Miles L. Epstein

HERG Channel Dysfunction in Human Long QT Syndrome INTRACELLULAR TRANSPORT AND FUNCTIONAL DEFECTS

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.

Immunohistochemical analysis of neuron types in the mouse small intestine

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.

Monoclonal Antibodies and Polyvalent Antiserum to Chicken Choline Acetyltransferase

Abstract: Monoclonal antibodies (mAbs) to chick cho‐line acetyltransferase (ChAT) were obtained from mouse‐hybridoma cultures after immunization with partially purified enzyme isolated from optic lobes. Antibodies that bound active enzyme were detected in 11 hybridoma cultures. The mAbs showed cross‐reactivity to ChAT from quail and beef but not to ChAT from several other species. An affinity column prepared with one of the mAbs was used to purify ChAT to apparent homogeneity. Polyclonal antiserum to mAb affinity‐purified ChAT was produced in a rabbit. This antiserum inhibited chick ChAT activity and quantitatively precipitated ChAT activity from solution. On immunoblots, the antiserum stained ChAT and two other proteins. After preadsorption of the antiserum with effluent from the mAb affinity column, the antiserum became monospecific for ChAT. This an‐tiserum was useful for immunocytochemical localization of ChAT. it selectively stained neuronal cell bodies in chick spinal cord and rat brain at locations known to contain cholinergic neurons.

THE SIGMA-1 RECEPTOR IS ENRICHED IN POSTSYNAPTIC SITES OF C-TERMINALS IN MOUSE MOTONEURONS. AN ANATOMICAL AND BEHAVIORAL STUDY

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.

Cholinergic amacrine cells of the chicken retina: A light and electron microscope immunocytochemical study

Cholinergic amacrine cells of the chicken retina were detected by immunohistochemistry using an antiserum against affinity-purified chicken choline acetyltransferase. Three populations of cells were detected: type I cholinergic amacrine cells had cell bodies on the border of the inner nuclear and inner plexiform layers and formed a prominent laminar band in sublamina 2 of the inner plexiform layer, while type II cholinergic amacrine cells had cell bodies in the ganglion cell layer, and formed a prominent laminar band in sublamina 4 of the inner plexiform layer. Type III cholinergic amacrine cell bodies were located towards the middle of the inner nuclear layer, and their processes were more diffusely distributed in sublaminas 1 and 3-5 of the inner plexiform layer. Type I and type II cells were present at densities of over 7000 cells/mm2 in central areas declining to less than 2000 cells/mm2 in the temporal retinal periphery. The cells were organized locally in a non-random mosaic, with regularity indices ranging from 3 peripherally to over 5 centrally. Neither at the light nor electron microscopic levels was a lattice of cholinergic dendrites of the kind reported by Tauchi and Masland [J. Neurosci. 5, 2494-2501 (1985)] detectable. Within the two prominent dendritic plexuses, a major feature of the synaptic interactions of the type I and type II cholinergic cells was extensive synaptic interaction between cholinergic processes. Apart from this, there was little, if any, input to cholinergic processes from non-cholinergic amacrine cells, but there was input from bipolar cells. Output from the cholinergic amacrine cell processes was directed towards non-cholinergic amacrine cells as well as other cholinergic amacrine cells, and ganglion cells.

Behavior of enteric neural crest‐derived cells varies with respect to the migratory wavefront

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 μm 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.

Cholinergic and acetylcholinesterase-containing neurons of the chicken retina.

In the chicken retina, choline acetyltransferase-like immunoreactivity (ChAT-LI) defines three populations of cholinergic amacrine cells and two terminal bands in the inner plexiform layer (IPL). Acetylcholinesterase (AChE) histochemistry defines two prominent bands within the IPL which corresponded to those containing ChAT. Other AChE-positive bands in the IPL are not associated with cholinergic transmission sites. Cholinergic cell bodies contain AChE, but the most intensely AChE-positive cells do not appear to be cholinergic. AChE histochemistry may be used to define the major cholinergic synaptic sites in the IPL, and may be a useful marker of IPL lamination.

Age-dependent changes in the gut environment restrict the invasion of the hindgut by enteric neural progenitors

The enteric nervous system (ENS) develops from neural crest cells (NCCs) that enter the foregut and hindgut to become enteric neural-crest-derived cells (ENCCs). When these cells of neural crest origin fail to colonize the terminal hindgut, this aganglionic region becomes non-functional and results in a condition in humans known as Hirschsprungs disease (HSCR). One of the genes associated with HSCR is endothelin receptor type B (Ednrb). To study the development of colonic aganglionosis we have utilized a novel knockout mouse (Ednrbflex3/flex3), in which the expression of a null Ednrb allele and YFP is confined to NCCs. We have identified two primary cellular defects related to defective EDNRB signaling. First, ENCC advance in Ednrbflex3/flex3 embryos is delayed shortly after NCCs enter the gut. Apart from this early delay, Ednrbflex3/flex3 ENCCs advance normally until reaching the proximal colon. Second, as Ednrbflex3/flex3 ENCCs reach the colon at E14.5, they display migratory defects, including altered trajectories and reduced speed, that are not dependent on proliferation or differentiation. We constructed grafts to test the ability of donor ENCCs to invade a recipient piece of aganglionic colon. Our results indicate that the age of the recipient, and not the age or genotype of donor ENCCs, determines whether the colon is invaded. We identify changes in laminin expression that are associated with the failure of ENCCs to invade recipient tissue. Together, our data suggest that a defect in pre-enteric Ednrbflex3/flex3 NCCs results in delayed colonic arrival, which, due to environment changes in the colon, is sufficient to cause aganglionosis.

Appearance of neurons and glia with respect to the wavefront during colonization of the avian gut by neural crest cells

The enteric nervous system is formed by neural crest cells that migrate, proliferate, and differentiate into neurons and glia distributed in ganglia along the gastrointestinal tract. In the developing embryo some enteric crest cells cease their caudal movements, whereas others continue to migrate. Subsequently, the enteric neurons form a reticular network of ganglia interconnected by axonal projections. We studied the developing avian gut to characterize the pattern of migration of the crest cells, and the relationship between migration and differentiation. Crest cells at the leading edge of the migratory front appear as strands of cells; isolated individual crest cells are rarely seen. In the foregut and midgut, these strands are located immediately beneath the serosa. In contrast, crest cells entering the colon appear first in the deeper submucosal mesenchyme and later beneath the serosa. As the neural crest wavefront passes caudally, the crest cell cords become highly branched, forming a reticular lattice that presages the mature organization of the enteric nervous system. Neurons and glia first appear within the strands at the advancing wavefront. Later neurons are consistently located at the nodes where branches of the lattice intersect. In the most rostral foregut and in the colon, some neurons initially appear in close association with extrinsic nerve fibers from the vagus and Remaks nerve, respectively. We conclude that crest cells colonize the gut as chains of cells and that, within these chains, both neurons and glia appear close to the wavefront.© 2002 Wiley‐Liss, Inc.

Cholinergic neurons of the chicken ciliary ganglion contain somatostatin.

Somatostatin immunoreactivity was studied in the avian ciliary ganglion by immunocytochemistry and radioimmunoassay. Immunoreactivity was localized to small diameter cell bodies of neurons from embryos, newly-hatched and adult preparations. Immunostaining of ganglia with a mixture of antisera to substance P and monoclonal antibody to somatostatin indicated that a number of somatostatin-immunoreactive neurons were surrounded by substance P-immunoreactive boutons, which characteristically terminate on choroidal neurons. Staining with a mixture of antisera to choline acetyltransferase and antibody to somatostatin showed that the somatostatin-immunoreactive neurons were less intensely-stained for choline acetyltransferase than were the neurons lacking somatostatin immunoreactivity. Bundles of nerve fibers showing somatostatin and choline acetyltransferase immunoreactivity were found in the choroid layers of the eye. Radioimmunoassay indicated the presence of somatostatin immunoreactivity in both chick and quail ganglia; the somatostatin immunoreactivity eluted from high pressure liquid chromatography in the same positions as authentic somatostatin 14 and 28. These results show that somatostatin is contained in cholinergic choroidal neurons in the chick and quail ciliary ganglion.