Characterization of retinal ganglion cell, horizontal cell, and amacrine cell types expressing the neurotrophic receptor tyrosine kinase Ret.

We report the retinal expression pattern of Ret, a receptor tyrosine kinase for the glial derived neurotrophic factor (GDNF) family ligands (GFLs), during development and in the adult mouse. Ret is initially expressed in retinal ganglion cells (RGCs), followed by horizontal cells (HCs) and amacrine cells (ACs), beginning with the early stages of postmitotic development. Ret expression persists in all three classes of neurons in the adult. Using RNA sequencing, immunostaining and random sparse recombination, we show that Ret is expressed in at least three distinct types of ACs, and ten types of RGCs. Using intersectional genetics, we describe the dendritic arbor morphologies of RGC types expressing Ret in combination with each of the three members of the POU4f/Brn3 family of transcription factors. Ret expression overlaps with Brn3a in 4 RGC types, with Brn3b in 5 RGC types, and with Brn3c in one RGC type, respectively. Ret+ RGCs project to the lateral geniculate nucleus (LGN), pretectal area (PTA) and superior colliculus (SC), and avoid the suprachiasmatic nucleus and accessory optic system. Brn3a+ Ret+ and Brn3c+ Ret+ RGCs project preferentially to contralateral retinorecipient areas, while Brn3b+ Ret+ RGCs shows minor ipsilateral projections to the olivary pretectal nucleus and the LGN. Our findings establish intersectional genetic approaches for the anatomic and developmental characterization of individual Ret+ RGC types. In addition, they provide necessary information for addressing the potential interplay between GDNF neurotrophic signaling and transcriptional regulation in RGC type specification.

Molecular Basis for Vitamin A Uptake and Storage in Vertebrates.

The ability to store and distribute vitamin A inside the body is the main evolutionary adaptation that allows vertebrates to maintain retinoid functions during nutritional deficiencies and to acquire new metabolic pathways enabling light-independent production of 11-cis retinoids. These processes greatly depend on enzymes that esterify vitamin A as well as associated retinoid binding proteins. Although the significance of retinyl esters for vitamin A homeostasis is well established, until recently, the molecular basis for the retinol esterification enzymatic activity was unknown. In this review, we will look at retinoid absorption through the prism of current biochemical and structural studies on vitamin A esterifying enzymes. We describe molecular adaptations that enable retinoid storage and delineate mechanisms in which mutations found in selective proteins might influence vitamin A homeostasis in affected patients.

The Effect of PKCα on the Light Response of Rod Bipolar Cells in the Mouse Retina.

PURPOSE: Protein kinase C α (PKCα) is abundantly expressed in rod bipolar cells (RBCs) in the retina, yet the physiological function of PKCα in these cells is not well understood. To elucidate the role of PKCα in visual processing in the eye, we examined the effect of genetic deletion of PKCα on the ERG and on RBC light responses in the mouse. METHODS: Immunofluorescent labeling was performed on wild-type (WT), TRPM1 knockout, and PKCα knockout (PKC-KO) retina. Scotopic and photopic ERGs were recorded from WT and PKC-KO mice. Light responses of RBCs were measured using whole-cell recordings in retinal slices from WT and PKC-KO mice. RESULTS: Protein kinase C alpha expression in RBCs is correlated with the activity state of the cell. Rod bipolar cells dendrites are a major site of PKCα phosphorylation. Electroretinogram recordings indicated that loss of PKCα affects the scotopic b-wave, including a larger peak amplitude, longer implicit time, and broader width of the b-wave. There were no differences in the ERG a- or c-wave between PKCα KO and WT mice, indicating no measurable effect of PKCα in photoreceptors or the RPE. The photopic ERG was unaffected consistent with the lack of detectable PKCα in cone bipolar cells. Whole-cell recordings from RBCs in PKC-KO retinal slices revealed that, compared with WT, RBC light responses in the PKC-KO retina are delayed and of longer duration. CONCLUSIONS: Protein kinase C alpha plays an important modulatory role in RBCs, regulating both the peak amplitude and temporal properties of the RBC light response in the rod visual pathway.

Repeated exposure to chlorpyrifos leads to prolonged impairments of axonal transport in the living rodent brain.

The toxicity of the class of chemicals known as the organophosphates (OP) is most commonly attributed to the inhibition of the enzyme acetylcholinesterase. However, there is significant evidence that this mechanism may not account for all of the deleterious neurologic and neurobehavioral symptoms of OP exposure, especially those associated with levels that produce no overt signs of acute toxicity. In the study described here we evaluated the effects of the commonly used OP-pesticide, chlorpyrifos (CPF) on axonal transport in the brains of living rats using manganese (Mn(2+))-enhanced magnetic resonance imaging (MEMRI) of the optic nerve (ON) projections from the retina to the superior colliculus (SC). T1-weighted MEMRI scans were evaluated at 6 and 24h after intravitreal injection of Mn(2+). As a positive control for axonal transport deficits, initial studies were conducted with the tropolone alkaloid colchicine administered by intravitreal injection. In subsequent studies both single and repeated exposures to CPF were evaluated for effects on axonal transport using MEMRI. As expected, intravitreal injection of colchicine (2.5µg) produced a robust decrease in transport of Mn(2+) along the optic nerve (ON) and to the superior colliculus (SC) (as indicated by the reduced MEMRI contrast). A single subcutaneous (s.c.) injection of CPF (18.0mg/kg) was not associated with significant alterations in the transport of Mn(2+). Conversely, 14-days of repeated s.c. exposure to CPF (18.0mg/kg/day) was associated with decreased transport of Mn(2+) along the ONs and to the SC, an effect that was also present after a 30-day (CPF-free) washout period. These results indicate that repeated exposures to a commonly used pesticide, CPF can result in persistent alterations in axonal transport in the living mammalian brain. Given the fundamental importance of axonal transport to neuronal function, these observations may (at least in part) explain some of the long term neurological deficits that have been observed in humans who have been repeatedly exposed to doses of OPs not associated with acute toxicity.

Localization of protein kinase C isoforms in the optic pathway of mouse embryos and their role in axon routing at the optic chiasm.

Protein kinase C (PKC) plays a key role in many receptor-mediated signaling pathways that regulate cell growth and development. However, its roles in guiding axon growth and guidance in developing neural pathways are largely unknown. To investigate possible functions of PKC in the growth and guidance of axons in the optic chiasm, we first determined the localization of major PKC isoforms in the retinofugal pathway of mouse embryos, at the stage when axons navigate through the midline. Results showed that PKC was expressed in isoform specific patterns in the pathway. PKC-α immunoreactivity was detected in the chiasm and the optic tract. PKC-ßΙΙ was strong in the optic stalk but was attenuated on axons in the diencephalon. Immunostaining for PKC-ε showed a colocalization in the chiasmatic neurons that express a surface antigen stage specific embryonic antigen-1 (SSEA-1). These chiasmatic neurons straddled the midline of the optic chiasm, and have been shown in earlier studies a role in regulation of axon growth and guidance. Expression levels of PKC-ßΙ, -δ and -γ were barely detectable in the pathway. Blocking of PKC signaling with Ro-32-0432, an inhibitor specific for PKC-α and -ß at nanomolar concentration, produced a dramatic reduction of ipsilateral axons from both nasal retina and temporal crescent. We conclude from these studies that PKC-α and -ßΙΙ are the predominant forms in the developing optic pathway, whereas PKC-ε is the major form in the chiasmatic neurons. Furthermore, PKC-α and -ßΙΙ are likely involved in signaling pathways triggered by inhibitory molecules at the midline that guide optic axons to the uncrossed pathway.

Visual circuit assembly requires fine tuning of the novel Ig transmembrane protein Borderless.

Establishment of synaptic connections in the neuropils of the developing nervous system requires the coordination of specific neurite-neurite interactions (i.e., axon-axon, dendrite-dendrite and axon-dendrite interactions). The molecular mechanisms underlying coordination of neurite-neurite interactions for circuit assembly are incompletely understood. In this report, we identify a novel Ig superfamily transmembrane protein that we named Borderless (Bdl), as a novel regulator of neurite-neurite interactions in Drosophila. Bdl induces homotypic cell-cell adhesion in vitro and mediates neurite-neurite interactions in the developing visual system. Bdl interacts physically and genetically with the Ig transmembrane protein Turtle, a key regulator of axonal tiling. Our results also show that the receptor tyrosine phosphatase leukocyte common antigen-related protein (LAR) negatively regulates Bdl to control synaptic-layer selection. We propose that precise regulation of Bdl action coordinates neurite-neurite interactions for circuit formation in Drosophila.

Two projection streams from macaque V1 to the pale cytochrome oxidase stripes of V2.

In the primate visual cortex, areas V1 and V2 distribute information they receive from the retina to virtually all extrastriate cortex, parsing this information into dorsal and ventral streams. Therefore, understanding the connectivity between V1 and V2 is crucial to understand visual cortical processing. Cytochrome oxidase staining in V2 reveals a repeating pattern of pale-thick-pale-thin stripes. V1 sends parallel output pathways to distinct V2 stripes. Previous models proposed either three or two parallel V1-to-V2 pathways in macaque, but both models viewed the two pale stripes within a single stripe cycle as a single compartment. However, recent studies have suggested that the two pale stripes may be functionally distinct, and in marmosets they also differ anatomically in the laminar origin of projections they receive from V1. Here we have asked whether the two pale stripes are also anatomically distinct in macaque. We made small retrograde tracer injections in different pale stripe types. We found that while both pale stripes receive a predominant V1 input from layers 2/3, only one set of pale stripes (pale lateral) receives significant projections from layer 4B, while the other set (pale medial) receives few or no layer 4B projections. Moreover, different tracer injections in nearby pale stripe types revealed that 97-99% of layer 2/3 cells only project to a single pale stripe type. These results demonstrate that in macaque, the two pale stripes are anatomically distinct compartments, and support the notion of two distinct projection streams from V1 to the two pale stripes of V2.

[Activity of cytochrome oxidase in centers of tectofugal and thalamofugal channels of the visual system of pigeon Columba livia].

By using a histochemical method of determination of activity of cytochrome oxidase (CO), the level of metabolic activity in pigeons has been shown to be higher in centers of the tectofugal visual channel (pretectal nuclei: Pr, SP, SP/IPS, thalamic nucleus Rot, telencephalic entopallidum) than in centers of the thalamofugal visual channel (GLd, visual area of the hyperpallium Wulst). These data agree with the concept of the dominating role of the tectofugal visual channel in organization of the bird everyday behavior. The high CO activity is also characteristic of the mesencephalic structures (EM, isthmus nuclei: IMc, IPc, SLu) modulating transduction of visual information in tectum, Rot and GLd. Similar differences in the metabolic activities between two visual system channels have been shown earlier in reptiles, which indicates the evolutionary conservatism of the tectofugal visual channel among the sauropside amniotes. However, in pigeons the level of the CO activity in some GLd nuclei approaches that in Rot, which allows us to suggest a rise in birds of the role of the thalamofugal channel in processing of information necessary for performance of complex visual functions.

Perturbations of microRNA function in mouse dicer mutants produce retinal defects and lead to aberrant axon pathfinding at the optic chiasm.

BACKGROUND: During development axons encounter a variety of choice points where they have to make appropriate pathfinding decisions. The optic chiasm is a major decision point for retinal ganglion cell (RGC) axons en route to their target in order to ensure the correct wiring of the visual system. MicroRNAs (miRNAs) belong to the class of small non-coding RNA molecules and have been identified as important regulators of a variety of processes during embryonic development. However, their involvement in axon guidance decisions is less clear. METHODOLOGY/PRINCIPAL FINDINGS: We report here that the early loss of Dicer, an essential protein for the maturation of miRNAs, in all cells of the forming retina and optic chiasm leads to severe phenotypes of RGC axon pathfinding at the midline. Using a conditional deletion approach in mice, we find in homozygous Dicer mutants a marked increase of ipsilateral projections, RGC axons extending outside the optic chiasm, the formation of a secondary optic tract and a substantial number of RGC axons projecting aberrantly into the contralateral eye. In addition, the mutant mice display a microphthalmia phenotype. CONCLUSIONS: Our work demonstrates an important role of Dicer controlling the extension of RGC axons to the brain proper. It indicates that miRNAs are essential regulatory elements for mechanisms that ensure correct axon guidance decisions at the midline and thus have a central function in the establishment of circuitry during the development of the nervous system.

Cyclical expression of Na+/K+-ATPase in the visual system of Drosophila melanogaster.

In the first (lamina) and second (medulla) optic neuropils of Drosophila melanogaster, sodium pump subunit expression changes during the day and night, controlled by a circadian clock. We examined alpha-subunit expression from the intensity of immunolabeling. For the beta-subunit, encoded by Nervana 2 (Nrv2), we used Nrv2-GAL4 to drive expression of GFP, and measured the resultant fluorescence in whole heads and specific optic lobe cells. All optic neuropils express the alpha-subunit, highest at the beginning of night in both lamina and medulla in day/night condition and the oscillation was maintained in constant darkness. This rhythm was lacking in the clock arrhythmic per(0) mutant. GFP driven by Nrv2 was mostly detected in glial cells, mainly in the medulla. There, GFP expression occurs in medulla neuropil glia (MNGl), which express the clock gene per, and which closely contact the terminals of clock neurons immunoreactive to pigment dispersing factor. GFP fluorescence exhibited circadian oscillation in whole heads from Nrv2-GAL4+UAS-S65T-GFP flies, although significant GFP oscillations were lacking in MNGl, as they were for both subunit mRNAs in whole-head homogenates. In the dissected brain tissues, however, the mRNA of the alpha-subunit showed a robust daily rhythm in concentration changes while changes in the beta-subunit mRNA were weaker and not statistically significant. Thus in the brain, the genes for the sodium pump subunits, at least the one encoding the alpha-subunit, seem to be clock-controlled and the abundance of their corresponding proteins mirrors daily changes in mRNA, showing cyclical accumulation in cells.

The activation of ERK1/2 and p38 mitogen-activated protein kinases is dynamically regulated in the developing rat visual system.

Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases that play an instrumental role in signal transduction from the cell surface to the nucleus. These enzymes are major intracellular mediators of developmental events and recently have been shown to control also synaptic plasticity processes [Sweatt, J.D., 2004. Mitogen-activated protein kinases in synaptic plasticity and memory. Curr. Opin. Neurobiol. 14, 311-317; Thomas, G.M., Huganir, R.L., 2004. MAPK cascade signalling and synaptic plasticity. Nat. Rev. Neurosci. 5, 173-183]. Mammalian members of this family are extracellular signal-regulated kinases 1/2 (ERK 1/2), c-Jun amino-terminal kinases or stress-activated protein kinases (JNK/SAPKs) and p38 kinases (p38(MAPK)). At the level of the visual system, it has been demonstrated that the ERK pathway regulates developmental plastic processes at both retino-thalamic and thalamo-cortical level and that p38(MAPK) controls a peculiar form of long-term depression in the visual cortex [Di Cristo, G., Berardi, N., Cancedda, L., Pizzorusso, T., Putignano, E., Ratto, G.M., Maffei, L., 2001. Requirement of ERK activation for visual cortical plasticity. Science 292, 2337-2340; Naska, S., Cenni, M.C., Menna, E., Maffei, L., 2004. ERK signaling is required for eye-specific retino-geniculate segregation. Development 131, 3559-3570; Xiong, W., Kojic, L.Z., Zhang, L., Prasad, S.S., Douglas, R., Wang, Y., Cynader, M.S., 2006. Anisomycin activates p38 MAP kinase to induce LTD in mouse primary visual cortex. Brain Res. 1085, 68-76]. Here, as a first approach to gain more insight on the role of two MAPKs - ERK1/2 and p38(MAPK) - in visual system maturation, we characterized by western blot the regulation of their phosphorylation/activation in rat retina, superior colliculus and visual cortex, during postnatal development from birth to adult age. Our main results show that: (i) in the retina p38(MAPK) activation peaks at P4, and then, from P15 to P45, both ERK1/2 and p38(MAPK) phosphorylation increases; (ii) in the superior colliculus phosphorylation of both MAPKs increases between P4 and P15; (iii) in the visual cortex ERK1/2 phosphorylation increases from P15 to P45, while phosphorylation of p38(MAPK) increases starting from P4. The present data demonstrate a distinct regulation of the activation of ERK1/2 and p38(MAPK) in the three visual areas analyzed which occurs in temporal correlation with critical events for visual system maturation. These results suggest an important role for ERK1/2 and p38(MAPK) in the postnatal development of the rat visual system.

A role for S1P signalling in axon guidance in the Xenopus visual system.

Sphingosine 1-phosphate (S1P), a lysophospholipid, plays an important chemotactic role in the migration of lymphocytes and germ cells, and is known to regulate aspects of central nervous system development such as neurogenesis and neuronal migration. Its role in axon guidance, however, has not been examined. We show that sphingosine kinase 1, an enzyme that generates S1P, is expressed in areas surrounding the Xenopus retinal axon pathway, and that gain or loss of S1P function in vivo causes errors in axon navigation. Chemotropic assays reveal that S1P elicits fast repulsive responses in retinal growth cones. These responses require heparan sulfate, are sensitive to inhibitors of proteasomal degradation, and involve RhoA and LIM kinase activation. Together, the data identify downstream components that mediate S1P-induced growth cone responses and implicate S1P signalling in axon guidance.

Matrix metalloproteinase-9 is involved in the development and plasticity of retinotectal projections in rats.

OBJECTIVE: During postnatal development, retinotectal projections undergo a process of misplaced axon elimination, leading to a topographical matching between the retinal surface and the superior colliculus. Matrix metalloproteinases (MMPs) have been implicated in the development and plasticity of the nervous system. We studied the expression and role of MMPs during normal development of retinotectal projections and after monocular enucleation-induced plasticity. MATERIAL AND METHODS: Lister hooded rats at different postnatal ages received subpial ethylene vinyl acetate 40W implants to deliver an MMP inhibitor or vehicle to the superior colliculus. Animals received intraocular injections of horseradish peroxidase for anterograde tracing of ipsilateral projections. For immunoblotting and zymography, colliculi were removed without fixation. RESULTS: We observed the highest MMP activity in the first postnatal week, with decreasing activity thereafter. Monocular enucleation at postnatal day 10 yielded a rapid increase in MMP activity, 24 h following denervation of the contralateral colliculus. Importantly, inhibition of MMP activity in vivo induced a marked delay of axonal clustering along the medial aspect of colliculus. CONCLUSIONS: Our data indicate that MMPs are crucial in retinotectal development concurring to the fine tuning of topographical order and synaptic specificity of these connections.

Neuronal death in the lateral geniculate nucleus of young ferrets following a cortical lesion: time-course, age dependence and involvement of caspases.

In humans and many other mammalian species, the behavioural consequences of a cortical lesion tend to be milder when it occurs early in life, and there is evidence that an important factor contributing to the behavioural sparing in the young is the formation of new thalamo-cortical connections by thalamic neurons initially connected with the lesioned area. However, this plasticity may be hindered by the secondary death of many of these neurons owing to the elimination by the primary lesion of their trophic support from the cortex. With the long-term aim of preventing this neuronal death, we have here characterised its timing in the lateral geniculate nucleus of ferrets following lesions of the visual cortex on postnatal days 5, 10, 20 or 35. After the earliest lesions (P5 or P10), this cell death began rapidly and occurred synchronously, being maximal at 48 h and declining to zero over the next few days. Following later lesions the cell death began more slowly and continued for longer. The dying neurons contained activated caspase-3 and fragmented DNA and their number 2 days after a P5 lesion was reduced by the broad-band caspase inhibitor z-VAD.fmk. These experiments open the way for a concerted effort to enhance adaptive plasticity by neuroprotection in the hours or days following a cortical lesion.

Distribution of soluble guanylyl cyclase in rat retina.

The nitric oxide (NO)-cGMP pathway is implicated in modulation of visual information processing in the retina. Despite numerous functional studies of this pathway, information about the retinal distribution of the major downstream effector of NO, soluble guanylyl cyclase (sGC), is very limited. In the present work, we have used immunohistochemistry and multiple labeling to determine the distribution of sGC in rat retina. sGC was present at high levels in inner retina but barely detectable in outer retina. Photoreceptors and horizontal cells, as well as Müller cells, were immunonegative, whereas retinal ganglion cells exhibited moderate staining for sGC. Strong immunostaining was found in subpopulations of bipolar and amacrine cells, but staining was weak in rod bipolar cells, and AII amacrine cells were immunonegative. Double labeling of sGC with neuronal nitric oxide synthase showed that the two proteins are generally located in adjacent puncta in inner plexiform layer, implying paracrine interactions. Our results suggest that the NO-cGMP pathway modulates the neural circuitry in inner retina, preferentially within the cone pathway.

Optic nerve transection affects development and use-dependent plasticity in neocortex of the rat: Quantitative acetylcholinesterase imaging.

We investigated the effects of neonatal optic nerve transection on cortical acetylcholinesterase (AChE) activity in hooded rats during postnatal development and following behavioral manipulation after weaning. AChE reaction product was quantified on digitized images of histochemically stained sections in layer IV of primary somatic sensory, primary visual and visual association cortex. Rats with optic nerve transection were compared to sham-operated littermates. In all cortical regions of both types of animal, AChE reaction product was increased to peak 2 weeks after birth and decreased thereafter, reaching adult levels at the end of the third postnatal week. During postnatal development, reaction product in primary visual cortex was lower in rats deprived of retinal input than in sham-operated littermates and the area delineated by reaction product was smaller. However, optic nerve transection did not modify the time course of postnatal development or statistically significantly diminish adult levels of AChE activity. Behavioral manipulations after weaning statistically significantly increased enzyme activity in sham-operated rats in all cortical areas examined. Compared with cage rearing, training in a discrimination task with food reward had a greater impact than environmental enrichment. By contrast, in the rats with optic nerve transection enrichment and training resulted in statistically significantly increased AChE activity only in lateral visual association cortex. Our findings provide evidence for intra- and supramodal influences of the neonatal removal of retinal input on neural activity- and use-dependent modifications of cortical AChE activity. The laminar distribution of the AChE reaction product suggests that the observed changes in AChE activity were mainly related to cholinergic basal forebrain afferents. These afferents may facilitate the stabilization of transient connections between the somatic sensory and the visual pathway.

Tyrosine hydroxylase immunoreactivity in the developing visual pathway of the zebrafish.

We analyzed the distribution of tyrosine hydroxylase immunoreactivity in the central nervous zones involved in the processing of visual information during zebrafish ontogeny, employing a segmental approach. In the retina, we observed immunolabeled cells in the inner nuclear layer after hatching. From the juvenile stages onwards, some of these cells presented two immunolabeled processes towards the inner and outer plexiform layers of the retina, which are identified as interplexiform cells. In the adult zebrafish retina, we have identified two cellular types displaying immunoreactivity for tyrosine hydroxylase: interplexiform and amacrine cells. In the optic tectum, derived from the mesencephalon, no immunolabeled neurons were observed in any of the stages analyzed. The periventricular gray zone and the superficial white zone display immunostained neuropile from the end of fry life onwards. At the 30-day postfertilization, the tyrosine hydroxylase immunoreactive neuropile in the optic tectum presents two bands located within the retinorecipient strata and deeper strata, respectively. All diencephalic regions, which receive direct retinal inputs, show immunolabeled cells in the preoptic area, in the pretectum, and in the ventral thalamus from embryonic stages onwards. During the fry development, the immunolabeled neurons can be observed in the periventricular pretectum from 15-days postfertilization and in both the ventrolateral thalamic nucleus and suprachiasmatic nucleus from 30-days postfertilization. The transient expression of tyrosine hydroxylase is observed in fibers of the optic tract during fry and juvenile development. The existence of immunolabeled neuropile in the zebrafish retinorecipient strata could be related to the turnover of retinotectal projections.

Role of the calcium modulated cyclases in the development of the retinal projections.

Transmembrane isoforms of adenylate cyclases (AC) integrate a wide variety of extracellular signals from neurotransmitters to morphogens and can also regulate cAMP production in response to calcium entry. Based on observations in the barrelless mouse strain, the Adcy1 gene (AC1) was involved in the segregation of binocular retinal inputs. To determine the potential role of other AC isoforms we localized the Adcy genes in the visual centres during development, using in situ hybridization. Six different AC subtypes were found in the developing retinal ganglion cell layer (RGC; AC1, AC2, AC3, AC5, AC8, and AC9), and three AC subtypes were expressed in the central brain targets, the dorsal lateral geniculate nucleus (AC1 and AC8), the ventral lateral geniculate nucleus (AC2 and AC8) and the superior colliculus (AC1, AC2, AC8). Using a genetic approach we tested the role of the calcium modulated cyclases AC1, AC5 and AC8 for the segregation retinal fibres. Ipsilateral retinal axons remained exuberant in the AC1(-/-) mice, with overlapping retinal projections from both eyes in the superior colliculus and the visual thalamus. These abnormalities were similar to those of barrelless mouse mutants. No abnormalities were detectable in the AC5(-/-) or the AC8(-/-) mice. Similar abnormalities were noted in the single AC1(-/-) and the AC1/AC8 double-knockout mice (DKO). Thus, only AC1 is required for the maturation of the retinal axon terminals whereas AC5 and AC8 are not needed. The specificity of AC1's action is linked to its cellular localization in the RGCs and to its distinctive functional profile, compared with the other cyclases expressed in the same cells.

Input to V2 thin stripes arises from V1 cytochrome oxidase patches.

Parallel streams from the primary visual cortex (V1) to the second visual area (V2) are thought to mediate different aspects of visual perception in primates. One hypothesis is that the projection from cytochrome oxidase patches to thin stripes is responsible for color, whereas a separate pathway from interpatches to pale stripes mediates form. Recently, the notion of segregated pathways has been challenged by a report showing that patches and interpatches project equally to thin stripes. We made injections of a retrograde tracer, cholera toxin-B (CTB-Au), into macaque V2 thin stripes and counted the number of labeled cells in patches versus interpatches in layer 2/3. Analysis of eight thin-stripe injections showed that a mean of 81% of labeled cells were located in patches (defined as 33% of the surface area of V1). This result confirms that the projection to thin stripes arises predominately from patches. To assess the segregation of patch and interpatch projections, we injected CTB-Au in a pale stripe and horseradish peroxidase in an adjacent thin stripe. In both successful cases, interdigitated fields of labeled cells were present in V1. Less than 1% of cells were double-labeled, indicating that the populations of cells supplying thin stripes and pale stripes are quite independent. This finding means that different signals are likely conveyed by patches and interpatches to V2.