Fast-Transported Glycoproteins and Nonglycosylated Proteins Contain Sulfate

35SO4-labeled fast-transported proteins of bullfrog dorsal root ganglion neurons were separated by two-dimensional gel electrophoresis, and their mobilities were compared to similar species labeled with [3H]mannose or [3H]fucose. Fluorography revealed regions of poorly resolved, high molecular weight material, likely to represent sulfated proteoglycans, as well as many well resolved spots that corresponded in mobility to individual [35S]methionine-labeled fast-transported proteins. The majority of these well resolved spots appeared as families, previously identified as glycoproteins based on their labeling with sugars. Thus, sulfate can be a contributor to the carbohydrate side-chain charge that underlies microheterogeneity. The most heavily 35SO4-labeled species, however, corresponded to fast-transported proteins that were not labeled by either sugar. The relative acid labilities of 35SO4 associated with individual species cut from the gel confirmed the assignments of these spots as glycoproteins or nonglycoproteins. A group of spots intermediate in their acid lability was also detected, suggesting that some proteins may contain sulfate linked to carbohydrate as well as to amino acid residues.     

Calmodulin-Binding Proteins Within the Slow Phase of Axonal Transport in the Rabbit Vagus Nerve Per Ekstrom and Martin Kanje

Calmodulin-binding proteins (CBPs) in the rabbit vagus nerve were studied by means of calmodulin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis. The soluble fraction (10(5) g supernatant) of a nerve homogenate contained four CBPs with molecular weights of 44, 55, 91, and 93 kD, respectively. Slowly transported proteins were recovered in the vagus 3 days after injection of [35S]methionine into the nodose ganglion. Four labelled CBPs with molecular weights of 44, 55, 69, and 83 kD, respectively were found. The nodose ganglion contained two labelled CBPs, 44 and 55 kD. The 55-kD CBP was identified as tubulin after immunoblotting. In separate experiments it was also shown that bovine brain tubulin bound to the calmodulin column.     

[32P]orthophosphate and [35S]methionine label separate pools of neurofilaments with markedly different axonal transport kinetics in mouse retinal ganglion cells in vivo

Newly synthesized neurofilament proteins become highly phosphorylated within axons. Within 2 days after intravitreously injecting normal adult mice with [³²P]orthophosphate, we observed that neurofilaments along the entire length of optic axons were radiolabeled by a soluble³²P-carrier that was axonally transported faster than neurofilaments.³²P-incorporation into neurofilament proteins synthesized at the time of injection was comparatively low and minimally influenced the labeling pattern along axons.³²P-incorporation into axonal neurofilaments was considerably higher in the middle region of the optic axons. This characteristic non-uniform distribution of radiolabel remained nearly unchanged for at least 22 days. During this interval, less than 10% of the total³²P-labeled neurofilaments redistributed from the optic nerve to the optic tract. By contrast, newly synthesized neurofilaments were selectively pulse-labeled in ganglion cell bodies by intravitreous injection of [³⁵S]methionine and about 60% of this pool translocated by slow axoplasmic transport to the optic tract during the same time interval. These findings indicate that the steady-state or resident pool of neurofilaments in axons is not identical to the newly synthesized neurofilament pool, the major portion of which moves at the slowest rate of axoplasmic transport. Taken together with earlier studies, these results support the idea that, depending in part on their phosphorylation state, transported neurofilaments can interact for short or very long periods with a stationary but dynamic neurofilament lattice in axons.Special issue dedicated to Dr. Sidney Ochs.     

Glycosylation as a Criterion for Defining Subpopulations of Fast-Transported Proteins

The role carbohydrate residues may play in the sorting of newly synthesized fast-transported proteins during the initiation of fast axonal transport has been examined by identifying individual fast-transported glycoproteins that contain either or both fucose and galactose. [3H]Fucose or [3H]galactose was incorporated together with [35S]methionine in vitro in bullfrog dorsal root ganglia. Fast-transported proteins that accumulated proximal to a ligature on the spinal nerve were separated via two-dimensional gel electrophoresis, and 92 gel spots were analyzed quantitatively for the presence of 35S and 3H. Of these spots, 56 (61%) contained either or both fucose and galactose. Glycomoieties were generally associated with families of charged spots whose isoelectric points could be altered with neuraminidase treatment. Single spots tended to be unglycosylated and were unaffected by neuraminidase. The prevalence of glycoproteins was considerably greater in the higher-molecular weight range. Of the 55 spots analyzed with molecular weight greater than approximately 35,000 daltons, 89% were glycosylated, whereas only 19% of the 37 spots with lower molecular weight contained sugar moieties. When considered in light of previous studies in which similar subpopulations have been described, the current findings suggest that the presence or absence of glycomoieties may represent another criterion by which proteins are sorted during the initiation of fast axonal transport.     

Circadian regulation of phospholipid metabolism in retinal photoreceptors and ganglion cells

The neural retina is a key component of the vertebrate circadian system that is responsible for synchronizing the central circadian pacemaker to external light-dark (LD) cycles. The retina is itself rhythmic, showing circadian cycles in melatonin levels and gene expression. We assessed the in vivo incorporation of 32P-phosphate and 3H-glycerol into phospholipids of photoreceptor cells (PRCs) and retina ganglion cells (GCs) from chicks in constant illumination conditions (dark: DD or light: LL) over a 24-h period. Our findings showed that in DD there was a daily oscillation in 32P-labeling of total phospholipids synthesized in GCs and axonally transported to the brain. This metabolic fluctuation peaked during the subjective night (zeitgeber time [ZT] 20), persisted for several hours well into the subjective day and declined at subjective dusk (ZT 10-12). PRCs also exhibited an in vivo rhythm of 32P-phospholipid synthesis in DD. This rhythm peaked around ZT 22, continued a few hours into the day and declined by the end of subjective dusk. The major individual species labeled 1 h after 32P administration was phosphatidylinositol (PI) in both PRCs and GCs. Rhythmic phospholipid biosynthesis was also observed in DD after 3H-glycerol administration, with levels in GCs elevated from midday to early night. PRCs exhibited a similar rhythmic profile with the lowest levels of labeling during midnight. Phosphatidylcholine (PC) accounted for the individual species with the highest ratio of 3H-glycerol incorporation in both cell populations at all phases examined. By contrast, in LL the rhythm of 3H-glycerol labeling of phospholipids damped out in both cell layers. Our findings support the idea that, in constant darkness, the metabolism of retinal phospholipids, including their de novo biosynthesis, is regulated by an endogenous circadian clock.     

Slow transport of the cytoskeleton after axonal injury

The delivery of cytoskeletal proteins to the axon occurs by slow axonal transport. We examined how the rate of slow transport was altered after axonal injury. When retinal ganglion cell (RGC) axons regenerated through peripheral nerve grafts, an increase in the rate of slow transport occurred during regrowth of the injured axons. We compared these results to axonal injury in the optic nerve where no substantial regrowth occurs and found a completely different response. Slow transport was decreased approximately tenfold in rate in the proximal segment of crushed optic nerves. This decreased rate of slow transport was not induced immediately, but occurred about 1 week after injury. To explore whether a decrease in the rate of slow transport was induced when the regeneration of peripheral nerves was physically blocked, we examined slow transport in motor neurons after the sciatic nerve was transected and ligated. In this case, no change in the rate of the comigrating tubulin and neurofilament (NF) radioactive peaks were observed. We discuss how the changes in the rate of slow transport may reflect different neuronal responses to injury and speculate about the possible molecular changes in the expression of tubulin which may contribute to the observed changes. © 1992 John Wiley & Sons, Inc.     

Cellular Origin and Biosynthesis of Rat Optic Nerve Proteins: A Two-Dimensional Gel Analysis

High resolution 2DGE (two-dimensional gel electrophoresis) was used to characterize neuronal and glial proteins of the rat optic nerve, to examine the phases of intraaxonal transport with which the neuronal proteins are associated, and to identify the ribosomal populations on which these proteins are synthesized. Neuronal proteins synthesized in the retinal ganglion cells were identified by injecting the eye with L-[35S]methionine, followed by 2DGE analysis of fast and slow axonally transported proteins in particulate and soluble fractions. Proteins synthesized by the glial cells were labeled by incubating isolated optic nerves in the presence of L-[35S]methionine and then analyzed by 2DGE. A number of differences were seen between filamentous proteins of neurons and glia. Most strikingly, proteins in the alpha- and beta-tubulin region of the 2D gels of glial proteins were distinctly different than was observed for axonal proteins. As expected, neurons but not glia expressed neurofilament proteins, which appeared among the slow axonally transported proteins in the particulate fraction; significant amounts of the glial filamentous protein, GFA, were also labeled under these conditions, which may have been due to transfer of amino acids from the axon to the glial compartment. The fast axonally transported proteins contained relatively large amounts of high-molecular-weight acidic proteins, two of which were shown to comigrate (on 2DGE) with proteins synthesized by rat CNS rough microsomes; this finding suggests that rough endoplasmic reticulum may be a major site of synthesis for fast transported proteins. In contrast, the free polysome population was shown to synthesize the principal components of slow axonal transport, including tubulin subunits, actin, and neurofilament proteins.     

Acrylamide Neuropathy: Changes in the Composition of Proteins of Fast Axonal Transport Resemble Those Observed in Regenerating Axons

Proteins conveyed by fast axonal transport along sensory and motor axons of rat sciatic nerve were labelled with L-[35S]methionine and characterized by one- and two-dimensional electrophoresis on polyacrylamide gels, followed by fluorography. Nerves from normal or bis-acrylamide-treated animals were compared with nerves from acrylamide-treated animals and nerves regenerating after a crush axotomy. In both sensory and motor axons significant changes in the pattern of labelled bands on one-dimensional gels occurred after 10 days of acrylamide treatment (50 mg/kg daily, i.p.). These changes resembled those seen in regenerating axons, but were less pronounced. No changes were detectable after shorter periods of treatment, even though the onset of the neuropathy, assessed by a behavioral test, occurred on days 4-6 of treatment. Two-dimensional separations of the labelled proteins revealed increased labelling of growth-associated protein 43 in acrylamide-treated animals, but again this was less pronounced than in regenerating nerves. Acrylamide treatment induces changes in composition of fast-transported protein that are qualitatively similar to those seen after axotomy. Since these changes are not detectable until the neuropathy is advanced, it is unlikely that they are causative factors. Instead, they are most likely a result of the cell body reaction previously observed in acrylamide intoxication, a reaction that resembles that produced by axotomy.     

Histofluorescence and ultrastructural observations of small intensely fluorescent (SIF) cells in the superior sympathetic ganglion of the guinea pig

Summary Amine-containing small intensely fluorescent (SIF) cells are ubiquitous in vertebrate sympathetic ganglia and, in some species, SIF cells have been identified as interneurons. The hypothesis proposed in this study is that SIF cells in superior sympathetic ganglia of the guinea pig function as interneurons, with efferent connections characteristic for the species. Fluorescence (catecholamine) microscopy and 5-hydroxydopamine marker for electron microscopy were used to study SIF cells, their processes and connections in this ganglion.Brightly fluorescent fibers were seen attached to virtually all SIF cells, and were of two types. The first type, single or arranged in cords, interconnected elements of the SIF-cell system; these apparent linkages joined individual SIF cells as well as adjacent clusters. The electron-microscopic evidence for synaptic contacts between SIF cells warrants the claim that integrated action is a presumed function of these elements. The second type of SIF-cell process was generally of greater length. These individual, branching fibers made presumed connections with dendrites of most principal ganglionic neurons. This arrangement suggested by histofluorescence preparations was confirmed by electron microscopy to involve synaptic connections, and the postsynaptic element was shown to be continuous with the perikaryon of the principal ganglionic neuron. Ultrastructural evidence that collections of dense-cored vesicles occur within processes of both principal ganglionic neurons and SIF cells, in proximity to unsheathed portions of plasma membrane, leads to the conclusion that interstitial diffusion of catecholamine from both may occur; the finding of SIF cell processes adjacent to fenestrated blood vessels suggests that catecholamine may also be transported through capillaries.     

Effect of Presynaptic P2 Receptor Stimulation on Transmitter Release

Because ATP is degraded to adenosine, its effect could be mediated by both P1 and P2 receptors. Hence, the actions of an ATP analogue, resistant to enzymatic breakdown (alpha, beta-methylene ATP), were studied on the resting and electrically evoked release of radioactivity from longitudinal muscle strips of guinea pig ileum, preloaded either with [3H]choline or with [3H]noradrenaline. Their effects were compared with the actions of adenosine and ATP. Although adenosine and ATP markedly decreased the [3H]acetylcholine release evoked by field stimulation, alpha,beta-methylene-ATP, a potent and selective agonist of P2x receptors, enhanced this release. However, 2-methyl-2-thio-ATP, an agonist of the P2y receptors, neither enhanced nor inhibited the [3H]-acetylcholine release. 8-Phenyltheophylline, an antagonist of P1 receptors, increased the stimulation-evoked release of acetylcholine, indicating that the release of acetylcholine is tonically controlled by endogenous adenosine via P1 receptors. When alpha,beta-methylene-ATP and 8-phenyltheophylline were added together, their potentiating effect on the acetylcholine release proved to be additive. Because alpha,beta-methylene-ATP failed to antagonize the presynaptic effect of adenosine on P1 purinoceptors, it seems very likely that its effect to enhance transmitter release is mediated via separate receptors, i.e., via P2x receptors, located on the axon terminals. Similarly, the stimulation-evoked release of [3H]noradrenaline was enhanced slightly by alpha,beta-methylene-ATP. Our results suggest that both cholinergic and noradrenergic axon terminals are equipped with P2 receptors through which the stimulation-evoked release of transmitter can be modulated by ATP in a positive manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exogenous nitric oxide causes collapse of retinal ganglion cell axonal growth cones in vitro

We show that nitric oxide (NO) from applied NO-donating chemicals induces collapse of ganglion cell axonal growth cones extending from explants of tadpole retina in culture. Peroxynitrite, a neurotoxic product of NO and superoxide reaction, did not induce collapse, and oxyhemoglobin, which binds NO, blocked the highly effective collapsing activity of the NO donor S-nitrosocysteine. Membrane-permeable analogs of cyclic guanosine monophosphate had no collapsing activity. Inhibitors of NO synthase did not induce collapse. NO is a potential retrograde messenger through which postsynaptic neurons signal to their inputs to modify synaptic efficacy following NMDA receptor activation. Our results suggest a role for NO as such a messenger during development of the retinotectal projection. © 1996 John Wiley & Sons, Inc.     

Substance P Synthesis and Transport in Explants of Nodose Ganglion/Vagus Nerve: Effects of Double Ligation, 2-Deoxyglucose, Veratridine, and Ouabain

Substance P (SP), the widely distributed undecapeptide, is synthesized in cell bodies of vagal sensory ganglia and transported bidirectionally toward the CNS and thoracic and abdominal viscera. In explants of the guinea pig inferior (nodose) vagal sensory ganglion and attached 2 cm of distal vagus nerve, SP is synthesized within the ganglion and transported predominantly distally. The quantity of distal transport is similar to that observed in vivo and provides an index of ongoing synthesis within the ganglion. In this report, the model is further characterized. Double ligation of the explant distal to the ganglion demonstrates that all the transported peptide is derived from the ganglion; there is no evidence of intraaxonal processing of peptide precursor. Approximately 50% of the peptide is in a rapid transport vs. an apparent stationary compartment. Not only transport, but also synthesis, of SP was blocked by 20 mM colchicine. Ongoing SP biosynthesis is dependent on a nutrient medium [medium 199 (M-199)] and is partially inhibited with added fetal bovine serum (FBS; 10%): total explant content in M-199/FBS vs. M-199, 1,785 +/- 101 (n = 8) vs. 2,254 +/- 123 pg (n = 9); p less than 0.02. Addition of 2-deoxyglucose (2-DG) decreased both total SP synthesis and transport (total explant content for 2-DG vs. control, 986 +/- 94 vs. 1,391 +/- 111; p less than 0.05). Medium supplemented with glucose to a final concentration of 600 mg/100 ml or with glucose (300 mg/100 ml) with or without insulin (50 ng/ml) did not alter explant SP content or transport. Veratridine (5 X 10(-6) M) inhibited both SP synthesis and transport; ouabain (10(-4) M) also inhibited synthesis, but less so transport. Tetrodotoxin reversed the effects of veratridine. These studies demonstrate the usefulness of this model, which can examine factors regulating both synthesis and transport of sensory neuropeptides in vitro. The results suggest that SP synthesis/transport may be under tonic inhibition, perhaps by both neural and humoral mechanisms.     

Misguidance and modulation of axonal regeneration by Stat3 and Rho/ROCK signaling in the transparent optic nerve

The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological blockade of ROCK, a key signaling component for myelin-associated growth inhibitors, reduced axonal U-turns and potentiated AAV2.Stat3-ca-induced regeneration. Similar results were obtained after the sustained delivery of CNTF in the axotomized retina. These results show the important role of Stat3 in the activation of the neuronal growth program for regeneration, and they reveal that axonal misguidance is a key limiting factor that can affect long-distance regeneration and target interaction after trauma in the CNS. The correction of axonal misguidance was associated with improved long-distance axon regeneration in the injured adult CNS.     

Altered Axonal Transport of Cytoskeletal Proteins in the Mutant Diabetic Mouse

Polypeptides in the motor axons of the sciatic nerve in 120-day-old normal and diabetic mice C57BL/Ks (db/db) were labeled by injection of [35S]methionine into the ventral horn of the spinal cord. At 8, 15, and 25 days after the injection, the distribution of radiolabeled polypeptides along the sciatic nerve was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four major radiolabeled polypeptides, tentatively identified as actin, tubulin, and the two lightest subunits of the neurofilament triplet, were studied in both diabetic and control mice. In the diabetic animals, the two polypeptides identified as actin and tubulin showed a reduction of average velocity of migration along the sciatic nerve, resulting in a higher fraction of radioactivity in the proximal part of the sciatic nerve, whereas the front of radioactivity (advancing at maximal velocity) moved at a normal rate. In contrast, both the average and maximal velocities of the two neurofilament subunits were slower in the diabetic mice than in the control mice. These results indicate that the axonal transport of the cytoskeletal proteins is differentially affected in the course of diabetic neuropathy, and may suggest that the impairment concerns mainly the proteins carried by the slowest component of axonal transport.     

Effects of exogenous triiodothyronine on fast axonal transport during tadpole metamorphosis

Bullfrog tadpoles at metamorphic stages V, X and XVIII were immersed in 25 nM triiodothyronine (T₃) to assess whether the 4–5 fold increase in fast axonal transport (FAxT) previously observed during this span of spontaneous metamorphosis (1) could be mimicked by precocious application of thyroid hormone. The trend initially observed was for T₃ to stimulate [³⁵S]methionine incorporation into lumbar DRG and inhibit incorporation in tail DRG. Both effects, however, appeared to be exerted primarily on satellite cells rather than neurons since most of the T₃-induced changes in DRG were of a similar magnitude to those in the respective nerve trunks. Findings consistent with this observation resulted from use of the retrogradely transported lectin, ricin₁₂₀, to determine the proportion of DRG incorporation occurring in neurons. When incorporation of [³⁵S]methionine in lumbar DRG neurons was examined, T₃ had no stimulatory effect at any of the metamorphic stages examined. When FAxT was assessed as [³⁵S]protein accumulating proximal to a nerve trunk ligature, and expressed as a percentage of newly-synthesized protein in lumbar DRG neurons, no stimulatory effect of T₃ was detected. The question remains whether the changes in FAxT in peripheral neurons observed during spontaneous metamorphosis may be induced by circulating hormones other than T₃ or are secondary to changes in the target tissues. Special issue dedicated to Dr. Marion E. Smith.     

ROCK2 is a major regulator of axonal degeneration,neuronal death and axonal regeneration in the CNS

The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the central nervous system. Several studies using pharmacological Rho-associated protein kinase (ROCK) inhibitors have shown positive effects on neurite regeneration and suggest additional pro-survival effects in neurons. However, as none of these drugs is completely target specific, it remains unclear how these effects are mediated and whether ROCK is really the most relevant target of the pathway. To answer these questions, we generated adeno-associated viral vectors to specifically downregulate ROCK2 and LIM domain kinase (LIMK)-1 in rat retinal ganglion cells (RGCs) in vitro and in vivo. We show here that specific knockdown of ROCK2 and LIMK1 equally enhanced neurite outgrowth of RGCs on inhibitory substrates and both induced substantial neuronal regeneration over distances of more than 5 mm after rat optic nerve crush (ONC) in vivo. However, only knockdown of ROCK2 but not LIMK1 increased survival of RGCs after optic nerve axotomy. Moreover, knockdown of ROCK2 attenuated axonal degeneration of the proximal axon after ONC assessed by in vivo live imaging. Mechanistically, we demonstrate here that knockdown of ROCK2 resulted in decreased intraneuronal activity of calpain and caspase 3, whereas levels of pAkt and collapsin response mediator protein 2 and autophagic flux were increased. Taken together, our data characterize ROCK2 as a specific therapeutic target in neurodegenerative diseases and demonstrate new downstream effects of ROCK2 including axonal degeneration, apoptosis and autophagy.     

Intraganglionic portal sinus located between small intensely fluorescent (SIF) cells and principal ganglionic neurons in the inferior mesenteric ganglion of the guinea pig

Summary The vascular system in the inferior mesenteric ganglion of the guinea pig was studied to clarify the transport pathway of transmitters released by the small intensely fluorescent (SIF) cells to the principal ganglionic neurons. Reconstruction of about 1500 1-m-thick serial sections of the ganglion demonstrated its portal system. SIF cells were tightly packed and formed two or three clusters under the capsule of the ganglion. Branches from the inferior mesenteric artery ran directly toward these clusters and broke up into a number of coiled and looped sinusoid capillaries among the SIF cells. They then drained into a large sinus surrounding the clusters in the ganglion. Capillaries were derived from this sinus and ramified among the principal ganglionic neurons. After supplying the neurons, these vessels drained into veins surrounding the ganglion. Therefore, as we observed two distinct groups of capillaries, we call this sinus the intraganglionic portal sinus. All the transmitters secreted from the SIF cells are collected into this intraganglionic portal sinus and are then conveyed through the capillaries to the principal ganglionic neurons.     

Transport of multiple nicotinic acetylcholine receptors in the rat optic nerve: high densities of receptors containing alpha6 and beta3 subunits

Neuronal nicotinic acetylcholine receptors (nAChRs) are abundant in the rat retina and at least seven heteromeric subtypes have been detected. Axons of retinal ganglion cells form the optic nerve and innervate areas of the brain important for visual processing, including the lateral geniculate nucleus, the superior colliculus, and the pretectal nucleus. Development of eye-specific layers in these projection areas are dependent upon retinal waves which are initially mediated by nAChRs [ Feller et al. , Science 272 (1996), 1182 ; Penn et al. , Science 279 (1998), 2108 ; Bansal et al. , J. Neurosci. 20 (2000), 7672 ]. Unilateral eye-enucleation studies in the rat indicate that nAChRs are on the terminals of optic nerve axons, where they may mediate influences of acetylcholine on visual pathways. In this study, we use radioligand binding and immunoprecipitation with subunit-selective antibodies to investigate the subunit composition of nAChRs in the rat optic nerve. We found multiple nAChR subtypes in the optic nerve, all of which contain the β2 subunit. Most of these receptors are mixed heteromeric subtypes, composed of at least three different subunits. Included among these subtypes is the highest percentage and density of α6- and β3-containing nAChRs of any area of the rat CNS that has been reported.     

A Battery of Cell- and Structure-specific Markers for the Adult Porcine Retina

The pig is becoming an increasingly used non-primate model in experimental studies of human retinal diseases and disorders. The anatomy, size, and vasculature of the porcine eye and retina closely resemble their human counterparts, which allows for application of standard instrumentation and diagnostics used in the clinic. Despite many reports that demonstrate immunohistochemistry as a useful method for exploring neuropathological changes in the mammalian central nervous system, including the pig, the porcine retina has been sparsely described. Hence, to facilitate further immunohistochemical analysis of the porcine retina, we report on the successful use of a battery of antibodies for staining of paraformaldehyde-fixed cryosectioned retina. The following antibodies were evaluated for neuronal cells and structures: recoverin (cones and rods), Rho4D2 (rods), transducin-γ (cones), ROM-1 (photoreceptor outer segments), calbindin (horizontal cells), PKC-α (bipolar cells), parvalbumin (amacrine and displaced amacrine cells), and NeuN (ganglion cells and displaced amacrines). For detecting synaptic connections in fiber layers, we used an antibody against synaptobrevin. For detecting retinal pigment epithelium, we studied antibodies against cytokeratin and RPE65, respectively. The glial cell markers used were bFGF (Müller cells and displaced amacrine cells), GFAP (Müller cells and astrocytes), and vimentin (Müller cells). Each staining effect was evaluated with regard to its specificity, sensitivity, and reproducibility in the identification of individual cells, specific cell structures, and fiber layers, respectively. The markers parvalbumin and ROM-1 were tested here for the first time for the porcine retina. All antibodies tested resulted in specific staining of high quality. In conclusion, all immunohistochemical protocols presented here will be applicable in fixed, cryosectioned pig retina. (J Histochem Cytochem 58:377–389, 2010)