Abundance and ultrastructural diversity of neuronal gap junctions in the OFF and ON sublaminae of the inner plexiform layer of rat and mouse retina

Abstract

Neuronal gap junctions are abundant in both outer and inner plexiform layers of the mammalian retina. In the inner plexiform layer (IPL), ultrastructurally-identified gap junctions were reported primarily in the functionally-defined and anatomically-distinct ON sublamina, with few reported in the OFF sublamina. We used freeze-fracture replica immunogold labeling and confocal microscopy to quantitatively analyze the morphologies and distributions of neuronal gap junctions in the IPL of adult rat and mouse retina. Under "baseline" conditions (photopic illumination/general anesthesia), 649 neuronal gap junctions immunogold-labeled for connexin36 were identified in rat IPL, of which 375 were photomapped to OFF vs. ON sublaminae. In contrast to previous reports, the volume-density of gap junctions was equally abundant in both sublaminae. Five distinctive morphologies of gap junctions were identified: conventional crystalline and non-crystalline "plaques" (71% and 3%), plus unusual "string" (14%), "ribbon" (7%) and "reticular" (2%) forms. Plaque and reticular gap junctions were distributed throughout the IPL. However, string and ribbon gap junctions were restricted to the OFF sublamina, where they represented 48% of gap junctions in that layer. In string and ribbon junctions, curvilinear strands of connexons were dispersed over 5 to 20 times the area of conventional plaques having equal numbers of connexons. To define morphologies of gap junctions under different light-adaptation conditions, we examined an additional 1150 gap junctions from rats and mice prepared after 30 min of photopic, mesopic and scotopic illumination, with and without general anesthesia. Under these conditions, string and ribbon gap junctions remained abundant in the OFF sublamina and absent in the ON sublamina. Abundant gap junctions in the OFF sublamina of these two rodents with rod-dominant retinas revealed previously-undescribed but extensive pathways for inter-neuronal communication; and the wide dispersion of connexons in string and ribbon gap junctions suggests unique structural features of gap junctional coupling in the OFF vs. ON sublamina.

Connexin36 vs. connexin32, “miniature” neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmatic nucleus

“…2C,E). Overall, immunopositive puncta were generally much smaller and were present in substantially lower density than in retina, inferior olivary nucleus, reticular thalamic nucleus and basomedial nucleus of the amygdala (Li et al, 2004;Rash et al, 2004b;Kamasawa et al, 2006), but were comparable in numbers to those observed in hippocampus and appeared several-fold greater than in cerebral cortex (Nagy, unpublished observations). Notably, the SCN contained a substantially greater density of Cx36-positive puncta than surrounding hypothalamic areas, and this feature alone was sufficient to distinguish SCN from surrounding tissue (Fig.…”

“…749.11, 2002) for immunocytochemical precedent, Long and coworkers (Long et al, 2005) proposed Cx36 as the primary or sole connexin in neuronal gap junctions in SCN, and suggested that Cx36 deletion disrupted circadian rhythms. Among the 21 connexins known to be present in mammals (Condorelli et al, 1998;Söhl and Willecke, 2003;Willecke et al, 2002), both Cx36 and Cx45 have been demonstrated in ultrastructurally-defined neuronal gap junctions (Rash et al, 2000(Rash et al, , 2001Fukuda et al, 2006;Kamasawa et al, 2006), and there is immunocytochemical evidence for neuronal expression of Cx50 and Cx57 in retina (Massey et al, 2003;Hombach et al, 2004;O'Brien et al, 2006). In view of controversies surrounding the existence and connexin composition of neuronal gap junctions in SCN, we used confocal immunofluorescence microscopy and freeze-fracture replica immunogold labeling (FRIL) to document gap junctions in SCN neurons and to identify the constituent connexins in neurons vs. glia, and whole-cell recordings from hypothalamic slices to re-assess tracer and electrotonic coupling.…”

“…Based on the near absence of immunofluorescent puncta in Cx36 KO mice, the rate of false-positive puncta is estimated to be very low. Conversely, with respect to false negatives, our recent quantitative confocal and FRIL studies of >2000 gap junctions varying from 4 to 3000 connexons in rodent retina suggest that the constraints of labeling and immunofluorescence imaging may limit light-microscopic detection of the smallest gap junctions (i.e., <30-50 connexons; Kamasawa et al, 2006). Thus, FRIL data suggesting that more than half of neuronal gap junctions in SCN have fewer than 50 connexons implies the possibility of additional "mini" gap junctions per neuron.…”

“…4B), one small "string" gap junction (not shown), and one small "meandering" gap junction (not shown). (For classification of gap junction morphologies in neurons, see Kamasawa et al, 2006). Overall in adult SCN, 92% (11/12) of labeled neuronal gap junctions had fewer than 100 connexons and 75% (9/12) contained fewer than 50 connexons.…”

“…100 "mini" gap junctions between spinal cord neurons in cervical, thoracic, and lumbosacral regions (Rash et al, 1996(Rash et al, , 1998a, with >50% of those smaller than 45 connexons (Rash et al, 1996). Subsequently, Cx36-fluorescence immunocytochemistry and FRIL revealed the abundance of similar neuronal "mini" gap junctions in several other regions of the CNS, including retina and locus coeruleus (Kamasawa et al, 2006;Rash et al, 2007). …”