Emerging evidence indicates rods can communicate with retinal ganglion cells (RGCs) via pathways that do not involve gap-junctions. Here we investigated the significance of such pathways for central visual responses, using mice lacking a key gap junction protein (Cx36−/−) and carrying a mutation that disrupts cone phototransduction (Gnat2cpfl3). Electrophysiological recordings spanning the lateral geniculate revealed rod-mediated ON and OFF visual responses in virtually every cell from all major anatomical sub-compartments of this nucleus. Hence, we demonstrate that one or more classes of RGC receive input from Cx36-independent rod pathways and drive extensive ON and OFF responses across the visual thalamus.
mouse; scotopic; melanopsin; electroretinogram; connexin
Neurons throughout the brain show spike activity that is temporally correlated to that expressed by their neighbors, yet the generating mechanism(s) remains unclear. In the retina, ganglion cells (GCs) show robust, concerted spiking that shapes the information transmitted to central targets. Here we report the synaptic circuits responsible for generating the different types of concerted spiking of GC neighbors in the mouse retina. The most precise concerted spiking was generated by reciprocal electrical coupling of GC neighbors via gap junctions, whereas indirect electrical coupling to a common cohort of amacrine cells generated the correlated activity with medium precision. In contrast, the correlated spiking with the lowest temporal precision was produced by shared synaptic inputs carrying photoreceptor noise. Overall, our results demonstrate that different synaptic circuits generate the discrete types of GC correlated activity. Moreover, our findings expand our understanding of the roles of gap junctions in the retina, showing that they are essential for generating all forms of concerted GC activity transmitted to central brain targets.
Parents and caregivers look to pediatric health care providers for guidance on feeding, safety issues, and child-care products for children, but trainees have infrequent first-hand exposure to child products marketed to parents.
To conduct a pilot study to assess an experiential field trip as a novel method of enhancing medical knowledge in ambulatory pediatric feeding and safety.
Resident physicians and medical students visited a local children's store, where they took part in an interactive store tour, product discussions, and product demonstrations led by a physician educator. Participants also completed a 20-question pretest and a 20-question posttest related to common ambulatory pediatric feeding and safety issues, based on recent American Academy of Pediatrics (AAP) policy statements and practice guidelines.
Sixty-seven medical students and resident physicians participated in the study. Overall, participants' short-term knowledge significantly increased from 9.9 ± 2.6 to 15.4 ± 2.2 questions correct (P = .001), with statistically significant gains (P < .001) on both the feeding and safety sections of the test. There were no differences in improvement based on participant's student or resident status, residency program type, program year, sex, or parental status. Ninety-five percent of the participants believed that their knowledge was enhanced by this approach, and participants uniformly agreed that this field trip was valuable to their pediatric training and that such field trip sessions should continue.
The inclusion of experiential learning through an interactive field trip in the curriculum of medical training was acceptable and feasible and showed short-term improvements in knowledge of AAP safety and feeding concepts.
An X-ray crystallographic structure is described for unliganded Vaccinia virus poly(A) polymerase monomer (VP55), showing the first domain-level structural isoforms among either VP55, it’s processivity factor VP39, or the VP55-VP39 heterodimer. The occurrence of domain-level motion specifically in monomeric VP55 is consistent with the finding that the monomeric protein undergoes saltatory translocation whereas the heterodimer does not.
Vaccinia virus poly(A) polymerase (VP55) is the only known polymerase that can translocate independently with respect to single-stranded nucleic acid (ssNA). Previously, its structure has only been solved in the context of the VP39 processivity factor. Here, a crystal structure of unliganded monomeric VP55 has been solved to 2.86 Å resolution, showing the first backbone structural isoforms among either VP55 or its processivity factor (VP39). Backbone differences between the two molecules of VP55 in the asymmetric unit indicated that unliganded monomeric VP55 can undergo a ‘rocking’ motion of the N-terminal domain with respect to the other two domains, which may be ‘rigidified’ upon VP39 docking. This observation is consistent with previously demonstrated experimental molecular dynamics of the monomer during translocation with respect to nucleic acid and with different mechanisms of translocation in the presence and absence of processivity factor VP39. Side-chain conformational changes in the absence of ligand were observed at a key primer contact site and at the catalytic center of VP55. The current structure completes the trio of possible structural forms for VP55 and VP39, namely the VP39 monomer, the VP39–VP55 heterodimer and the VP55 monomer.
VP55; Vaccinia virus poly(A) polymerase; polymerases
We describe a model for cortical development that resolves long-standing difficulties of earlier models. It is proposed that, during embryonic development, synchronous firing of neurons and their competition for limited metabolic resources leads to selection of an array of neurons with ultra-small-world characteristics. Consequently, in the visual cortex, macrocolumns linked by superficial patchy connections emerge in anatomically realistic patterns, with an ante-natal arrangement which projects signals from the surrounding cortex onto each macrocolumn in a form analogous to the projection of a Euclidean plane onto a Möbius strip. This configuration reproduces typical cortical response maps, and simulations of signal flow explain cortical responses to moving lines as functions of stimulus velocity, length, and orientation. With the introduction of direct visual inputs, under the operation of Hebbian learning, development of mature selective response “tuning” to stimuli of given orientation, spatial frequency, and temporal frequency would then take place, overwriting the earlier ante-natal configuration. The model is provisionally extended to hierarchical interactions of the visual cortex with higher centers, and a general principle for cortical processing of spatio-temporal images is sketched.
synchronous oscillation; cortical development; synaptic organization; cortical response properties; cortical information flow
Dexmedetomidine is an α2-receptor agonist commonly used for sedation and analgesia in ICU patients. Dexmedetomidine is known to provide sympatholysis and also to have direct atrioventricular and sinoatrial node inhibitory effects. In rare instances, orthotopic lung transplantation has been associated with disruption of autonomic innervation of the heart. The combination of this autonomic disruption and dexmedetomidine may be associated with severe bradycardia and/or asystole. Since orthotopic lung transplant patients with parasympathetic denervation will not respond with increased heart rate to anticholinergic therapy, bradyarrhythmias must be recognized and promptly treated with direct acting beta agonists to avoid asystolic cardiac events.
Maternal race/ethnicity, age, and socioeconomic status (SES) are important factors determining birth outcome. Previous studies have demonstrated that, teenagers, and mothers with advanced maternal age (AMA), and Black/Non-Hispanic race/ethnicity can independently increase the risk for a poor pregnancy outcome. Similarly, public insurance has been associated with suboptimal health outcomes. The interaction and impact on the risk of a pregnancy resulting in a NICU admission has not been studied. Our aim was, to analyze the simultaneous interactions of teen/advanced maternal age (AMA), race/ethnicity and socioeconomic status on the odds of NICU admission.
The Consortium of Safe Labor Database (subset of n = 167,160 live births) was used to determine NICU admission and maternal factors: age, race/ethnicity, insurance, previous c-section, and gestational age.
AMA mothers were more likely than teenaged mothers to have a pregnancy result in a NICU admission. Black/Non-Hispanic mothers with private insurance had increased odds for NICU admission. This is in contrast to the lower odds of NICU admission seen with Hispanic and White/Non-Hispanic pregnancies with private insurance.
Private insurance is protective against a pregnancy resulting in a NICU admission for Hispanic and White/Non-Hispanic mothers, but not for Black/Non-Hispanic mothers. The health disparity seen between Black and White/Non-Hispanics for the risk of NICU admission is most evident among pregnancies covered by private insurance. These study findings demonstrate that adverse pregnancy outcomes are mitigated differently across race, maternal age, and insurance status.
Depression affects a significant proportion of the population, with 1-year and lifetime prevalence of 3–5% and 10–30% respectively. Full remission is achieved in only a third of patients following treatment with first-line antidepressant. There is a need for novel treatments for treatment-resistant depression (TRD). Dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis has been described in patients with depression. There is persistent rise in the levels of cortisol (end product of the HPA axis) and impairment of the negative feedback inhibition mechanism of the HPA axis. Dysregulation of the HPA axis has been found to be linked to nonresponse to antidepressants and relapse following successful treatment. The efficacy of pharmacological agents that intervene with the mechanisms involved in dysregulation of cortisol synthesis and release are being explored in depression, particularly in TRD. Studies have been carried out with these drugs as augmenting agents for antidepressants or as monotherapy. The strongest evidence has come from studies using metyrapone, a cortisol synthesis inhibitor, and this has been described in detail in this review. The most robust evidence for its antidepressant efficacy in depression comes from a double-blind, randomized, placebo-controlled study of augmentation of serotonergic antidepressants with metyrapone. A 3-week augmentation of serotonergic antidepressants with 1 g metyrapone daily was shown to be superior to placebo in reducing the Montgomery–Asberg Depression Rating Scale by 50%, 5 weeks following initiation of treatment. The mechanism of the antidepressant action of metyrapone is not clear but the evidence for various potential mechanisms is discussed.
antidepressant; antiglucocorticoid; depression; hypothalamic–pituitary–adrenal axis; metyrapone; treatment resistant
CNS glia exhibit a variety of gap junctional interactions: between neighboring astrocytes, between neighboring oligodendrocytes, between astrocytes and oligodendrocytes, and as ‘reflexive’ structures between layers of myelin in oligodendrocytes. Together, these junctions are thought to form a network facilitating absorption and removal of extracellular K+ released during neuronal activity. In mice, loss of the two major oligodendrocyte connexins causes severe demyelination and early mortality, while loss of the two major astrocyte connexins causes mild dysmyelination and sensorimotor impairment, suggesting that reflexive and/or oligo-oligo coupling may be more important for the maintenance of myelin than other forms. To further explore the functional relationships between glial connexins, we generated double knockout mice lacking one oligodendrocyte and one astrocyte connexin. Cx32-Cx43 dKO animals develop white matter vacuolation without obvious ultrastructural abnormalities in myelin. Progressive loss of astrocytes but not oligodendrocytes or microglia accompanies sensorimotor impairment, seizure activity and early mortality at around 16 weeks of age. Our data reveal an unexpected role for connexins in the survival of white matter astrocytes, requiring the expression of particular isoforms in both oligodendrocytes and astrocytes.
Gap junction; connexin; myelin; astrocyte; oligodendrocytes; Cx47; Cx32; Cx43
Counterintuitive dynamics of various biological phenomena occur when composite system dynamics differ qualitatively from that of their component systems. Such composite systems typically arise when modelling situations with time-varying biotic or abiotic conditions, and examples range from metapopulation dynamics to population genetic models. These biological, and related physical, phenomena can often be modelled as simple financial games, wherein capital is gained and lost through gambling. Such games have been developed and used as heuristic devices to elucidate the processes at work in generating seemingly paradoxical outcomes across a spectrum of disciplines, albeit in a field-specific, ad hoc fashion. Here, we propose that studying these simple games can provide a much deeper understanding of the fundamental principles governing paradoxical behaviours in models from a diversity of topics in evolution and ecology in which fluctuating environmental effects, whether deterministic or stochastic, are an essential aspect of the phenomenon of interest. Of particular note, we find that, for a broad class of models, the ecological concept of equilibrium reactivity provides an intuitive necessary condition that must be satisfied in order for environmental variability to promote population persistence. We contend that further investigations along these lines promise to unify aspects of the study of a range of topics, bringing questions from genetics, species persistence and coexistence and the evolution of bet-hedging strategies, under a common theoretical purview.
stochastic dynamics; persistence; coexistence; paradoxical games; reversal behaviour; reactivity
The mechanisms regulating assembly of gap junctions are not well defined. It is shown that assembly of gap junctions containing Cx50 requires both a PDZ domain–binding motif at the connexin C-terminus and the scaffolding protein ZO-1.
The three connexins expressed in the ocular lens each contain PDZ domain–binding motifs directing a physical association with the scaffolding protein ZO-1, but the significance of the interaction is unknown. We found that Cx50 with PDZ-binding motif mutations did not form gap junction plaques or induce cell–cell communication in HeLa cells, whereas the addition of a seven–amino acid PDZ-binding motif restored normal function to Cx50 lacking its entire C-terminal cytoplasmic domain. C-Terminal deletion had a similar although weaker effect on Cx46 but little if any effect on targeting and function of Cx43. Furthermore, small interfering RNA knockdown of ZO-1 completely inhibited the formation of gap junctions by wild-type Cx50 in HeLa cells. Thus both a PDZ-binding motif and ZO-1 are necessary for Cx50 intercellular channel formation in HeLa cells. Knock-in mice expressing Cx50 with a PDZ-binding motif mutation phenocopied Cx50 knockouts. Furthermore, differentiating lens fibers in the knock-in displayed extensive intracellular Cx50, whereas plaques in mature fibers contained only Cx46. Thus normal Cx50 function in vivo also requires an intact PDZ domain–binding motif. This is the first demonstration of a connexin-specific requirement for a connexin-interacting protein in gap junction assembly.
Human genetic diseases and mouse knockouts illustrate that the maintenance of central nervous system myelin requires connexin expression by both astrocytes and oligodendrocytes. Because these cell types express nonoverlapping sets of connexins, the intercellular channels formed between them must be asymmetric with regard to connexin content, defined as heterotypic. Here, we show that oligodendrocyte Cx47 can form heterotypic channels with astrocyte Cx43 or Cx30 but not Cx26, whereas oligodendrocyte Cx32 can functionally interact with astrocyte Cx30 or Cx26 but not Cx43. Thus, as many as four types of intercellular channels could be formed between astrocytes and oligodendrocytes.
gap junction; connexin; myelin; glia
Hepatitis C virus (HCV) is an important human pathogen, persistently infecting more than 170 million individuals worldwide. Studies of the HCV life cycle have become possible with the development of cell culture systems supporting the replication of viral RNA and the production of infectious virus. However, the exact functions of individual proteins, especially of nonstructural protein 4B (NS4B), remain poorly understood. NS4B triggers the formation of specific, vesicular membrane rearrangements, referred to as membranous webs, which have been reported to represent sites of HCV RNA replication. However, the mechanism of vesicle induction is not known. In this study, a panel of 15 mutants carrying substitutions in the highly conserved NS4B C-terminal domain was generated. Five mutations had only a minor effect on replication, but two of them enhanced assembly and release of infectious virus. Ten mutants were replication defective and used for selection of pseudoreversions. Most of the pseudoreversions also localized to the highly conserved NS4B C-terminal domain and were found to restore replication competence upon insertion into the corresponding primary mutant. Importantly, pseudoreversions restoring replication competence also restored heterotypic NS4B self-interaction, which was disrupted by the primary mutation. Finally, electron microscopy analyses of membrane alterations induced by NS4B mutants revealed striking morphological abnormalities, which were restored to wild-type morphology by the corresponding pseudoreversion. These findings demonstrate the important role of the C-terminal domain in NS4B self-interaction and the formation of functional HCV replication complexes.
Background: Type 2 diabetes is characterized by glucose intolerance and insulin resistance. Obesity is the leading cause of type 2 diabetes. Growing evidence suggests that chronic exposure to inorganic arsenic (iAs) also produces symptoms consistent with diabetes. Thus, iAs exposure may further increase the risk of diabetes in obese individuals.
Objectives: Our goal was to characterize diabetogenic effects of iAs exposure and high-fat diet (HFD) in weaned C57BL/6 mice.
Methods: Mice were fed HFD or low-fat diet (LFD) while exposed to iAs in drinking water (25 or 50 ppm As) for 20 weeks; control HFD and LFD mice drank deionized water. Body mass and adiposity were monitored throughout the study. We measured glucose and insulin levels in fasting blood and in blood collected during oral glucose tolerance tests (OGTT) to evaluate the diabetogenic effects of the treatment.
Results: Control mice fed HFD accumulated more fat, had higher fasting blood glucose, and were more insulin resistant than were control LFD mice. However, these diabetes indicators decreased with iAs intake in a dose-dependent manner. OGTT showed impaired glucose tolerance for both control and iAs-treated HFD mice compared with respective LFD mice. Notably, glucose intolerance was more pronounced in HFD mice treated with iAs despite a significant decrease in adiposity, fasting blood glucose, and insulin resistance.
Conclusions: Our data suggest that iAs exposure acts synergistically with HFD-induced obesity in producing glucose intolerance. However, mechanisms of the diabetogenic effects of iAs exposure may differ from the mechanisms associated with the obesity-induced type 2 diabetes.
arsenic; diabetes; diet-induced obesity; glucose intolerance; high-fat diet; insulin resistance
A double-blind, randomized clinical trial was conducted to determine the effect of consumption of supplemental whey protein (WP), soy protein (SP), and an isoenergetic amount of carbohydrate (CHO) on body weight and composition in free-living overweight and obese but otherwise healthy participants. Ninety overweight and obese participants were randomly assigned to 1 of 3 treatment groups for 23 wk: 1) WP; 2) SP (each providing ~56 g/d of protein and 1670 kJ/d); or 3) an isoenergetic amount of CHO. Supplements were consumed as a beverage twice daily. Participants were provided no dietary advice and continued to consume their free-choice diets. Participants’ body weight and composition data were obtained monthly. Dietary intake was determined by 24-h dietary recalls collected every 10 d. After 23 wk, body weight and composition did not differ between the groups consuming the SP and WP or between SP and CHO; however, body weight and fat mass of the group consuming the WP were lower by 1.8 kg (P < 0.006) and 2.3 kg (P < 0.005), respectively, than the group consuming CHO. Lean body mass did not differ among any of the groups. Waist circumference was smaller in the participants consuming WP than in the other groups (P < 0.05). Fasting ghrelin was lower in participants consuming WP compared with SP or CHO. Through yet-unknown mechanisms, different sources of dietary protein may differentially facilitate weight loss and affect body composition. Dietary recommendations, especially those that emphasize the role of dietary protein in facilitating weight change, should also address the demonstrated clinical potential of supplemental WP.
To use clinical vignettes to understand antimicrobial prescribing practices in neonatal intensive care units (NICUs).
Four tertiary care NICUs.
Antibiotic prescribers in NICUs.
Clinicians from 4 tertiary care NICUs completed an anonymous survey containing 12 vignettes that described empiric, targeted, or prophylactic antibiotic use. Responses were compared with Centers for Disease Control and Prevention guidelines for appropriate use.
Overall, 161 (59% of 271 eligible respondents) completed the survey, 37% of whom had worked in NICUs ≥7 years. Respondents were more likely to appropriately identify use of targeted therapy for methicillin-susceptible Staphylococcus aureus, i.e., use of oxacillin rather than vancomycin, than for E. coli, i.e., use of first generation rather than third generation cephalosporin, (p<0.01). Increased experience significantly predicted appropriate prescribing (p=.02). The proportion of respondents choosing appropriate duration of post-surgical prophylaxis (p<.01) and treatment for necrotizing enterocolitis differed by study site (p=.03).
The survey provides insight into antibiotic prescribing practices and informs the development of future antibiotic stewardship interventions for NICUs.
survey; stewardship; guidelines
Previous laboratory studies have shown that exposures to inorganic As (iAs) disrupt insulin production or glucose metabolism in cellular and animal models. Epidemiological evidence has also linked chronic human exposures to iAs to an increased risk of diabetes mellitus, a metabolic disease characterized by impaired glucose tolerance and insulin resistance. We have recently shown that arsenite and its methylated metabolites inhibit insulin-stimulated glucose uptake in cultured adipocytes by disrupting insulin-activated signal transduction pathway and preventing insulin-dependent translocation of GLUT4 transporters to the plasma membrane. Here, we present results of follow-up studies using male C57BL/6 mice chronically exposed to arsenite (1 to 50 ppm As) or to its metabolite methylarsonite (0.1 to 5 ppm As) in drinking water for 8 weeks. Results of these studies show that only the exposure to arsenite at the highest level of 50 ppm As produces symptoms attributable to impaired glucose tolerance. Notably, tissue concentrations of iAs and its methylated metabolites in pancreas and in major glucose metabolizing tissues in mice in this exposure group were comparable to the concentrations of total As reported in livers of Bangladeshi residents exposed to much lower concentrations of iAs in drinking water. These results suggest that because mice clear iAs and its metabolites more rapidly than humans, much higher exposure levels may be needed in mouse studies to produce the diabetogenic effects of iAs commonly found in human populations exposed to iAs from environmental sources.
arsenic; insulin signaling; glucose tolerance; B6 mice; diabetes mellitus
Conditional inactivation of connexin43 (Cx43) in the pigmented epithelium of the mouse eye results in a reduction in aqueous humor production and complete loss of the vitreous chamber. It was proposed that gap junctions between pigmented and nonpigmented epithelia of the ciliary body are critical for the production of the aqueous humor. To form such junctions, Cx43 in the pigmented epithelium must interact with connexin(s) present in the adjacent cells of the nonpigmented epithelium. The importance of Cx43 expression in the nonpigmented epithelium for the establishment of gap junctions and the regulation of intraocular pressure was tested.
To inactivate Cx43 in the nonpigmented epithelium of the mouse eye, a mouse line was crossed with a floxed Cx43 locus (Cx43flox/flox) and a transgenic mouse line expressing cre recombinase under the control of the Pax6α promoter. General eye structure was evaluated by light microscopy, gap junctions were analyzed by electron microscopy, and intraocular pressure was directly assessed with micropipettes.
In Pax6α-cre/Cx43flox/flox mice, Cx43 was partially inactivated in the nonpigmented epithelium of the ciliary body and iris. Animals developed dilatations between the pigmented and nonpigmented epithelia and displayed a significant reduction in intraocular pressure. However, gap junctions between the ciliary epithelial layers were decreased but not eliminated.
Cx43 expression in the nonpigmented epithelium of the ciliary body contributes to the formation of gap junctions with the cells of the pigmented epithelium. These gap junctions play a critical role in maintaining the physical integrity of the ciliary body epithelium. Although the partial loss of Cx43 from the nonpigmented epithelium was correlated with a measurable drop in intraocular pressure, possible changes in Cx43 in the aqueous outflow pathway may provide an additional contribution to the observed phenotype.
Converging evidence indicates that electrical synaptic transmission via gap junctions plays a crucial role in signal processing in the retina. In particular, amacrine and ganglion cells express numerous gap junctions, resulting in extensive electrical networks in the proximal retina. Both connexin36 (Cx36) and connexin45 (Cx45) subunits are widely distributed in the inner plexiform layer (IPL) and therefore are likely contribute to gap junctions formed by a number of ganglion cell subtypes. In the present study, we used the gap junction-permeant tracer Neurobiotin to compare the coupling pattern of different ganglion cell subtypes in wild-type (WT) and Cx36 knockout (KO) mouse retinas. We found that homologous ganglion-to-ganglion cell coupling was lost for two subtypes after deletion of Cx36, whereas two other ganglion cell subtypes retained homologous coupling in the KO mouse. In contrast, deletion of Cx36 resulted in a partial or complete loss of ganglion-to-amacrine cell heterologous coupling in 9 of 10 ganglion cell populations studied. Overall, our results indicate that Cx36 is the predominant subunit of gap junctions in the proximal mouse retina, expressed by most ganglion cell subtypes, and thereby likely plays a major role in the concerted activity generated by electrical synapses.
tracer coupling; gap junction; ganglion cell; amacrine cell; mouse
To examine the functions of electrical synapses in the transmission of signals from rod photoreceptors to ganglion cells, we generated connexin36 knockout mice. Reporter expression indicated that connexin36 was present in multiple retinal neurons including rod photoreceptors, cone bipolar cells, and AII amacrine cells. Disruption of electrical synapses between adjacent AIIs and between AIIs and ON cone bipolars was demonstrated by intracellular injection of Neurobiotin. In addition, extracellular recording in the knockout revealed the complete elimination of rod-mediated, on-center responses at the ganglion cell level. These data represent direct proof that electrical synapses are critical for the propagation of rod signals across the mammalian retina, and they demonstrate the existence of multiple rod pathways, each of which is dependent on electrical synapses.
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.
connexin; gap junction; amacrine cell
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 ~1 log unit. In addition, OFF signaling uses a tertiary rod pathway that is ~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.
convergence; gap junction; retina; retinal ganglion cell; rod; vision
Neurons of the inferior olivary nucleus (IO) form the climbing fibers that excite Purkinje cells of the cerebellar cortex. IO neurons are electrically coupled through gap junctions, and they generate synchronous, subthreshold oscillations of membrane potential at ~5–10 Hz. Experimental and theoretical studies have suggested that both the rhythmicity and synchrony of IO neurons require electrical coupling. We recorded from pairs of IO neurons in slices of mouse brainstem in vitro. Most pairs of neurons from wild-type (WT) mice were electrically coupled, but coupling was rare and weak between neurons of knock-out (KO) mice for connexin36, a neuronal gap junction protein. IO cells in both WT and KO mice generated rhythmic membrane fluctuations of similar frequency and amplitude. Oscillations in neighboring pairs of WT neurons were strongly synchronized, whereas the oscillations of KO pairs were uncorrelated. Spontaneous oscillations in KO neurons were not blocked by tetrodotoxin. Spontaneously oscillating neurons of both WT and KO mice generated occasional action potentials in phase with their membrane rhythms, but only the action potentials of WT neuron pairs were synchronous. Harmaline, a β-carboline derivative thought to induce tremor by facilitating rhythmogenesis in the IO, was injected systemically into WT and KO mice. Harmaline-induced tremors were robust and indistinguishable in the two genotypes, suggesting that gap junction-mediated synchrony does not play a role in harmaline-induced tremor. We conclude that electrical coupling is not necessary for the generation of spontaneous subthreshold oscillations in single IO neurons, but that coupling can serve to synchronize rhythmic activity among IO neurons.
inferior olive; electrical coupling; connexin36; gap junction; harmaline; rhythms; synchrony
Gap junctions between neurons function as electrical synapses, and are present in all layers of mammalian and teleost retina. These synapses are largest and most prominent between horizontal cells where they function to increase the receptive field of a single neuron beyond the width of its dendrites. Receptive field size and the extent of gap junctional coupling between horizontal cells is regulated by ambient light levels and may mediate light/dark adaptation. Furthermore, teleost horizontal cell gap junction hemichannels may facilitate a mechanism of feedback inhibition between horizontal cells and cone photoreceptors. As a prelude to using mouse genetic models to study horizontal cell gap junctions and hemichannels, we sought to determine the connexin complement of mouse horizontal cells. Cx36, Cx37, Cx43, Cx45 and Cx57 mRNA could be detected in mouse retina by RT-PCR. Microscopy was used to further examine the distribution of Cx26 and Cx36. Cx26 immunofluorescence and a β-gal reporter under regulatory control of the Cx36 promoter did not colocalize with a horizontal cell marker, indicating that these genes are not expressed by horizontal cells. The identity of the connexin(s) forming electrical synapses between mouse horizontal cells and the connexin that may form hemichannels in the horizontal cell telodendria remains unknown.
connexin26; connexin36; horizontal cell; gap junction; hemichannel
Gap junctions are aggregates of intercellular channels that permit direct cell–cell transfer of ions and small molecules. Initially described as low-resistance ion pathways joining excitable cells (nerve and muscle), gap junctions are found joining virtually all cells in solid tissues. Their long evolutionary history has permitted adaptation of gap-junctional intercellular communication to a variety of functions, with multiple regulatory mechanisms. Gap-junctional channels are composed of hexamers of medium-sized families of integral proteins: connexins in chordates and innexins in precordates. The functions of gap junctions have been explored by studying mutations in flies, worms, and humans, and targeted gene disruption in mice. These studies have revealed a wide diversity of function in tissue and organ biology.
Gap junctions that allow material to move between cells are composed of connexins. The choice of connexin affects the permeability and other features of these junctions.