Biomedical subjects

David L Paul

Publications and source records attributed to David L Paul.

At least 19 recordsLinked to original sources

PubMed2006

Genetic and physiological evidence that oligodendrocyte gap junctions contribute to spatial buffering of potassium released during neuronal activity.

Mice lacking the K+ channel Kir4.1 or both connexin32 (Cx32) and Cx47 exhibit myelin-associated vacuoles, raising the possibility that oligodendrocytes, and the connexins they express, contribute to recycling the K+ evolved during neuronal activity. To study this possibility, we first examined the effect of neuronal activity on the appearance of vacuoles in mice lacking both Cx32 and Cx47. The size and number of myelin vacuoles was dramatically increased when axonal activity was increased, by either a natural stimulus (eye opening) or pharmacological treatment. Conversely, myelin vacuoles were dramatically reduced when axonal activity was suppressed. Second, we used genetic complementation to test for a relationship between the function of Kir4.1 and oligodendrocyte connexins. In a Cx32-null background, haploinsufficiency of either Cx47 or Kir4.1 did not affect myelin, but double heterozygotes developed vacuoles, consistent with the idea that oligodendrocyte connexins and Kir4.1 function in a common pathway. Together, these results implicate oligodendrocytes and their connexins as having critical roles in the buffering of K+ released during neuronal activity.

Animals↗

PubMed2006

Connexin43 is required for production of the aqueous humor in the murine eye.

Connexin43 is a major component of the gap junctions between pigmented and non-pigmented cells of the double-layered epithelium in the ciliary body of the eye. We directly tested the hypothesis that gap junctions play a crucial role in the production of the aqueous humor by inactivating the GJA1 (connexin43) gene in the pigmented epithelium with cre-loxP technology. To accomplish this, we crossed a line expressing cre recombinase driven by the nestin promoter and a line with floxed connexin43 alleles. Resultant lines exhibited loss of connexin43 from the pigmented epithelium, iris, retinal pigment epithelium and the lens. We observed plasma proteins in the aqueous humor and pathological changes consistent with a loss of intraocular pressure. As the ciliary body is responsible for aqueous humor production, these data support the hypothesis that the gap junctions between pigmented and non-pigmented epithelium are necessary for production of the aqueous humor that is in turn required for the generation of normal intraocular pressure and nourishment of the postnatal lens. The loss of connexin43 expression in the iris correlated with a separation of the posterior pigmented epithelium from the anterior myoepithelium and with meiosis, possibly resulting from a loss of function of the dilator pupillae.

Animals↗

PubMed2006

Study of claudin function by RNA interference.

Claudins are tight junction proteins that play a key selectivity role in the paracellular conductance of ions. Numerous studies of claudin function have been carried out using the overexpression strategy to add new claudin channels to an existing paracellular protein background. Here, we report the systematic knockdown of endogenous claudin gene expression in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells using small interfering RNA against claudins 1-4 and 7. In MDCK cells (showing cation selectivity), claudins 2, 4, and 7 are powerful effectors of paracellular Na+ permeation. Removal of claudin-2 depressed the permeation of Na+ and resulted in the loss of cation selectivity. Loss of claudin-4 or -7 expression elevated the permeation of Na+ and enhanced the proclivity of the tight junction for cations. On the other hand, LLC-PK1 cells express little endogenous claudin-2 and show anion selectivity. In LLC-PK1 cells, claudin-4 and -7 are powerful effectors of paracellular Cl- permeation. Knockdown of claudin-4 or -7 expression depressed the permeation of Cl- and caused the tight junction to lose the anion selectivity. In conclusion, claudin-2 functions as a paracellular channel to Na+ to increase the cation selectivity of the tight junction; claudin-4 and -7 function either as paracellular barriers to Na+ or as paracellular channels to Cl-, depending upon the cellular background, to decrease the cation selectivity of the tight junction.

Animals↗

PubMed2006

Connexin29 is highly expressed in cochlear Schwann cells, and it is required for the normal development and function of the auditory nerve of mice.

Connexins (Cxs) are a family of protein subunits constituting gap junctions, which facilitate exchanges of molecules important for cellular signaling and metabolic activities intercellularly or between different regions of the cytoplasm in the same cells. Mutations in Cxs are the major cause of nonsyndromic childhood deafness, which are mostly found in Cx26 and Cx30 expressed in cochlear supporting cells and fibrocytes. So far, little is known about the functional contribution of Cxs in other types of cochlear cells. Here, we show that Cx29 was highly expressed in the cochlea. The developmental expression time course of Cx29 was similar to that of a myelin marker [myelin associate glycoprotein (MAG)]. Immunolabeling identified Cx29 exclusively in the Schwann cells myelinating the soma and fiber of spiral ganglion (SG) neurons. The absence of the Cx29 gene in mice (Cx29(-/-) mice), with a penetrance of approximately 50%, caused a delay in the maturation of hearing thresholds, an early loss of high-frequency sensitivities, a prolongation in latency and distortion in the wave I of the auditory brainstem responses, and elevated sensitivity to noise damages. The morphology of sensory hair cells and otoacoustic emissions that depend on the integrity of hair cells were normal in Cx29(-/-) mice. In contrast, decreases in MAG expression and severe demyelination at the soma of SG neurons were found in Cx29(-/-) mice. Our findings demonstrated the requirement of Cx29 for normal cochlear functions and suggest that Cx29 is a new candidate gene for studying the auditory neuropathy.

Animals↗

PubMed2005

Morphology and tracer coupling pattern of alpha ganglion cells in the mouse retina.

Alpha cells are a type of ganglion cell whose morphology appears to be conserved across a number of mammalian retinas. In particular, alpha cells display the largest somata and dendritic arbors at a given eccentricity and tile the retina as independent on- (ON) and off-center (OFF) subtypes. Mammalian alpha cells also express a variable tracer coupling pattern, which often includes homologous (same cell type) coupling to a few neighboring alpha cells and extensive heterologous (different cell type) coupling to two to three amacrine cell types. Here, we use the gap junction-permeant tracer Neurobiotin to determine the architecture and coupling pattern of alpha cells in the mouse retina. We find that alpha cells show the same somatic and dendritic architecture described previously in the mammal. However, alpha cells show varied tracer coupling patterns related to their ON and OFF physiologies. ON alpha cells show no evidence of homologous tracer coupling but are coupled heterologously to at least two types of amacrine cell whose somata lie within the ganglion cell layer. In contrast, OFF alpha cells are coupled to one another in circumscribed arrays as well as to two to three types of amacrine cell with somata occupying the inner nuclear layer. We find that homologous coupling between OFF alpha cells is unaltered in the connexin36 (Cx36) knockout (KO) mouse retina, indicating that it is not dependent on Cx36. However, a subset of the heterologous coupling of ON alpha cells and all the heterologous coupling of OFF alpha cells are eliminated in the KO retina, suggesting that Cx36 comprises most of the junctions made with amacrine cells.

Amacrine Cells↗

PubMed2005

Paracellin-1 and the modulation of ion selectivity of tight junctions.

Tight junctions play a key selectivity role in the paracellular conductance of ions. Paracellin-1 is a member of the tight junction claudin protein family and mutations in the paracellin-1 gene cause a human hereditary disease, familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) with severe renal Mg2+ wasting. The mechanism of paracellin-1 function and its role in FHHNC are not known. Here, we report that in LLC-PK1 epithelial cells paracellin-1 modulated the ion selectivity of the tight junction by selectively and significantly increasing the permeability of Na+ (with no effects on Cl-) and generated a high permeability ratio of Na+ to Cl-. Mutagenesis studies identified a locus of amino acids in paracellin-1 critical for this function. Mg2+ flux across cell monolayers showed a far less-pronounced change (compared to monovalent alkali cations) following exogenous protein expression, suggesting that paracellin-1 did not form Mg2+-selective paracellular channels. We hypothesize that in the thick ascending limb of the nephron, paracellin-1 dysfunction, with a concomitant loss of cation selectivity, could contribute to the dissipation of the lumen-positive potential that is the driving force for the reabsorption of Mg2+.

Amino Acid Sequence↗

PubMed2005

Amphetamine withdrawal produces region-specific and time-dependent changes in connexin36 expression in rat brain.

Repeated amphetamine treatment produces a long-lasting augmentation of locomotor behavior in rats, a phenomenon known as behavioral sensitization. This process is thought to be a correlate of the addictive process in humans, and it is believed that there are drug-induced neuroadaptations that underlie these behavioral changes. One mechanism by which amphetamine can alter brain function is by affecting direct intercellular communication between neurons via gap junctions. The purpose of the present study was to examine the effect of an amphetamine treatment regimen known to produce changes in dye coupling between neurons, a functional correlate of gap junction function, on the expression of the neuronal gap junction-forming protein, connexin36. Here we report that withdrawal from an extended amphetamine regimen produces region-specific and time-dependent changes in connexin36 expression in rat nucleus accumbens and prefrontal cortex, brain regions known play roles in sensitization and addiction. This is, to our knowledge, the first demonstration of pharmacological manipulation of connexin36 in vivo.

Amphetamine↗

PubMed2005

Atrioventricular nodal reverse facilitation in connexin40-deficient mice.

BACKGROUND: Facilitation is an important physiologic property of the atrioventricular (AV) node. Previous studies demonstrated abnormal AV conduction in connexin (Cx)40-deficient mice. OBJECTIVES: We hypothesize that Cx40-deficient mice display altered patterns of AV nodal facilitation compared with wild-type mice. METHODS: Sixteen 36-week-old mice (eight Cx40(-/-) mice and eight Cx40(+/+) controls) underwent in vivo closed chest electrophysiologic study. A 2Fr octapolar catheter was advanced into the right ventricle to record a His-bundle electrogram. A special facilitation stimulation protocol was performed in each mouse to evaluate facilitation. Following atrial drive pacing (S1S1) at 150 ms, a facilitating beat S2 was delivered prior to the test beat S3. S3H3 was measured for varying S1S2 values at fixed H2S3 intervals. RESULTS: Progressive shortening of S1S2 (from 150 ms to 130, 110, and 90 ms) resulted in gradual prolongation of S2H2. The prolongation was more pronounced in Cx40(-/-) mice for each S1S2 compared with wild-type mice (P <.001). In each wild-type mouse, for a given H2S3 interval, this gradual increase in S2H2 produced progressive shortening of S3H3, so-called AV nodal facilitation phenomenon. However, in each Cx40(-/-) mouse, facilitation was seen only at S1S2 of 130 ms (P <.001 vs S1S2 of 150 ms). Evidence of reverse facilitation was documented at S1S2 of 110 and 90 ms. CONCLUSION: Facilitation is observed in wild-type mice. With similar S1S2 intervals in Cx40-deficient mice, facilitation is seen only at longer S1S2 intervals, whereas reverse facilitation is seen at shorter S1S2 intervals, suggesting that Cx40 is involved in the generation of AV nodal facilitation.

Animals↗

PubMed2005

Connexin 40, a target of transcription factor Tbx5, patterns wrist, digits, and sternum.

Haploinsufficiency of T-box transcription factor 5 (TBX5) causes human Holt-Oram syndrome (HOS), a developmental disorder characterized by skeletal and heart malformations. Mice carrying a Tbx5 null allele (Tbx5(+/Delta)) have malformations in digits, wrists, and sternum joints, regions where Tbx5 is expressed. We demonstrate that mice deficient in connexin 40 (Cx40), a Tbx5-regulated gap junction component, shared axial and appendicular skeletal malformations with Tbx5(+/Delta) mice. Although no role in skeleton patterning has been described for gap junctions, we demonstrate here that Cx40 is involved in formation of specific joints, as well as bone shape. Even a 50% reduction in either Tbx5 or Cx40 produces bone abnormalities, demonstrating their crucial control over skeletal development. Further, we demonstrate that Tbx5 exerts in part its key regulatory role in bone growth and maturation by controlling via Cx40 the expression of Sox9 (a transcription factor essential for chondrogenesis and skeleton growth). Our study strongly suggests that Cx40 deficiency accounts for many skeletal malformations in HOS and that Tbx5 regulation of Cx40 plays a critical role in the exquisite developmental patterning of the forelimbs and sternum.

Animals↗

PubMed2004

Convergence and segregation of the multiple rod pathways in mammalian retina.

Using a multidisciplinary approach, we demonstrate that three different pathways are responsible for the transmission of rod signals across the mouse retina. Each pathway serves a primarily nonoverlapping range of stimulus intensities, with ganglion cells receiving either segregated or convergent inputs. For both on-center (ON) and off-center (OFF) ganglion cells, the primary rod pathway carries signals with the lowest threshold, whereas the secondary rod pathway is less sensitive by approximately 1 log unit. In addition, OFF signaling uses a tertiary rod pathway that is approximately 1 log unit less sensitive than the secondary. Although some ganglion cells received rod inputs exclusively from one of the pathways, others showed convergent inputs. Using pharmacological and genetic approaches, we defined classes of ON and OFF ganglion cells for which the scotopic inputs derive only from the primary pathway or from both primary and secondary pathways. In addition, we observed a class of OFF ganglion cell receiving mixed input from primary and tertiary pathways. Interestingly, OFF ganglion cells receiving convergent inputs from all three rod pathways or from the secondary and tertiary pathways together were never observed. Overall, our data show a complex arrangement of convergence and segregation of rod inputs to ganglion cells in the mammalian retina.

Action Potentials↗

PubMed2004

Four classes of intercellular channels between glial cells in the CNS.

Astrocytes form extensive gap junctions with other astrocytes and with oligodendrocytes. Junctional communication between CNS glia is likely of critical importance because loss of the gap junction channel-forming proteins, connexins Cx32 and Cx47, result in severe demyelination. However, CNS glia express at least six connexins, and the cellular origins and relationships of these proteins have not been determined. We produced a Cx29 reporter mouse in which the connexin coding sequence was replaced with a histological marker, which was used to demonstrate that Cx29, Cx32, and Cx47 are expressed specifically in oligodendrocytes. To determine the relationships between astrocyte and oligodendrocyte connexins, we used double- and triple-immunofluorescence microscopy using semithin sections (<1 microm) of adult mouse spinal cord. Astrocytes form two distinct classes of gap junctions with each other; those composed of Cx26 and those composed of Cx43 and Cx30. In addition, astrocytes establish two classes of intercellular channels with oligodendrocytes, heterotypic Cx26-Cx32 channels and heterotypic Cx30/Cx43-Cx47 channels that may also be heteromeric. In contrast, Cx29 does not colocalize with any of the other five connexins. The data provide the first in vivo demonstration of heterotypic intercellular channels and reveal an unexpected complexity in the composition of glial gap junctions.

Animals↗

PubMed2004

Unique distributions of the gap junction proteins connexin29, connexin32, and connexin47 in oligodendrocytes.

Oligodendrocytes of adult rodents express three different connexins: connexin29 (Cx29), Cx32, and Cx47. In this study, we show that Cx29 is localized to the inner membrane of small myelin sheaths, whereas Cx32 is localized on the outer membrane of large myelin sheaths; Cx29 does not colocalize with Cx32 in gap junction plaques. All oligodendrocytes appear to express Cx47, which is largely restricted to their perikarya. Cx32 and Cx47 are colocalized in many gap junction plaques on oligodendrocyte somata, particularly in gray matter. Cx45 is detected in the cerebral vasculature, but not in oligodendrocytes or myelin sheaths. This diversity of connexins in oligodendrocytes (in different populations of cells and in different subcellular compartments) likely reflects functional differences between these connexins and perhaps the oligodendrocytes themselves.

Animals↗

PubMed2004

Connexins: functions without junctions.

Emerging studies indicate that connexins have activities completely unrelated to gap junctions and, conversely, that non-connexin proteins can form gap junction channels.

Bacteria↗

PubMed2003

Connexins are critical for normal myelination in the CNS.

Mutations in Cx32, a gap-junction channel-forming protein, result in X-linked Charcot-Marie-Tooth disease, a demyelinating disease of the peripheral nervous system. However, although oligodendrocytes express Cx32, central myelination is unaffected. To explore this discrepancy, we searched for additional oligodendrocyte connexins. We found Cx47, which is expressed specifically in oligodendrocytes, regulated in parallel with myelin genes and partially colocalized with Cx32 in oligodendrocytes. Mice lacking either Cx47 or Cx32 are viable. However, animals lacking both connexins die by postnatal week 6 from profound abnormalities in central myelin, characterized by thin or absent myelin sheaths, vacuolation, enlarged periaxonal collars, oligodendrocyte cell death, and axonal loss. These data provide the first evidence that gap-junction communication is crucial for normal central myelination.

Animals↗

PubMed2003

Central role of connexin40 in the propagation of electrically activated vasodilation in mouse cremasteric arterioles in vivo.

When a short segment of arteriole is stimulated, vasomotor responses spread bidirectionally along the vessel axis purportedly via gap junctions. We used connexin40 knockout (Cx40-/-) mice to study vasomotor responses induced by 10-second trains of electrical stimulation (30 Hz, 1 ms, 30 to 50 V) in 2nd or 3rd order arterioles of the cremaster muscle. Measurements were made at the stimulation site (local) and at conducted sites (500, 1000, and 2000 microm upstream). In wild-type (Cx40+/+) animals, electrical stimulation evoked a local vasoconstriction and a conducted vasodilation that spread very rapidly along the vessel length without detectable decay. In Cx40-/- mice, the conducted dilation was converted into either vasoconstriction or a slowly developing vasodilation that decayed along the vessel length. Tetrodotoxin (TTX, 1 micromol/L) had no effect on the local vasoconstriction in either Cx40+/+ or Cx40-/- mice, but enhanced the conducted vasodilation in Cx40+/+ animals. In Cx40-/- mice, TTX abolished the conducted vasoconstriction when present and revealed a small vasodilation that decayed with distance. In the group of Cx40-/- mice in which electrical stimulation elicited a conducted vasodilation, TTX had no effect. Immunocytochemistry revealed Cx40 only in the endothelial layer of arterioles from Cx40+/+ mice and complete elimination of this connexin in the Cx40-/- animals. These results indicate that focal current stimulation causes vasoconstriction by a combination of perivascular nerve stimulation and smooth muscle activation. Moreover, electrical stimulation activates a nonneuronal, Cx40-dependent vasodilator response that spreads along the vessel length without decay.

Abdominal Muscles↗

PubMed2003

Oligodendrocyte progenitor enrichment in the connexin32 null-mutant mouse.

Before the establishment of chemical synapses, neural progenitors are often coupled by connexin-mediated gap junctions providing a robust mechanism for cell-cell communication in developing brain. The present study was undertaken to determine whether alterations in junctional coupling also affect neural progenitor proliferation, survival, and differentiation in adult brain. We localized the connexin32 gap junction protein to a subset of NG2+ and platelet-derived growth factor alpha receptor+ early oligodendrocyte progenitors in the dentate gyrus of adult mice. In connexin32-deficient mice, we found an increase in the total number of proliferating nestin+ and NG2+ progenitors in the subgranular zone, hilus, and polymorphonuclear layer of the dentate gyrus in vivo and in the total number of nestin+ progenitors capable of clonogenic expansion in vitro. By bromodeoxyuridine labeling, lineage analysis, and terminal deoxynucleotidyl nick end labeling, we demonstrate that turnover of these cells is constitutively enhanced in the connexin32 knock-out dentate gyrus reflecting a dynamic defect in oligodendrogenesis in this population. Analyses of surviving bromodeoxyuridine-labeled cells at 1, 3, 7, and 28 d after injection demonstrate that this transient amplifying population fails to terminally differentiate and is deleted by an apoptotic-like mechanism within 3 d of labeling. These data provide empirical evidence to support the hypothesis that connexin expression influences adult progenitor number and specifically implicate connexin32-mediated signaling in the activation, survival, and differentiation of a subset of early oligodendrocyte progenitors in postnatal brain.

Animals↗

PubMed2003

Multiple connexins contribute to intercellular communication in the Xenopus embryo.

To explore the role of gap junctional intercellular communication (GJIC) during Xenopus embryogenesis, we utilized the host-transfer and antisense techniques to specifically deplete Cx38, the only known maternally expressed connexin. Cx38-depleted embryos developed normally but displayed robust GJIC between blastomeres at 32-128 cell stages, suggesting the existence of other maternal connexins. Analysis of embryonic cDNA revealed maternal expression of two novel connexins, Cx31 and Cx43.4, and a third, Cx43, that had been previously identified as a product of zygotic transcription. Thus, the early Xenopus embryo contains at least four maternal connexins. Unlike Cx38, expression of Cx31, Cx43 and Cx43.4 continue zygotically. Of these, Cx43.4 is the most abundant, accumulating significantly in neural structures including the brain, the eyes and the spinal cord.

Animals↗

PubMed2003

Nkx2.5 homeoprotein regulates expression of gap junction protein connexin 43 and sarcomere organization in postnatal cardiomyocytes.

Nkx2.5, an evolutionarily conserved homeodomain containing transcription factor, is one of the earliest cardiogenic markers. Although its expression continues through adulthood, its function in adult cardiomyocytes is not well understood. To examine the effect of Nkx2.5 in terminal differentiated postnatal cardiomyocytes, we generated transgenic mice expressing either wild-type Nkx2.5 (TG-wild), a putative transcriptionally active mutant (carboxyl-terminus deletion mutant: TG-DeltaC) or a DNA non-binding point mutant of Nkx2.5 (TG-I183P) under alpha-myosin heavy chain promoter. Most TG-wild and TG-DeltaC mice died before 4 months of age with heart failure associated with conduction abnormalities. Cardiomyocytes expressing wild-type Nkx2.5 or a putative transcriptionally active mutant (DeltaC) had dramatically reduced expression of connexin 43 and changed sarcomere structure. Wild-type Nkx2.5 adenovirus-infected adult cardiomyocytes demonstrated connexin 43 downregulation as early as 16 h after infection, indicating that connexin 43 downregulation is due to Nkx2.5 overexpression but not due to heart failure phenotype in vivo. These studies indicate that overexpression of Nkx2.5 in terminally differentiated cardiomyocytes dramatically alters cardiac cell structure and function.

Adenoviridae↗