The Retinal TNAP

Accumulating evidence from recent literature underline the important roles of tissue non specific alkaline phosphatase (TNAP) in diverse functions as well as diseases of the nervous system. Exploration of TNAP in well characterized neural circuits such as the retina, might significantly advance our understanding regarding neural TNAP’s roles. This chapter reviews the scarce literature as well as our findings on retinal TNAP. We found that retinal TNAP activity was preserved and followed diverse patterns throughout vertebrate evolution. We have consistently observed TNAP activity (1) in retinal vessels, (2) in photoreceptors and (3) in the majority of the studied species in the outer (OPL) and inner plexiform layers (IPL), where synaptic transmission occurs. Importantly, in some species the IPL exhibits several TNAP positive strata. These strata exactly corresponded those seen after quadruple immunohistochemistry with four canonical IPL markers (tyrosine hydroxylase, choline acetyltransferase, calretinin, protein kinase C α). Diabetes results in diminishing retinal TNAP activity before changes in canonical markers

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

A unique role for Kv3 voltage-gated potassium channels in starburst amacrine cell signaling in mouse retina.

Direction-selective retinal ganglion cells show an increased activity evoked by light stimuli moving in the preferred direction. This selectivity is governed by direction-selective inhibition from starburst amacrine cells occurring during stimulus movement in the opposite or null direction. To understand the intrinsic membrane properties of starburst cells responsible for direction-selective GABA release, we performed whole-cell recordings from starburst cells in mouse retina. Voltage-clamp recordings revealed prominent voltage-dependent K(+) currents. The currents were mostly blocked by 1 mm TEA, activated rapidly at voltages more positive than -20 mV, and deactivated quickly, properties reminiscent of the currents carried by the Kv3 subfamily of K+ channels. Immunoblots confirmed the presence of Kv3.1 and Kv3.2 proteins in retina and immunohistochemistry revealed their expression in starburst cell somata and dendrites. The Kv3-like current in starburst cells was absent in Kv3.1-Kv3.2 knock-out mice. Current-clamp recordings showed that the fast activation of the Kv3 channels provides a voltage-dependent shunt that limits depolarization of the soma to potentials more positive than -20 mV. This provides a mechanism likely to contribute to the electrical isolation of individual starburst cell dendrites, a property thought essential for direction selectivity. This function of Kv3 channels differs from that in other neurons where they facilitate high-frequency repetitive firing. Moreover, we found a gradient in the intensity of Kv3.1b immunolabeling favoring proximal regions of starburst cells. We hypothesize that this Kv3 channel gradient contributes to the preference for centrifugal signal flow in dendrites underlying direction-selective GABA release from starburst amacrine cell

Strategic Positioning of Connexin36 Gap Junctions Across Human Retinal Ganglion Cell Dendritic Arbors

Connexin36 (Cx36) subunits form gap junctions (GJ) between neurons throughout the central nervous system. Such GJs of the mammalian retina serve the transmission, averaging and correlation of signals prior to conveying visual information to the brain. Retinal GJs have been exhaustively studied in various animal species, however, there is still a perplexing paucity of information regarding the presence and function of human retinal GJs. Particularly little is known about GJ formation of human retinal ganglion cells (hRGCs) due to the limited number of suitable experimental approaches. Compared to the neuronal coupling studies in animal models, where GJ permeable tracer injection is the gold standard method, the post-mortem nature of scarcely available human retinal samples leaves immunohistochemistry as a sole approach to obtain information on hRGC GJs. In this study Lucifer Yellow (LY) dye injections and Cx36 immunohistochemistry were performed in fixed short-post-mortem samples to stain hRGCs with complete dendritic arbors and locate dendritic Cx36 GJs. Subsequent neuronal reconstructions and morphometric analyses revealed that Cx36 plaques had a clear tendency to form clusters and particularly favored terminal dendritic segments

Physicochemical Characterization and Cyclodextrin Complexation of the Anticancer Drug Lapatinib

Lapatinib (LAP), the tyrosine kinase inhibitor drug with moderate bioavailability, was characterized in terms of physicochemical properties: acid-base characteristics, lipophilicity, and solubility. The highly lipophilic nature of the drug and its extremely low water solubility (S0=0.82 nM) limit the development of a parenteral formulation. In order to enhance solubility and bioavailability, inclusion complex formation with cyclodextrins (CDs) is a promising method of choice. Therefore, LAP-CD interactions were also studied by a multianalytical approach. The stability constants of LAP with native cyclodextrins, determined by UV spectroscopy, identified the seven-membered β-CD as the most suitable host. Continuous variation method (Job’s plot) by 1H NMR showed a 1 : 1 stoichiometry for the complexes. The geometry of the complex was elucidated by 2D ROESY NMR measurements and molecular modeling, indicating that the partial molecular encapsulation includes the fluorophenyl ring of LAP. Phase-solubility studies with four CDs, β-CD, (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD), randomly methylated-β- (RAMEB-) cyclodextrin, and sulfobutylether-β-cyclodextrin (SBE-β-CD), show an AL type diagram and highly increased solubility via CD complexation. The results are especially promising with SBE-β-CD, exerting more than 600-fold gain in solubility. The equilibrium and structural information presented herein can offer the molecular basis for an improved drug formulation with enhanced bioavailability

Gap Junctions Are Essential for Generating the Correlated Spike Activity of Neighboring Retinal Ganglion Cells

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

Connexin36 Expression in the Mammalian Retina: A Multiple-Species Comparison

Much knowledge about interconnection of human retinal neurons is inferred from results on animal models. Likewise, there is a lack of information on human retinal electrical synapses/gap junctions (GJ). Connexin36 (Cx36) forms GJs in both the inner and outer plexiform layers (IPL and OPL) in most species including humans. However, a comparison of Cx36 GJ distribution in retinas of humans and popular animal models has not been presented. To this end a multiple-species comparison was performed in retinas of 12 mammals including humans to survey the Cx36 distribution. Areas of retinal specializations were avoided (e.g., fovea, visual streak, area centralis), thus observed Cx36 distribution differences were not attributed to these species-specific architecture of central retinal areas. Cx36 was expressed in both synaptic layers in all examined retinas. Cx36 plaques displayed an inhomogenous IPL distribution favoring the ON sublamina, however, this feature was more pronounced in the human, swine and guinea pig while it was less obvious in the rabbit, squirrel monkey, and ferret retinas. In contrast to the relative conservative Cx36 distribution in the IPL, the labels in the OPL varied considerably among mammals. In general, OPL plaques were rare and rather small in rod dominant carnivores and rodents, whereas the human and the cone rich guinea pig retinas displayed robust Cx36 labels. This survey presented that the human retina displayed two characteristic features, a pronounced ON dominance of Cx36 plaques in the IPL and prevalent Cx36 plaque conglomerates in the OPL. While many species showed either of these features, only the guinea pig retina shared both. The observed similarities and subtle differences in Cx36 plaque distribution across mammals do not correspond to evolutionary distances but may reflect accomodation to lifestyles of examined species