical vein rings display rhythmic contractions occurring with a frequency of 1.47+/-0.01 min(-1) and 274+/-2.2 mg (n=211) of amplitude, which corresponds to 11.1+/-0.4% of the maximal KCl contracture. Vasomotion waves were recorded for up to 8 h; their amplitude and duration was larger in umbilical veins than arteries or chorionic vessels (p<0.001), vasomotion frequency was indistinguishable among these vessels. Segments of the umbilical vein closer to the fetus showed larger amplitudes and longer-lasting waves. Gap junction blockers, including peptide Gap 27, 18alpha-glycyrrhetinic acid, hexanol, heptanol or octanol, reduced the amplitude but not the frequency of vasomotion; all these drugs, in addition, decreased tissue basal tension. The role of intracellular calcium stores was evidenced using calcium-free buffer, which reduced oscillation amplitude and tissue basal tension. Cyclopiazonic acid increased wave amplitude and tissue basal tension, reducing oscillatory frequency. We propose that biological oscillators localized in the smooth muscle layer of the umbilical cord, trigger vasomotion waves, which are synchronized and propagated via gap junctions; internal calcium reservoirs are essential for their maintenance. These myogenic oscillations may be relevant for maternal-fetus blood flow and contribute to fetal nutrition and development.

Biological Clocks↗

PubMed2005

Dissociation, culture and morphologic changes of interstitial cells of Cajal in vitro.

AIM: To study the method of dissociation, culture and investigate its morphologic changes in vitro of interstitial cells of Cajal (ICC). METHODS: Enzymatic digestion and Ficoll density centrifugation were used to dissociate ICC from the ileal segment of mice. Factors including contamination, Ca(2+), Mg(2+) and collagenase, and stem cell factor, etc., were investigated. ACK2, the antibody of c-kit, was used to identify the cultured ICC. Both light microscope and fluorescence microscope were used to observe the changes of ICC in vitro. RESULTS: The method for dissociation and culture of ICC in vitro was successfully established. After 24 h, cultured ICC exhibited a few axis-cylinders, and longer axis-cylinders were observed to form synapse of each other after 3 d. More widespread connections formed within 7 d in vitro. The changes of its morphologic character were obvious within 7 d; however, there were no obvious morphologic changes after 30 d. CONCLUSION: Many factors can influence the dissociation and culture of ICC.

Animals↗