Immunohistochemical Analysis of the Developing Inner Plexiform Layer in Postnatal Rat Retina

Abstract

purpose. To investigate the development from early postnatal life to adulthood of neural cell processes that establish the circuitry of the inner plexiform layer (IPL). Emphasis was focused on the ontogeny of subsets of cGMP- and protein kinase C (PKC)-immunoreactive amacrine and bipolar cells.

methods. Paraformaldehyde-fixed postnatal and adult retinas were used for light microscopic analysis of immunohistochemical labeling of cryo-sections. Synthesis of cGMP in neural structures was achieved by means of an in vitro stimulation with a well-established nitric oxide donor.

results. In vitro stimulation of postnatal and mature retina with the nitric oxide donor results in NO-activated cGMP synthesis in subsets of bipolar and amacrine cells. NO-activated cGMP immunoreactivity is expressed in specific cell populations during the first postnatal week. Other cell subsets, consisting of amacrine cells and rod bipolar cells, express PKC immunoreactivity during postnatal development. An increasing number of rod bipolar cells start to exhibit cGMP labeling after eye opening, and a colocalization with PKC is established in adult retinas. Processes from these cell populations terminate in several sublaminas in the developing IPL, but cGMP- and PKC-labeled terminals appear to be confined to ON-lamina as the retina matures.

conclusions. The development of cGMP- and PKC-labeled fibers within the IPL appears to be in concert with events of neural differentiation and synaptogenesis. These results suggest that the nitric oxide/cGMP signaling pathway and PKC may participate in activity-dependent processes during development that establish the mature circuitry of synaptic contacts within the IPL. The presence of cGMP in mature rod bipolar cells suggests a role in the signal transduction of rod bipolar cell–AII amacrine cell pathway.

In addition to synaptic transmission and neuromodulation, accumulating evidence suggests that neurotransmitters may act as developmental mediators that influence the final architecture of the nervous system. ¹ One argument for such a role is the early expression of the transmitters, receptors, and transporter proteins within immature and developing cell populations. Acetylcholine,γ -aminobutyric acid (GABA), and glutamate represent transmitter candidates that have been suggested to be involved in early migration, process outgrowth, synaptogenesis, and dendritic pruning in the growing retina. ² Indeed, recent morphologic analysis reveal that immature rat retina display immunoreactivities to amino acid neurotransmitters ^{3 4} as well as to choline acetyltransferase, vesicular acetylcholine transporter, ⁵ and a variety of glutamate receptors. ^{6 7 8 9} Collectively, these studies imply that a fixed timing of transmitters and/or receptors expression during early postnatal development seems to be a prerequisite for the establishment of functional neural circuits present in the mature retina. The final shaping of the retina may include an upregulation of certain receptors ⁹ or transmitter downregulation ¹⁰ during the first postnatal weeks. 

One part of neural development results from a number of complex events that include neurite outgrowth and the establishment and shaping of synaptic contacts. Modifications of the neural cytoskeleton by phosphorylation have been attributed as an important event in neurite development, a process that involves the presence of protein kinase C (PKC). ¹¹ The novel messenger nitric oxide (NO) also may act in developmental events such as synaptic differentiation and establishment of synaptic connections ^{12 13} and maturation of neurons. ^{14 15} The enzyme responsible for NO production, nitric oxide synthase (NOS), has been identified by means of NOS immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry in almost all regions of the central nervous system, including the retina. ^{16 17 18 19 20} NO activates soluble guanylate cyclase in target cells, which results in a synthesis of cyclic guanosine 3′,5′-monophosphate (cGMP). ²¹ The cGMP content of a cell is thus a function of actions of NO in the tissue, which we have taken advantage of in this study. 

For a number of reasons, the rat retina was used for examining the potential functions of the NO/cGMP and PKC systems during development. First, it has been demonstrated that amacrine cells in rat retina display NADPH-diaphorase activity ²² as well as NOS immunoreactivity. ^{16 18 23} Ontogenetic studies show that NOS immunoreactive– and NADPH-labeled amacrine cells appear proximally in the inner nuclear layer around postnatal day 3 (P3), and labeling in the inner plexiform layer (IPL) can be distinguished at P11 to P12. ^{18 22} Second, bipolar cells and amacrine cell processes establish synaptic connections in the IPL during the second postnatal week. Immunohistochemically detectable accumulation of cGMP in cone bipolar cells of NO-stimulated rabbit retina ²⁴ and the selective staining of rod bipolar cells by PKC antibodies ²⁵ allow a morphologic analysis of the developing bipolar cell populations. Also, recent data show that bipolar and amacrine cells in the rabbit retina contain soluble guanylate cyclase ¹⁹ and different PKC isoforms. ²⁶ Finally, outgrowth of axons and dendrites during development include activation of intracellular messengers and phosphorylation of neural cytoskeletal molecules by PKC. 

The aim of this study was mainly to examine the immunohistochemical localization of NO/cGMP pathway and PKC in the bipolar cell population and in subsets of amacrine cells. Special attention was paid to the ontogeny of these cells and their role the development of the IPL in postnatal retina. A preliminary report of this work has appeared in abstract form. ²⁷  

Materials and Methods

Animals

Adult female and postnatal pigmented PVG rats were used throughout this study. The day of birth was designated P0. Adult animals were killed with carbon dioxide before decapitation, and young animals (newborn to 2 weeks old) were killed by decapitation. The eyes were enucleated and further processed depending on the experimental protocol. Experiment and animal care was according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the Swedish Government Committee for Animal Experimentation Ethics. 

NO Stimulation In Vitro

To obtain normal tissue for NO stimulation, the eyes were removed and the anterior segments were discarded. Neural retinas attached to the sclera were either free-floating or flat-mounted on a Millipore filter (Bedford, MA) and kept in CO₂-independent medium (Gibco, Paisley, UK) before being used. Retinas from P5, P10, P15, and P25 animals as well as from adult animals were examined. 

The specimens were preincubated for 30 minutes at 37°C with Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO) containing 1 mM isobutyl methylxanthine (IBMX; Sigma) and 0.1 mM zaprinast (May and Baker Ltd, Dagenham, UK). NO stimulation was performed by adding 1 mM sodium nitroprusside (Nipride; Roche, Basel, Switzerland) for 10 minutes, while control retinas were kept in the preincubation medium as described previously. ²⁸ Finally, all specimens were immersed directly in cold fixative (see below). 

Antibodies

For the visualization of cGMP enrichment in bipolar cells of NO-stimulated retinas, we used a well-characterized polyclonal sheep anti-cGMP antiserum. ²⁹ A polyclonal rabbit anti-human PKC antibody (Chemicon, Temecula, CA) was used as rod bipolar cell marker. ²⁵  

Immunohistochemistry

All specimens were fixed using 4% paraformaldehyde diluted in 0.1 M phosphate buffer (PB) for 4 hours at 4°C and then rinsed with PB overnight. After infiltration with 25% sucrose in PB for 2 to 3 days at 4°C, sections were cut at 10 to 12 μm. 

After cryo-sectioning the specimens were washed in phosphate-buffered saline containing 0.25% Triton X-100 (PBST) for approximately 1 hour at room temperature. The sections were incubated for 20 to 24 hours at 4°C either with anti-cGMP (1:3000) or anti-PKC (1:4000) diluted in PBST containing 1% bovine serum albumin (BSA; Sigma). After several washes in PBST, the sections were incubated with appropriate secondary antiserum for 45 to 60 minutes at room temperature. A Texas Red–conjugated donkey anti-sheep IgG (Jackson Laboratories, West Grove, CA) or fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgM/IgG (Southern Biotechnology Associates, Birmingham, AL) were used to visualize cGMP and PKC, respectively. The secondary antisera were diluted 1:200 in PBST containing 1% BSA. After rinses in PBST, the sections were coverslipped using anti-fading mounting medium (0.1% 1,4-phenylenediamine in 45 ml glycerin and 5 ml PBS). 

In double-labeling experiments, sections of NO-stimulated retinas were first incubated with anti-cGMP antibody, whereupon Texas Red–conjugated donkey anti-sheep IgG was applied. After several washes, incubation with anti-PKC antibody followed. This antibody was visualized with either an FITC-conjugated swine anti-rabbit IgG (Sigma) or FITC-conjugated goat anti-rabbit IgM/IgG. Incubation times, washes, and antibody concentration were as above. 

Microscopy and Figure Preparation

Micrographs were obtained using a digital camera system (model E400 microscope; Nikon, Tokyo, Japan; and model DEI-750 camera; Optronix Engineering, Goletta, CA), and Photoshop (Adobe Photosystems, San Jose, CA) was used for image handling. The pictures were taken using appropriate filter blocks for FITC and Texas Red; three color channels were handled separately and only the background level, contrast, and brightness of the entire image were changed. 

Results

During the first 2 weeks of postnatal development, the retina undergoes a considerable neural differentiation from immature neuroblastic layering to adult appearance. Although the P11 retina appears similar to the adult, synapses of amacrine cells and bipolar cells usually can be observed first at P11 and 13, respectively. ^{30 31} Because we are interested in the development of neural elements that are involved in the neural circuitry of the IPL, the development of bipolar and amacrine cells from P5 through P10, P15, and P25 to adult was investigated. When examined by cGMP immunohistochemistry after NO stimulation, flat-mounted specimens showed the best results, whereas free-floating retinas occasionally showed variations in cGMP labeling. This feature can probably be explained by an uneven exposure to the NO donor during stimulation because of folds in the tissue. cGMP- and PKC-labeled bipolar cells were identified according to the morphologic classification provided by Euler and Wässle. ²⁵  

cGMP Immunoreactivity

Adult Retina.

In sections of adult retinas that were exposed to the NO donor, most immunolabeling (both in terms of cell numbers and labeling intensity) occurred in a mixed population of rod bipolar cells and ON-center cone bipolar cells (Fig. 1A ). Typical morphologic features were as follows for cGMP immunoreactive rod bipolar cells in the adult. They generally displayed a strong intracellular immunolabeling throughout their entire length from their dendritic branches in the outer plexiform layer (OPL) to their axon terminals at the vitreal border of the IPL. Their round somata were located close to the OPL and a distinct dendritic tree ramified within the OPL. The axon projected through the retinal layers and terminated with large lobulated terminals in sublamina 5 of the IPL, at the ganglion cell layer. Each cGMP rod bipolar cell developed only some few synaptic end-bulbs, which all were closely associated with each other. 

We also observed flat, unlobulated axon terminals in sublaminas 3 and 4 that originated from somata located approximately at the same level or slightly proximal to the rod bipolar cell bodies somata (see Figs. 3D 4C ). This suggests that cGMP also accumulates in a subpopulation of cone bipolar cells, mainly of type 6 and perhaps also type 5. cGMP labeling also was observed in axon terminals with small lobulated terminals that were distinguished from the large terminals of rod bipolar cells (see Figs. 3D 4C ). This indicates the presence of type 8 cone bipolar cells. ²⁵ Some very few amacrine cells with terminals at the distal border (sublamina 1) of the IPL as well as large cells in the ganglion cell layer (GCL) also displayed cGMP immunoreactivity (see Figs. 1A 3D 4C ). The cGMP-immunoreactive processes of the IPL of the adult animal thus predominantly arise from a mixed population of both rod and type 6 cone bipolar cells. Type 5 and type 8 cone bipolar cells as well as amacrine cells and cells in the ganglion cell layer also contribute with cGMP-labeled fibers. Unstimulated retina incubated only with phosphodiesterase inhibitors, IBMX, and zaprinast, were devoid of cGMP labeling (not illustrated). 

Postnatal Retina.

In the developing neural retina, immunoreactivity to cGMP after NO stimulation was at P5 evident in subpopulations of immature nerve cells. Several cGMP-immunoreactive cells with elongated somata were found in the developing INL close to the immature IPL. At this stage, a thin process extended from the somata into the IPL, where two faint laminas were formed very close to the presumptive GCL (Fig. 1B) . 

At P10, numerous positive cell somata were found in the INL, and their shape varied from round to elongated. Most of the cell somata were scattered within the inner two thirds of the INL and displayed different intensities of immunolabeling. We observed fine processes from elongated and heavily labeled presumptive amacrine cell somata that formed 2 or 3 laminas in the middle of the IPL (Fig. 1C) . Dendritic arborizations from presumptive bipolar cells terminating into the OPL also were evident. 

At P15, cGMP immunoreactivity accumulated in response to NO donor stimulation in cell types similar to the ones seen in P10 retinas. With respect to the number of immunolabeled laminas in the IPL, a distinct difference was noticed. The IPL became thicker at P15 and up to four distinct laminas were clearly discernible, most likely derived from both bipolar and amacrine cGMP-positive cells (Fig. 1D) . A few large cells in the GCL with processes in the IPL also showed cGMP immunoreactivity. Immunolabeled cells with different labeling intensities were localized within the INL, but it was not possible to determine whether the number of labeled cells differed between P10 and P15 (Fig. 1D) . Presumptive somata of amacrine and bipolar cells were at this stage close to their mature position close to the IPL (Figs. 1D 4A) . Several of these cells projected radial through the retina and showed morphologic characteristics of bipolar cells. We observed several type 6 cone bipolar cells with typical flat axonal arborizations that stratified in sublamina 4 IPL (see Fig. 3A ). 

By P25, the cGMP-positive lamination within the IPL remained but displayed an intermediate appearance similar to both P15 and adult retinas. The two outermost sublaminas (1 and 2) were difficult to distinguish (Figs. 1E 3B 3C) , whereas sublaminas 3 and 4 were innervated by flat terminals derived from type 5 and type 6 cone bipolar cells. Axon terminals of the cGMP-labeled cone bipolar cells had dendritic processes in the OPL, and their somata now occupied the position seen in mature retinas. Numerous lobulated axon terminals of varying maturity (i.e., differences in size and labeling intensity) and were seen in sublamina 5 and in the GCL (Figs. 1E 4B) . Because of the morphology of their axon terminals and the identification of somata with prominent dendritic trees close to the OPL, we assume that this population represents rod bipolar cells. There were also a low number of cGMP-labeled amacrine cell somata in the proximal part of the INL (Fig. 1E) . Some of the amacrine cells were multistratifying, sending terminals in either the ON- or OFF-region of the IPL (see Figs. 3B 3C ). 

PKC Immunoreactivity

In the adult rat retina, the polyclonal PKC antibody used in this study labels a large population of rod bipolar cells with large lobulated axon terminals within the IPL. A small subset of somata in the GCL resembling amacrine cells also displayed PKC immunoreactivity (Figs. 2A 4B ). These observations are in good agreement with previous studies. ^{25 32}  

We were not able to observe PKC immunoreactivity in P0 retina. However, at P3, a set of PKC-labeled cells with large, round somata with processes were found in the GCL and vitreally in the neuroblastic layer (= immature inner nuclear layer), close to the developing IPL. A short and rather coarse process extended from the somata and formed two distinct laminas at the inner and outer limits of the IPL (Fig. 2B) . From the morphology and location of the labeled somata and their processes, we assume that this set of nerve cells is developing amacrine cells. After the first postnatal week, the labeling of the two laminas had increased and reached its optimum at P7 (Fig. 2C) . From then and onwards the labeling of the two laminas decreased considerably, and they were not easily distinguished later in development (P10–P15), even though amacrine cell somata were still distinguishable (Figs. 2D 2E 2F) . Although PKC-labeled laminas derived from amacrine cells were not observed in the adult retina, we have not excluded the possibility that the heavily labeled bipolar cells may have masked more weakly labeled laminas derived from amacrine cells. 

There were no PKC-immunoreactive rod bipolar cells during the first postnatal days. At P7, scattered elongated immature rod bipolar cell somata became distinguishable in approximately the middle of the developing INL (Fig. 2C) . Subsequently, there was a dramatic increase in the number of PKC-immunoreactive rod bipolar cells, which attained their adult positions and morphology during the second postnatal week (Figs. 2D 2E 2F) . Some somata had fine dendritic and axonal processes that projected toward the outer and inner plexiform layers, respectively (Fig. 2C) . From P7 to P10, axonal processes of bipolar cells extended vitreally and their terminals developed small lobulated terminals adjacent to the GCL. At P10, most of the somata were round, occupied their adult position in the outer part of the INL, and possessed a distinctly labeled dendritic process that terminated within the OPL (Fig. 2D) . Bipolar cells at P15 were similar to bipolar cells at P10, except that the axon terminals showed a more mature morphologic appearance (Figs. 2E 2F) . The most prominent morphologic change in the PKC-labeled rod bipolar cells from P10 to adult retina was the continuous development of the axonal end bulbs and dendritic trees within the OPL (Figs. 2A 2D 2E 2F 4A 4B 4C) . 

Double Labeling

Immunocytochemical double labeling of cGMP and PKC was used for a simultaneous examination of the bipolar cell populations at P15, P25, and in adults. We used two different FITC-conjugated secondary antisera to rule out any cross-reactions between the cGMP- and PKC-labeled structures. 

cGMP and PKC immunoreactivities were clearly seen to be localized to separate bipolar cell populations in the P15 retinas (see Fig. 4A ). Somata of the PKC-labeled rod bipolar cells and the cGMP-labeled cone bipolar cells were also distributed at separate levels in the INL. That both rod and cone bipolar cells were labeled was further demonstrated by the stratification patterns of their axon terminals in the IPL; PKC-labeled terminals distributed in the innermost sublamina (sublamina 5), whereas cGMP-labeled terminals were located in sublaminas 3 and 4 (Figs. 3A 4A ). At P25, rod bipolar cell terminals were lobulated, and some had a yellowish color, indicating a colocalization of PKC and cGMP labeling (Fig. 4B) . Although PKC has a membranous localization and cGMP is cytosolic, colabeled rod bipolar cell somata were observed close to the OPL (Fig. 4B) . The majority of the cGMP- and PKC-labeled somata were separate. Examinations of adult retinas showed that the number of colabeled bipolar cell somata and axon terminals increased with age from P25 and onwards (Fig. 4C) . Distinct colabeled axon terminals were easily observed (Fig. 4C) . Although most PKC-labeled bipolar cells also expressed cGMP labeling in the adult retina, it was possible to observe bipolar cells that only expressed cGMP labeling. 

Discussion

Our study supports previous findings that a mixed population of neurons in the inner retina can be labeled by an antiserum raised against cGMP after exposure to the NO donor. ^{24 33 34} Examinations of the developing retina demonstrated that neural processes from cGMP- and PKC-positive cells stratified within the IPL at the end of the first postnatal week and that additional cGMP-labeled laminas developed with age until the time for eye opening. However, this stratification pattern with four distinct laminas was not easily observed in the mature retina. 

Adult Retina

Morphologic Considerations.

Exogenous applied NO-stimulated cGMP synthesis in a mixed neural population consisting mainly of rod and cone bipolar cells and also minor populations of amacrine and ganglion cells. It seems reasonable to assume that NO-activated soluble guanylate cyclase in these cell populations, which are known to contain detectable levels of the enzyme in soluble form. ^{19 35} Colabeling experiments with PKC in the adult retina demonstrated that cGMP was synthesized in a large number of rod bipolar cells. We also observed cGMP accumulation in a subpopulation of cone bipolar cells, which agrees with observations in rabbit. ²⁴ The cGMP-labeled cone bipolar cells in the rat stratified in the inner part of the IPL and are considered to be ON-bipolar cells (see ^{Refs. 25} and ³⁶ ). Our morphologic data supports previous findings ^{37 38} of a NO-activated guanylate cyclase that catalyzes the synthesis of cGMP in ON-bipolar cells. 

Functional Considerations.

Rod and OFF-/ON- cone bipolar cells establish the through pathway of the mature retina, having dendrites in the OPL on photoreceptor terminals and their axon terminals in various laminas in the IPL. Axon terminals of cone bipolar cells establish synaptic contacts on ganglion cell dendrites within the IPL, whereas rod bipolar cells contact ganglion cells through gap junctions between AII amacrine cells and cone bipolar cells. ^{39 40} An interesting finding in the present study was the presence of cGMP labeling in both rod and cone bipolar cells of the ON pathway, indicating a role for NO/cGMP in two separate processes of signal transduction. First, ON-cone bipolar cells contact ganglion cells and are also coupled to AII amacrine cells by gap junctions. ^{36 41 42} A recent study by Mills and Massey ⁴³ showed that gap junction conductance between these cells can be modified by cGMP, and they presumed that cGMP acts on the bipolar cell side in these junctions. It is tempting to speculate that cGMP is chemically rectifying in these heterotypic gap junctions and modulates the intercellular flow from the AII amacrine cell to the bipolar cell. The second scenario might be that NO liberated from NOS-positive amacrine cells stimulates cGMP synthesis of the rod bipolar cells and activates cyclic nucleotide–gated channels, which results in a depolarization. Stimulation of metabotropic glutamate receptors hyperpolarize rod bipolar cells by a cGMP-inhibiting phosphodiesterase. ^{44 45 46} Thus, cGMP may be involved in the regulation of the glutamatergic responses in the ON-bipolar cell population. 

We also observed cGMP labeling of some large somata in the GCL, which we interpreted as ganglion cells. As mentioned above, transcripts for soluble guanylate cyclase have been detected in somata in the GCL, and previous work has proposed the presence of a cGMP-gated cation channel in ganglion cells. ⁴⁷  

Postnatal Retina

We here demonstrate that separate populations of the cGMP- and PKC-labeled bipolar cells can be observed in the inner retina at the end of the first postnatal week. At this time, cGMP was activated in type 5 and 6 ON-cone bipolar cells. Judging from the presence NOS ¹⁸ and NO-induced cGMP immunoreactivity, the ON-cone bipolar cell–AII circuit appears to be functional early in the immature retina. By modulating gap junction conductance between these cell types, a selective communication can be established between different neurons in the developing retina as well. Gap junction conductance between neocortical neurons during processes of circuit formation has been observed ⁴⁸ and is known to be modulated by NO/cGMP. ^{49 50}  

The numerous cGMP-positive amacrine cells terminating in both the ON and OFF layers also indicate that neural interactions can occur before the presence of visual stimuli and before mature neural connections have been established. Early neural signaling involving the NO/cGMP pathway has been described in immature cerebellar slices, ⁵¹ which suggests that interactions between specific cell populations can occur in developing nerve tissue and hence also infetal retina. As recently reviewed, ⁵² the mammalian nervous system has been suggested to employ an activity-dependent control of the development of neural connections. For instance, NO has been proposed to regulate activity-dependent stimulation of synaptogenesis in developing olfactory receptor neurons. ⁵³ Similar events also have become evident in the visual system, especially the development of retinotectal and retinogeniculate projections. When NO synthesis was inhibited in the visual system of embryonic chick, ⁵⁴ developmental refinement of eye-specific projections was inhibited. Further, inhibition of NOS in late postnatal ferrets, ⁵⁵ disrupted the formation of sublaminas in the lateral geniculate nucleus. We therefore interpret our data to suggest that the NO/cGMP system is important in the early postnatal retina, participating in activity-dependent events that occur during development. 

Axon terminals of PKC-labeled rod bipolar cells developed typical lobules before eye opening, and their maturation paralleled the formation of synapses within the IPL. ³¹ Although most PKC-positive rod bipolar cells appeared morphologically mature after eye opening, an increasing number of them began to express cGMP labeling from P25 into adulthood. This finding imply a role for PKC in morphologic differentiation and that PKC may have a role in adult rod bipolar cells as well. Interestingly, the ability to synthesize cGMP does not coincide with morphologic maturation of the PKC-positive rod bipolar cells. If we assume that the ability to synthesize cGMP is of functional importance, rod bipolar cells become functionally mature late in postnatal development. Thus, there seems to be a variability in maturation of bipolar cell in the rat retina: one that is completed before eye-opening (type 6 cone bipolar cells) and one that continues after P15 (rod bipolar cells). 

Subsets of Amacrine Cells Display cGMP and PKC Immunoreactivities during Development

This study shows that distinct subpopulations of PKC- and cGMP-labeled putative amacrine cells show specific postnatal immunostaining patterns that differ from the adult one. The morphologic features of these cells and their differentiation patterns indicate that they most likely correspond to amacrine cells. ⁵⁶ In addition, amacrine cells displaying immunoreactivity to subspecies of PKC have been observed in rabbit retina. ²⁶ We here show that the position and immunoreactivity for cGMP- and PKC-labeled amacrine cells are established during postnatal development, and that their optimal arborization patterns within the IPL differ in time. This is particularly illustrated by the PKC-positive amacrine cells, which are numerous during the first postnatal week and develop sublaminas within the IPL. However, the sublaminas are not clearly distinguished after 10 to 12 days of development and have thus started to decline before synaptogenesis. In contrast, the sublamination established by cGMP-labeled amacrine cell processes in the IPL appears to be correlated with events of synapse formation and dendritic organization of ganglion cells, which occurs at the end of the second postnatal week. ^{30 31 57 58}  

PKC and Neural Development

The expression of PKC in subsets of amacrine cells before eye opening is of particular interest and suggests that aspects of protein phosphorylation by PKC may be important in axon and/or dendrite development and may also participate in synaptogenesis during development. Elongation and branching of axons and/or dendrites are events in which phosphorylation of the cytoskeleton by a protein kinase is important for the regulation of neuronal morphology. Stimulatory effects on neural processes in various cell types can occur, either by inhibition or by activation of PKC. For example, PKC activation mediated via NMDA receptors stimulate neurite outgrowth from cerebellar granule cells. ⁵⁹ In cultured hippocampal neurons incubated with PKC inhibitors, initiation of neurite outgrowth and branching of axons was inhibited, whereas dendrites remained unaffected. ^{60 61} In this study, the appearance of PKC immunoreactivity is seen to coincide with the branching and positioning of certain amacrine cells and PKC-mediated effects thus appear important for the development of amacrine cell neurites. If the decreasing PKC immunoreactivity in amacrine cells is correlated with decreasing stability of the cytoskeleton can only be speculated on. 

The difference in the appearance of PKC in amacrine cells and bipolar cells shown in this study is worth noticing. PKC is present not only during development but also in adult rod bipolar cells. This illustrates that PKC is likely to fulfill different functions in different cells. From other studies it appears likely that PKC is in mature cells involved in activity-dependent events, such as long-term potentiation or some similar increase in synaptic efficacy. ⁶² The observations in this study suggest that PKC may participate both in cellular aspects during development of neural branches as well as in function(s) of mature axons and dendrites. At the different stages, PKC appears to encompass events such as phosphorylation of cytoskeleton components, transmitter release, and structural maintenance. 

 Supported by Grant 14X-2321 and 13012-01A from the Swedish Medical Research Council, the Foundation Fighting Blindness, Grant BMH 4-CT-1593 from EU BioMed2, the Knut and Alice Wallenberg, and the Crafoord and the Maggie Stephens Foundations.

 Submitted for publication February 3, 1999; revised July 8, 1999; accepted August 17, 1999.

 Commercial relationships policy: N.

 

Figure 1.

 

(A) through (E). Micrographs of NO-activated cGMP immunoreactivity in adult and postnatal retina. (A) Adult retina. cGMP accumulates in cone bipolar cells (arrows) with lobulated axon terminals in sublamina 5 of the inner plexiform layer (IPL) and in amacrine cells (arrowheads) in the adult retina. Note also labeling in sublaminas 3 and 4. (B) At P5 numerous cell somata in the neuroblastic layer (NBL) express cGMP labeling. Two faint laminae (arrows) derived from presumptive amacrine cells (arrowheads) are established within the IPL. (C) In P10 retina, the number of cGMP-labeled bipolar cells (arrows) and amacrine cells (arrowheads) has increased. The lamination within the IPL is distinct. (D) Somata of cGMP-labeled cone bipolar cells (arrows) and amacrine cells (arrowheads) occupy a mature position at P15. Within the IPL there is cGMP labeling in sublaminas 1 to 4. (E) The IPL of the P25 retina display mature characteristics with cGMP labeling predominantly in sublaminas 3 to 5. The cone bipolar cells (arrows) terminate in sublamina 4, and some have developed lobulated terminals that terminate in sublamina 5. Amacrine cells (arrowheads) with different labeling characteristics can be observed. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NBL, neuroblastic layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars, (A) through (E) 15 μm.

Figure 2.

 

(A) through (F) Micrographs showing the distribution of PKC-immunoreactive neurons in adult and postnatal retina. (A) Adult retina. PKC labeling is seen in rod bipolar cells (arrows). Few PKC-labeled amacrine cell somata (arrowheads) are observed in the GCL and INL. (B) At P3, PKC-labeled amacrine cells (arrowheads) have developed and establish 2 laminas within the IPL. (C) At P7, numerous PKC-labeled amacrine cell somata appear in the GCL and INL and their processes are distributed in two distinct laminas within the IPL. Somata of rod bipolar cells (arrows) have become discernible in the INL at this stage. (D) In the P10 retina, the PKC-labeled rod bipolar cells have attained their mature morphology and display lobulated terminals close to the GCL. A few less stained amacrine cell somata (arrowheads) are present in the GCL and INL. (E, F) P12 (E) and P15 (F) retinas show similar morphologies with numerous rod bipolar cells and few amacrine cell somata (arrowheads). Rod bipolar cell terminals close to the GCL become more distinguished at P15. Scale bars, (A) through (F) 15 μm.

Figure 3.

 

(A) through (D) Immunolabeling for cGMP in P15, P25, and adult retina showing various cell types that respond to NO-stimulation. (A) This picture was generated from less P15-stained retina to show the detailed morphology of labeled cells. Most but not all the cGMP-labeled cone bipolar cells (arrows) terminate in sublamina 4 of the IPL. Lobulated terminals are not seen at this stage. An amacrine cell (arrowhead) terminating in sublamina 1 is also seen (B, C). At P25, processes of most labeled amacrine cells (arrowheads) terminate in sublaminas 3 and 4 of the IPL. (D) Adult retina showing cGMP-immunolabeled bipolar cells (arrows) and their processes with lobulated terminals in the IPL, and less stained amacrine cells (arrowheads). A large labeled ganglion cell soma is seen in the GCL. Inset: large cGMP-labeled ganglion cell extending processes (arrowheads) into the IPL. Scale bars, (A) through (D) 15 μm.

Figure 4.

 

(A) through (C) Double labeling of cGMP and PKC immunoreactivity in P15, P25, and adult retina. (A) At P15, cGMP-labeled amacrine and bipolar cells (red) are clearly separated from the population of PKC-labeled cells (green). Note also the separate localization of cGMP- and PKC-labeled terminals in the IPL. (B) In the P25 retina, some bipolar cell somata in the OPL display yellow color that indicates colocalization of cGMP and PKC. Lobulated bipolar cell terminals (arrows) also were colabeled. (C) Several of the rod bipolar cell terminals (arrows) express both cGMP and PKC. Note also the well-developed dendritic arbors in the OPL. Abbreviations are as in Figure 1A . Scale bars, (A) through (C) 10 μm.

The authors thank Karin Arnér and Katarzyna Said for skillful technical expertise.