Morphological characterization of mouse ganglion cells
In this study we injected 210 ganglion cells with Neurobiotin to reveal the soma/dendritic morphology of the distinct sub-types in the wildtype mouse retina. Using a number of morphometric features, we distinguished 22 subpopulations of ganglion cells (summarized in ) and then determined the tracer coupling of each (). It is important to note that the coupling pattern of a ganglion cell was never used to classify it and thus was treated as a variable independent of a cell’s morphological identity. Still, we found that the tracer coupling pattern of each distinct ganglion cell subpopulation was stereotypic across the dark-adapted retinas we studied.
A number of previous studies, using a variety of morpho-metric methods, have described the different ganglion cell subtypes in the mouse retina. Although we classified our cells independent of these studies, they still form important frameworks in which to compare and incorporate our ganglion cell subtypes (). Sun et al. (2002)
initially characterized 17 ganglion cell subtypes using classical morphological criteria including soma and dendritic size, soma/dendritic architecture, and dendritic stratification in the IPL. Three subsequent studies used cluster analyses to sort murine ganglion cells in a multidimensional space. These studies distinguished 12 (Badea and Nathans, 2004
), 11 (Kong et al., 2005
), and 19 (Coombs et al., 2006
) distinct ganglion cell morphological clusters. Overall, 17 of our 22 ganglion cell subtypes could be clearly matched to the 17 ganglion cell subpopulations characterized by Sun et al. (2002)
. The remaining five subtypes appear to be rare ganglion cells that were not encountered in the Sun et al. (2002)
study. In addition, most of our ganglion cell subtypes were described in the cluster analysis studies, including four subtypes which had no counterparts in the description by Sun et al. (2002)
. However, whereas we found one-to-one correlations between 17 of our ganglion cell sub-types with those described by Sun et al. (2002)
, the relationship of our cells to those reported in the cluster analysis studies was more equivocal. First, some of our ganglion cell subtypes appeared to match cells in more than one cluster. For example, the G5
cells resembled ganglion cells in both monostratified 2 and monostratified 4 clusters of Badea and Nathans (2004)
. Second, a number of our ganglion cell sub-types did not appear to be included in one or more of these clustering schemes. Our G19
ganglion cell had no counterpart in any of the previous studies. Third, cells in certain clusters resembled more than one of our ganglion cell subpopulations. For example, descriptions of cluster 1 cells in the scheme provided by Kong et al. (2005)
, and G13
cells in our sample. This disparity between the different datasets, including our own, is likely due to the different labeling and analyzing techniques, but also suggests that some clusters incorporate multiple ganglion cell subtypes. It should be noted that some of our morphometric values (e.g., soma size, dendritic fields diameter) were 25–50% smaller than those given for counterpart ganglion cell subtypes in the previous studies (Sun et al., 2002
; Badea and Nathans, 2004
; Kong et al., 2005
; Coombs et al., 2006
). This discrepancy is most likely due to tissue shrinkage resulting from the dehydration process in our histological protocol.
Tracer-coupling patterns of mouse ganglion cells
This article is the first comprehensive study to describe and compare ganglion cell coupling across the entire population in the wildtype mouse retina. Overall, our study indicates that the different ganglion cell subtypes display stereotypic tracer coupling patterns in the mouse retina (summarized in ). Thus, the tracer coupling pattern can be a useful and additional feature to distinguish the different ganglion cell sub-types. However, it is important to point out that, despite the standardized experimental conditions used here, we did observe some variability in the absolute number of cells coupled to ganglion cells of the same subtype (see ). While this observation may reflect biological variability, we believe it more likely reflects a technical variability related to inconsistency in the amount of tracer delivered via electrodes and its diffusion across the coupled network of cells. Despite this variability in the absolute number of coupled cells, the overall coupling pattern remained stereotypic for each ganglion cell subtype.
Figure 14 Schematic summarizing the tracer coupling patterns of the 22 ganglion cell subtypes in the mouse retina. In addition to coupling, some morphological features of both injected and coupled cells are also shown, including dendritic and axonal (if any) stratification (more ...)
We found that 16 of the 22 morphologically distinct ganglion cell subpopulations displayed homologous coupling to ganglion cell neighbors and/or heterologous coupling to nearby amacrine cells. Although varied, we found that the ganglion cell coupling in the mouse retina was governed by some general rules. First, ON ganglion cells were coupled to amacrine cells with somata displaced to the GCL, whereas OFF ganglion cells were coupled to amacrine cells with somata lying in the INL. Second, most ON ganglion cells were coupled to polyaxonal amacrine cells (G1,G6, and G10; but not G2), whereas OFF ganglion cells were coupled to wide-field amacrine cells (G3 and G7). Third, homologous ganglion-toganglion cell coupling occurred only among cells whose dendrites stratified at least partially in sublamina-a, which included ganglion cells with diffuse (G13 cells), monostratified (G3,G6,G11, and G18 cells) and bistratified dendritic arbors (G16 and G17 cells). These are presumably OFF and ON-OFF physiological subtypes. Ganglion cells whose dendritic arbor was restricted to sublamina-b, presumably ON cells, never showed homologous coupling. Fourth, of the ganglion cells that showed homologous coupling, only bistratified ganglion cells (G16 and G17 cells) were not coupled to amacrine cells as well.
We found that seven ganglion cell populations displayed homologous coupling with their ganglion cell neighbors. In many G3,G7,G17, and G18 cell injections, not only somata, but proximal dendrites or even the entire dendritic arbor of homologously coupled ganglion cells were visible as well. In all these cases, soma/dendritic morphologies of coupled and injected ganglion cells were identical. This finding indicates that mouse ganglion cells only couple to nearby like-type ganglion cells. However, it remains to be determined if inter-typical coupling can also be ruled out for G11,G13, and G16 cells where labels were confined to coupled somata.
We found that 14 ganglion cell populations showed heterologous coupling to amacrine cells, whose arbors were often visible, allowing for examination of their soma/dendritic morphologies. Interestingly, all coupled amacrine cells observed in this study were either polyaxonal or wide-field amacrine cells, whereas we found no evidence of coupling between ganglion cells and narrow-field amacrine cells. However, labels of many coupled amacrine cells were restricted to their soma and yet to be morphologically characterized.
Role of ganglion cell coupling
Overall, our results indicate that nearly three-quarters of the ganglion cells in the mouse retina are coupled to ganglion cell and/or amacrine cell neighbors. This extensive coupling in the inner mouse retina is consistent with findings in other mammals (Vaney, 1991
; Xin and Bloomfield, 1997
; Hoshi et al., 2007
). At first glance, this widespread coupling is disconcerting, as it suggests a significant lateral spread of visual signals as they leave the retina, thereby substantially reducing spatial acuity and blurring the image transmitted to higher visual centers. However, previous morphological studies have shown that the tracer coupling patterns of ganglion cells are restricted to small local groups of nearest neighbors (Vaney, 1991
; Xin and Bloomfield, 1997
). This circumscribed pattern of tracer movement was particularly true for the homologous coupling we observed here for murine ganglion cells. Likewise, the receptive fields of individual ganglion cells have been shown to match closely the size of their dendritic fields, irrespective of the presence of tracer coupling (Bloom-field and Xin, 1997
; Völgyi et al., 2000
). Taken together, these results suggest that electrical synapses formed by ganglion cells do not underlie a significant spread of current across the IPL, and thus their function must be quite different from that of the extensively coupled horizontal cell syncytia in the outer retina.
One proposed function for ganglion cell electrical coupling is to provide for correlated activity of neighboring cells. Correlated firing is believed to compress information for efficient transmission and thereby enhance bandwidth of the optic nerve (Meister and Berry, 1999
). In this scheme, synchronous activity can provide additional information to the brain by multiplexing with asynchronous signals from individual ganglion cells. Concerted spike activity is also thought to enhance the saliency of visual signals by increasing temporal summation at central targets (Alonso et al., 1996
; Stevens and Zador, 1998
; Usrey and Reid, 1999
). In this way, concerted ganglion cell activity may provide the temporal precision by which retinal signals are reliably transmitted to central targets (Singer, 1999
). In fact, correlated spiking may account for up to one-half of all retinal spike activity, largely a result of the extensive coupling between ganglion and amacrine cells in the IPL.
Concerted activity among ganglion cells can range from relatively loose correlations, reflected in cross-correlograms spanning tens of milliseconds, to narrowly synchronized spikes with latencies <3 msec (Arnett and Spraker, 1981
; Mastronarde, 1983a
; Brivanlou et al., 1998
; DeVries, 1999
; Hu and Bloomfield, 2003
). The short latency correlations are believed to reflect direct electrical coupling between neighboring ganglion cells, whereas the somewhat broader intermediate correlations may be generated by ganglion cells forming electrical synapses with a common population of amacrine cells (Brivanlou et al., 1998
; DeVries, 1999
; Hu and Bloomfield, 2003
). Interestingly, we found that about one-third of our ganglion cell subtypes displayed homologous coupling to their neighbors, whereas nearly two-thirds showed heterologous coupling to amacrine cells. Our results thus suggest that a range of concerted spike activity for ganglion cells is present in the mouse retina, but intermediate latency correlations should be the most prevalent. This idea is consistent with the findings Schnitzer and Meister (2003)
indicating that the intermediate latency correlation is the most common form of concerted spiking in the vertebrate retina. Clearly, physiological studies are called for to examine the concerted activity of murine ganglion cells to elucidate the functional roles of the different patterns of coupling expressed in the IPL of the mouse retina.