Patrick W. Keeley

Changes in levels of D1, D2, or NMDA receptors during withdrawal from brief or extended daily access to IV cocaine

We previously reported that brief (1 h), but not extended (6 h), daily access to cocaine results in a sensitized locomotor response to cocaine and in elevated c-Fos immunoreactivity and DAT binding in the nucleus accumbens (N.Acc) core. In order to better our understanding of the neural adaptations mediating the transition from controlled drug use to addiction, the current experiments were set to further explore the neural adaptations resulting from these two access conditions. Rats received either brief daily access to saline or cocaine, or brief daily access followed by extended daily access to cocaine. Subjects were then sacrificed either 20 min, or 14 or 60 days, after the last self-administration session. Samples of the ventral tegmental area (VTA), N.Acc core and shell, dorsal striatum, and medial prefrontal cortex (mPFC) were taken for analysis of D1 ([3H]SCH-23390), D2 ([3H]Spiperone), and NMDA ([3H]MK-801) receptor binding (using the method of receptor autoradiography). At 20 min into withdrawal, D2 receptors were elevated and NMDA receptors were reduced in the mPFC of the brief access animals while D1 receptors were elevated in the N.Acc shell of the extended access animals, compared to saline controls. D2 receptors were reduced in the N.Acc shell of the brief access animals compared to saline controls after 14 days, and compared to extended access animals after 60 days of withdrawal. In summary, extended access to cocaine resulted in only transient changes in D1 receptors binding. These results suggest that the development of compulsive drug use is largely unrelated to changes in total binding of D2 or NMDA receptors.

Spatial patterning of cholinergic amacrine cells in the mouse retina

The two populations of cholinergic amacrine cells in the inner nuclear layer (INL) and the ganglion cell layer (GCL) differ in their spatial organization in the mouse retina, but the basis for this difference is not understood. The present investigation examined this issue in six strains of mice that differ in their number of cholinergic cells, addressing how the regularity, packing, and spacing of these cells varies as a function of strain, layer, and density. The number of cholinergic cells was lower in the GCL than in the INL in all six strains. The nearest neighbor and Voronoi domain regularity indexes as well as the packing factor were each consistently lower for the GCL. While these regularity indexes and the packing factor were largely stable across variation in density, the effective radius was inversely related to density for both the GCL and INL, being smaller and more variable in the GCL. Consequently, despite the lower densities in the GCL, neighboring cells were more likely to be positioned closer to one another than in the higher‐density INL, thereby reducing regularity and packing. This difference in the spatial organization of cholinergic cells may be due to the cells in the GCL having been passively displaced by fascicles of optic axons and an expanding retinal vasculature during development. In support of this interpretation, we show such displacement of cholinergic somata relative to their dendritic stalks and a decline in packing efficiency and regularity during postnatal development that is more severe for the GCL. J. Comp. Neurol. 508:1–12, 2008.

Dendritic spread and functional coverage of starburst amacrine cells.

The network of starburst amacrine cells plays a fundamental role in the neural circuitry underlying directional selectivity within the retina. Individual sectors of the starburst dendritic field are directionally selective by virtue of a mutually inhibitory relationship between starburst amacrine cells with overlapping dendrites. These features of the starburst amacrine cell network suggest that starburst cells regulate their dendritic overlap to ensure a uniform coverage of the retinal surface. The present study has compared the dendritic morphology of starburst amacrine cells in two different strains of mice that differ in starburst amacrine cell number. The A/J (A) strain contains about one‐quarter fewer starburst amacrine cells than does the C57BL/6J (B6) strain, although the mosaics of starburst amacrine cells in both strains are comparably patterned. Dendritic field size, however, does not compensate for the difference in density, the A strain having a slightly smaller dendritic field relative to the B6 strain, yielding a significantly larger dendritic coverage factor for individual cells in the B6 strain. The area of the distal (output) annulus of the dendritic field occupies a comparable proportion of the overall field area in the two strains, but overlapping annuli establish a finer meshwork of co‐fasciculating processes in the B6 strain. These results would suggest that the architecture of the dendritic network, rather than the overall size of the dendritic field, is dependent on the density of starburst amacrine cells. J. Comp. Neurol. 505:539–546, 2007.

Morphology of Dopaminergic Amacrine Cells in the Mouse Retina: Independence from Homotypic Interactions

To determine the role of homotypic interactions between neighboring dopaminergic amacrine (DA) cells upon dendritic morphogenesis, the morphology of single cells was examined relative to the positioning of all neighboring homotypic cells. For each labeled cell, the dendritic field was reconstructed, its Voronoi domain was calculated, and the two were related. The dendritic fields of DA cells were observed to be large, sparse, and highly irregular. Dendrites readily overlapped those of neighboring cells, showing no evidence for dendritic tiling or inter‐digitation consistent with homotypic repulsion or avoidance. Furthermore, a direct comparison of dendritic field area with the Voronoi domain area of the same cell showed no evidence for dendritic growth being constrained or biased by the local distribution of homotypic neighbors in wild‐type retinas. A comparison of the processes of adjacent filled cells confirmed their immediate proximity to one another within the inner plexiform layer, indicating that they do not engage in mutual avoidance by coursing at different depths. Together, these results suggest that the morphogenesis of DA cells is independent of homotypic interactions. However, in the absence of the pro‐apoptotic Bax gene, which yields a fourfold increase in DA cell number, a small but significant reduction in dendritic field size was obtained, although not so great as would be predicted by the increase in density. The present results are considered in light of recent studies on the role of cell adhesion molecules expressed by developing DA cells. J. Comp. Neurol. 518:1220–1231, 2010.

Role of Afferents in the Differentiation of Bipolar Cells in the Mouse Retina

To establish dendritic arbors that integrate properly into a neural circuit, neurons must rely on cues from the local environment. The neurons presynaptic to these arbors, the afferents, are one potential source of these cues, but the particular dendritic features they regulate remain unclear. Retinal bipolar cells can be classified by the type of photoreceptor, cone or rod, forming synaptic contacts with their dendrites, suggesting a potential role of these afferents in shaping the bipolar cell dendritic arbor. In the present investigation, the role of photoreceptors in directing the differentiation of bipolar cells has been studied using two genetically modified “coneless” and “conefull” mice. Single cone (Type 7/CB4a) and rod bipolar cells were labeled with DiI to reveal the entire dendritic arbor and subsequently analyzed for several morphological features. For both cone and rod bipolar cells, the dendritic field area, number of dendritic terminals, and stratification of terminals in the outer plexiform layer were comparable among coneless, conefull, and wild-type retinas, and the overall morphological appearance of each type of cell was essentially conserved, indicating an independence from afferent specification. The presence of normal afferents was, however, found to be critical for the proper spatial distribution of dendritic terminals, exhibiting a clustered distribution for the cone bipolar cells and a dispersed distribution for the rod bipolar cells. These results demonstrate a selectivity in the afferent dependency of bipolar cell differentiation, their basic morphogenetic plan commanded cell intrinsically, and their fine terminal connectivity directed by the afferents themselves.

Apoptosis Regulates ipRGC Spacing Necessary for Rods and Cones to Drive Circadian Photoentrainment

The retina consists of ordered arrays of individual types of neurons for processing vision. Here, we show that such order is necessary for intrinsically photosensitive retinal ganglion cells (ipRGCs) to function as irradiance detectors. We found that during development, ipRGCs undergo proximity-dependent Bax-mediated apoptosis. Bax mutant mice exhibit disrupted ipRGC spacing and dendritic stratification with an increase in abnormally localized synapses. ipRGCs are the sole conduit for light input to circadian photoentrainment, and either their melanopsin-based photosensitivity or ability to relay rod/cone input is sufficient for circadian photoentrainment. Remarkably, the disrupted ipRGC spacing does not affect melanopsin-based circadian photoentrainment but severely impairs rod/cone-driven photoentrainment. We demonstrate reduced rod/cone-driven cFos activation and electrophysiological responses in ipRGCs, suggesting that impaired synaptic input to ipRGCs underlies the photoentrainment deficits. Thus, for irradiance detection, developmental apoptosis is necessary for the spacing and connectivity of ipRGCs that underlie their functioning within a neural network.

Design principles and developmental mechanisms underlying retinal mosaics.

Most structures within the central nervous system (CNS) are composed of different types of neuron that vary in both number and morphology, but relatively little is known about the interplay between these two features, i.e. about the population dynamics of a given cell type. How such arrays of neurons are distributed within a structure, and how they differentiate their dendrites relative to each other, are issues that have recently drawn attention in the invertebrate nervous system, where the genetic and molecular underpinnings of these organizing principles are being revealed in exquisite detail. The retina is one of the few locations where these principles have been extensively studied in the vertebrate CNS, indeed, where the design principles of ‘mosaic regularity’ and ‘uniformity of coverage’ were first explicitly defined, quantified, and related to each other. Recent studies have revealed a number of genes that influence the formation of these histotypical features in the retina, including homologues of those invertebrate genes, although close inspection reveals that they do not always mediate comparable developmental processes nor elucidate fundamental design principles. The present review considers just how pervasive these features of ‘mosaic regularity’ and ‘uniform dendritic coverage’ are within the mammalian retina, discussing the means by which such features can be assessed in the mature and developing nervous system and examining the limitations associated with those assessments. We then address the extent to which these two design principles co‐exist within different populations of neurons, and how they are achieved during development. Finally, we consider the neural phenotypes obtained in mutant nervous systems, to address whether a prospective gene of interest underlies those very design principles.

Development and plasticity of outer retinal circuitry following genetic removal of horizontal cells.

The present study examined the consequences of eliminating horizontal cells from the outer retina during embryogenesis upon the organization and assembly of the outer plexiform layer (OPL). Retinal horizontal cells exhibit a migration defect in Lim1-conditional knock-out (Lim1-CKO) mice and become mispositioned in the inner retina before birth, redirecting their dendrites into the inner plexiform layer. The resultant (mature) OPL, developing in the absence of horizontal cells, shows a retraction of rod spherules into the outer nuclear layer and a sprouting of rod bipolar cell dendrites to reach ectopic ribbon-protein puncta. Cone pedicles and the dendrites of type 7 cone bipolar cells retain their characteristic stratification and colocalization within the collapsed OPL, although both are atrophic and the spatial distribution of the pedicles is disrupted. Developmental analysis of Lim1-CKO retina reveals that components of the rod and cone pathways initially co-assemble within their normal strata in the OPL, indicating that horizontal cells are not required for the correct targeting of photoreceptor terminals or bipolar cell dendrites. As the rod spherules begin to retract during the second postnatal week, rod bipolar cells initially show no signs of ectopic growth, sprouting only subsequently and continuing to do so well after the eighth postnatal week. These results demonstrate the critical yet distinctive roles for horizontal cells on the rod and cone pathways and highlight a unique and as-yet-unrecognized maintenance function of an inhibitory interneuron that is not required for the initial targeting and co-stratification of other components in the circuit.

Neuronal clustering and fasciculation phenotype in Dscam- and Bax-deficient mouse retinas.

Individual types of retinal neurons are distributed to minimize proximity to neighboring cells. Many of these same cell types extend dendrites to provide coverage of the retinal surface. These two cardinal features of retinal mosaics are disrupted, for certain cell types, in mice deficient for the Down syndrome cell adhesion molecule, Dscam, exhibiting an aberrant clustering of somata and fasciculation of dendrites. The Dscam mutant mouse retina also exhibits excess numbers of these same cell types. The present study compared these two features in Dscam mutant retinas with the Bax knockout retina, in which excess numbers of two of these cell types, the melanopsin‐positive retinal ganglion cells (MRGCs) and the dopaminergic amacrine cells (DACs), are also present. Whole retinas were immunolabeled for both populations, and every labeled soma was plotted. For the MRGCs, we found a gene dosage effect for Dscam, with the Dscam+/− retinas showing smaller increases in cell number, clustering, and fasciculation. Curiously, Bax−/− retinas, showing numbers of MRGCs intermediate to those found in the Dscam−/− and Dscam+/− retinas, also had clustering and fasciculation phenotypes that were intermediate to retinas with those genotypes. DACs, by comparison, showed changes in both the Dscam−/− and the Bax−/− retinas that did not correlate with their increases in DAC number. The fasciculation phenotype in the Dscam−/− retina was particularly prominent despite only modest clustering. These results demonstrate that the somal clustering and fasciculation observed in the Dscam mutant retina are not unique to Dscam deficiency and are manifested distinctively by different retinal cell types. J. Comp. Neurol. 520:1349–1364, 2012.

Independent Genomic Control of Neuronal Number across Retinal Cell Types

The sizes of different neuronal populations within the CNS are precisely controlled, but whether neuronal number is coordinated between cell types is unknown. We examined the covariance structure of 12 different retinal cell types across 30 genetically distinct lines of mice, finding minimal covariation when comparing synaptically connected or developmentally related cell types. Variation mapped to one or more genomic loci for each cell type, but rarely were these shared, indicating minimal genetic coregulation of final number. Multiple genes, therefore, participate in the specification of the size of every population of retinal neuron, yet genetic variants work largely independent of one another during development to modulate those numbers, yielding substantial variability in the convergence ratios between pre- and postsynaptic populations. Density-dependent cellular interactions in the outer plexiform layer overcome this variability to ensure the formation of neuronal circuits that maintain constant retinal coverage and complete afferent sampling.