The definition of the nerve cell types of the myenteric plexus of the mouse small intestine has become important, as more researchers turn to the use of mice with genetic mutations to analyze roles of specific genes and their products in enteric nervous system function and to investigate animal models of disease. We have used a suite of antibodies to define neurons by their shapes, sizes, and neurochemistry in the myenteric plexus. Anti-Hu antibodies were used to reveal all nerve cells, and the major subpopulations were defined in relation to the Hu-positive neurons. Morphological Type II neurons, revealed by anti-neurofilament and anti-calcitonin gene-related peptide antibodies, represented 26% of neurons. The axons of the Type II neurons projected through the circular muscle and submucosa to the mucosa. The cell bodies were immunoreactive for choline acetyltransferase (ChAT), and their terminals were immunoreactive for vesicular acetylcholine transporter (VAChT). Nitric oxide synthase (NOS) occurred in 29% of nerve cells. Most were also immunoreactive for vasoactive intestinal peptide, but they were not tachykinin (TK)-immunoreactive, and only 10% were ChAT-immunoreactive. Numerous NOS terminals occurred in the circular muscle. We deduced that 90% of NOS neurons were inhibitory motor neurons to the muscle (26% of all neurons) and 10% (3% of all neurons) were interneurons. Calretinin immunoreactivity was found in a high proportion of neurons (52%). Many of these had TK immunoreactivity. Small calretinin neurons were identified as excitatory neurons to the longitudinal muscle (about 20% of neurons, with ChAT/calretinin/+/- TK chemical coding). Excitatory neurons to the circular muscle (about 10% of neurons) had the same coding. Calretinin immunoreactivity also occurred in a proportion of Type II neurons. Thus, over 90% of neurons in the myenteric plexus of the mouse small intestine can be currently identified by their neurochemistry and shape.

Cell-to-cell communication was characterized in cumulus-oocyte complexes from rat ovarian follicles before and after ovulation. Numerous, small gap junctional contacts were present between cumulus cells and oocytes before ovulation. The gap junctions are formed on the oocyte surface by cumulus cell processes that traverse the zona pellucida and contact the oolemma. The entire cumulus mass was also connected by gap junctions via cumulus-cumulus interactions. In the hours preceding ovulation, the frequency of gap junctional contacts between cumulus cells and the oocyte was reduced, and the cumulus was disorganized. Electrophysiological measurements indicated that bidirectional ionic coupling was present between the cumulus and oocyte before ovulation. In addition, iontophoretically injected fluorescein dye was tranferrred between the oocyte and cumulus cells. Examination of the extent of ionic coupling in cumulus-oocyte specimens before and after ovulation revealed that ionic coupling between the cumulus and oocyte progressively decreased as the time of ovulation approached. In postovulatory specimens, no coupling was detected. Although some proteolytic mechanism may be involved in the disintegration of the cumulus-oocyte complex, neither the cumulus cells nor the oocyte produced detectable levels of plasminogen activator, a protease which is synthesized by membrana granulosa cells.In summary, cell communication is a characteristic feature of the cumulusoocyte complex, and this communication is terminated near the time of ovulation. This temporal pattern of the termination of communication between the cumulus and the oocyte may indicate that communication provides a mechanism for regulating the maturation of the oocyte during follicular development before ovulation.KEY WORDS cell communication gap junctions -ionic (electrotonic) coupling dye transfer cumulus-oocyte complex ovulationThe avascular compartment of the mammalian ovarian follicle contains several different types of cells whose metabolism and function must be precisely controlled during the hours preceding ovulation. Since a number of known hormonedependent metabolic and morphologic changes occur during this rather brief time period (4), the follicle represents a unique system to study the regulation of heterologous cell interactions during development and differentiation. The major population of cells within the follicular antrum are epithelial granulosa cells. Within 58J. CELL BIOLOGY 9 The Rockefeller University Press 9

Mutations in HERG are associated with human chromosome 7-linked congenital long QT (LQT-2) syndrome. We used electrophysiological, biochemical, and immunohistochemical methods to study the molecular mechanisms of HERG channel dysfunction caused by LQT-2 mutations. Wild type HERG and LQT-2 mutations were studied by stable and transient expression in HEK 293 cells. We found that some mutations (Y611H and V822M) caused defects in biosynthetic processing of HERG channels with the protein retained in the endoplasmic reticulum. Other mutations (I593R and G628S) were processed similarly to wild type HERG protein, but these mutations did not produce functional channels. In contrast, the T474I mutation expressed HERG current but with altered gating properties. These findings suggest that the loss of HERG channel function in LQT-2 mutations is caused by multiple mechanisms including abnormal channel processing, the generation of nonfunctional channels, and altered channel gating.The congenital long QT syndrome is a disorder associated with delayed cardiac repolarization and prolonged electrocardiographic QT intervals and the development of ventricular arrhythmias (torsades de pointes) and sudden death (1). One cause of congenital long QT syndrome is mutation in the human ether-a-go-go-related gene (HERG) producing chromosome 7-linked congenital long QT syndrome (LQT-2) 1 (2). HERG encodes a voltage-gated potassium channel (3). HERG channel current has been shown to have properties similar to the rapidly activating delayed rectifier K ϩ current (I Kr ), and it plays an important role in cardiac action potential repolarization in the mammalian heart (4 -6). HERG channels are also an important target for block by many drugs, and suppression of HERG current causes action potential prolongation and cardiac arrhythmias (5-12). Therefore, HERG channels have emerged as an important cardiac ion channel.More than 30 HERG mutations have been identified in LQT-2 patients (2, 13-18). The electrophysiological properties of a few LQT-2 mutations have been studied in Xenopus oocytes, where they have been shown to result in reduced or absent HERG current (19). Although the molecular basis for some congenital human diseases is known to involve multiple mechanisms including defective protein processing and abnormal protein function, the molecular basis for long QT syndrome has not been studied. In the present work, we used electrophysiological, biochemical, and immunohistochemical methods to study intracellular protein processing and functional properties of wild type and five LQT-2 mutant HERG channels. Our findings show that some mutant HERG proteins are not processed to the mature form of the channel. Other mutant HERG proteins undergo normal processing but do not form functional channels, or they gate abnormally. These findings provide new information about the molecular mechanisms for the failure of mutant LQT-2 channels to generate normal HERG current. Table I were generated by site-directed mutagenesis using the Altered Site II in vi...

The sigma-1 receptor regulates various ion channel activity and possesses protein chaperone function. Using an antibody against the full sequence of the sigma-1 receptor we detected immunostaining in wild type but not in knockout mice. The receptor was found primarily in motoneurons localized to the brainstem and spinal cord. At the subcellular level the receptor is restricted to large cholinergic postsynaptic densities on the soma of motoneurons and is colocalized with the Kv2.1 potassium channel and the muscarinic type 2 cholinergic receptor. Ultrastructural analysis of the neurons indicates that the immunostained receptor is located close but separate from the plasma membrane, possibly in subsurface cisternae formed from the endoplasmic reticulum (ER), which are a prominent feature of cholinergic postsynaptic densities. Behavioral testing on a rotorod revealed that Sigma-1 receptor knockout mice remained on the rotorod for significantly less time (a shorter latency period) compared to the wild type mice. Together these data indicate that the sigma-1 receptor may play a role in the regulation of motor behavior.

Neural crest cells leave the hindbrain, enter the gut mesenchyme at the pharynx, and migrate as strands of cells to the terminal bowel to form the enteric nervous system. We generated embryos containing fluorescent enteric neural crest-derived cells (ENCCs) by mating Wnt1-Cre mice with Rosa-floxed-YFP mice and investigated ENCC behavior in the intact gut of mouse embryos using time-lapse fluorescent microscopy. With respect to the entire gut, we have found that ENCCs in the cecum and proximal colon behave uniquely. ENCCs migrating caudally through either the ileum, or caudal colon, are gradually advancing populations of strands displaying largely unpredictable local trajectories. However, in the cecum, advancing ENCCs pause for approximately 12 h, and then display an invariable pattern of migration to distinct regions of the cecum and proximal colon. In addition, while most ENCCs migrating through other regions of the gut remain interconnected as strands; ENCCs initially migrating through the cecum and proximal colon fragment from the main population and advance as isolated single cells. These cells aggregate into groups isolated from the main network, and eventually extend strands themselves to reestablish a network in the mid-colon. As the advancing network of ENCCs reaches the terminal bowel, strands of sacral crest cells extend, and intersect with vagal crest to bridge the small space between. We found a relationship between ENCC number, interaction, and migratory behavior by utilizing endogenously isolated strands and by making cuts along the ENCC wavefront. Depending on the number of cells, the ENCCs aggregated, proliferated, and extended strands to advance the wavefront. Our results show that interactions between ENCCs are important for regulating behaviors necessary for their advancement.

Studies have shown that ovarian failure is a common side-effect of chemotherapy treatment; however, continuation of regular menses post-treatment does not necessarily imply that the ovaries have escaped damage. This animal study measures directly the primordial follicle (PMF) loss following exposure to chemotherapy and evaluates reproductive outcome following significant destruction of the PMF population. Inbred Balb/c mice aged 5-6 weeks were administered different doses of an alkylating agent, cyclophosphamide, and the total number of PMF remaining in both ovaries was counted. Results show that cyclophosphamide causes PMF destruction in proportion to increasing dose (P = 0.0001). Reproductive performance was assessed after exposure to 75 mg/kg cyclophosphamide, a dose which destroys approximately 50% of PMF reserve, by evaluation of ovulation, mating and pregnancy rates. Reproductive potential of treated mice was not affected compared with controls despite the significant loss of PMF. Our results indicate that reproductive performance is not an accurate parameter for assessing ovarian injury. Rather, histological counting of PMF number more directly reflects the damage caused by chemotherapy to the ovary. This method can be used as a sensitive, inexpensive tool to gauge the damage to fertility caused by new chemotherapy agents or protocols.

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Neural crest-derived cells colonize the entire gastrointestinal tract. The migration of these enteric neural crest-derived cells (ENCCs) occurs by their formation of cellular strands that extend into the intestinal mesenchyme. We have studied the behavior of crest cells that underlies the formation and extension of these strands by time-lapse microscopy. ENCCs expressing fluorescent marker molecules were visualized in situ in the embryonic mouse and chick gut. The major contributor to strand extension is from cells located within a region approximately 300 m behind (rostral to) the most caudal cells in the migratory wavefront. Cells in the region immediately behind the leading cell of the strand either move intermittently in parallel with the leading cell, or advance caudally toward the wavefront over other ENCCs. Another addition to the strands arises from isolated cells located caudal to the wavefront. These cells showed a range of behavior including attachment and separation from the strands. The extending strands converged to form nodes, and then diverged along independent paths to form new strands, a behavior suggestive of attraction and repulsion. This behavior is probably responsible for the unique reticulated arrangement of ganglia in the enteric nervous system. As cells become positioned farther behind the wavefront, they exhibit more restricted movement and varied trajectories. We conclude that ENCCs exhibit different behaviors, depending on their position with respect to the wavefront. These different behaviors suggest a critical role for cell-cell interaction in the migratory process. Developmental Dynamics 236:84 -92, 2007.