Comparison of electron microscopy and silver staining for the detection of the first entorhinal synapses to develop in the dentate gyrus

Summary The development of the projection from the entorhinal cortex to the dentate gyrus (perforant path) has been studied with the electron microscope. The projection was lesioned in baby rats 5–13 days old and the dentate gyrus examined after 6–72 hr. Degenerating synapses first appeared in small numbers in the dentate neuropile at 7 days and in greater numbers in progressively older animals. There was a sixteen-fold increase in the number of synapses undergoing degeneration between 7 and 13 days. This investigation provides a calibration for the reduced silver method which has been used to trace developing axons (Singh, 1977). By this method the first signs of Wallerian degeneration, after cutting axons in the perforant path, were seen in the dentate neuropile at 9 days.     

An electrophysiological analysis of chronic ethanol neurotoxicity in the dentate gyrus: Distribution of entorhinal afferents

Summary This study investigated the persistent effects of chronic ethanol consumption on the distribution of entorhinal afferents to stratum moleculare of the dentate gyrus. Rats were maintained on ethanol, or sucrose-containing liquid diets, for a period of 20 weeks but were withdrawn from the special diet for at least 8 weeks prior to acute electrophysiologic recordings. One-dimensional laminar analyses were obtained by stepping a microelectrode in 25 m increments across both the dorsal and ventral blades of the dentate gyrus and sampling the field potentials at each point. Current-source density (CSD) was calculated from the field potential data.Electrical stimulation of the angular bundle elicited a short-latency, negative field potential covering the outer 2/3 of the molecular layer. CSD analysis revealed a major current sink in stratum moleculare bounded by a major current source, localized to the hilus, granule cell layer, and proximal stratum moleculare, and a minor current source localized to the outer molecular layer. Chronic ethanol treatment resulted in (1) a significant shrinkage of the spatial extent of the current sink in stratum moleculare, (2) a significant reduction in the distance from the peak inward synaptic current to the granule cell layer and (3) no change in the distance from the proximal inversion point to the granule cell layer. Taken together, these results indicate a loss of entorhinal afferents in the outer molecular layer. Coupled with available anatomical evidence, these results suggest that chronic ethanol treatment produces a preferential loss of lateral entorhinal afferents to the dentate gyrus.Supported by funds from the Medical Research Service of the Veterans Administration, NIAAA Grant AA-00200 to Don W. Walker, NIAAA predoctoral fellowship AA-05153 to WCA and NINCDS grant NS-10229 to William Brownell     

The role of excitatory amino acid receptors in the propagation of epileptiform discharges from the entorhinal cortex to the dentate gyrus in vitro

Summary The relationship between epileptiform events in the medial entorhinal cortex (MEC) and the dentate gyrus was investigated using a slice preparation from rat brain. Simultaneous intracellular recordings were made from neurones in layer II of the MEC and neurones in the granule cell layer of the dentate gyrus (DGC). Epileptiform activity was induced by perfusion with Mg⁺⁺-free medium or GABA_A-receptor blockers. Epileptiform discharges in MEC cells were reflected on a one-to-one basis and at a latency of 1–3 ms by depolarizing events in DGC. The latter rarely gave rise to action potentials. Bath perfusion of the N-methyl-D-aspartate (NMDA) receptor blocker, 2-aminophosphonovalerate (2-AP5) abolished the Mg⁺⁺-free induced events in MEC cells and the corresponding depolarizations in the DGC but local application of 2-AP5 to the dentate gyrus only reduced the depolarizations. The non-NMDA-receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), whether bath applied or applied locally to the DG, had little effect on the cortical events but strongly reduced the depolarizations of the DGC. The discharges induced in MEC cells by GABA-blockers were reduced by bath applied 2-AP5 but abolished by CNQX. These effects were mirrored in the dentate gyrus by a reduction in the depolarizing events by 2-AP5 and their abolition by CNQX. Local application of either antagonist to the dentate gyrus reduced but did not abolish the depolarizations. Thus, Mg⁺⁺-free induced events in MEC depend mainly on enhanced NMDA-receptor activity, while events induced by bicuculline are primarily dependant on non-NMDA receptors. The depolarizing events in the DGC which reflect the activity in the EC are mediated by both types of receptor, although non-NMDA receptors play a much greater role.     

A reevaluation of excitatory amino acid-mediated synaptic transmission in rat dentate gyrus

1. Intracellular recordings were made from granule cells in combined slices of the hippocampus and parahippocampal cortex from adult Wistar rats. The neurons had a mean resting membrane potential (EM) of -85.1 +/- 0.7 (SE) mV, input resistance (Rin) of 30.9 +/- 1.5 M omega and action potential (AP) amplitude of 79.9 +/- 1.06 mV measured from the threshold potential. The threshold for AP generation was approximately 40 mV positive to EM. 2. The passive current-voltage relationship showed anomalous rectification, with Rin increasing by 34% on average at modest depolarizations. With large excursions of the EM (by +/- 30 mV or more), there was a marked fall in Rin. 3. With extracellular recording, a monophasic, positive-going field potential of 5-15 mV was evoked by stimulation of the perforant path (PP). Intracellularly, an excitatory postsynaptic potential (EPSP) of up to 40 mV in size was recorded. It was unusual to evoke an AP on orthodromic stimulation. Perfusion with picrotoxin (PTX, up to 20 microM) had small and variable effects on the EPSP, which implies that GABAergic inhibition does not play a major role. 4. Tonic depolarization reduced the EPSP. Hyperpolarization either had no effect or again decreased the EPSP. 5. The role of excitatory amino acid (EAA) receptor subtypes in mediation of the EPSP was investigated. Perfusion with the non-N-methyl-D-aspartate (NMDA) receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) caused a dose-dependent reduction of the EPSP with a shift to the right of the input-output relationship. The ED50 for this effect was approximately 2 microM. 6. In the presence of 5-10 microM CNQX a small component of the EPSP usually remained, which could be blocked by the NMDA receptor antagonist +/- 2-amino-5-phosphonovaleric acid (APV, 20-50 microM). This depolarizing component was markedly enhanced during perfusion with Mg2(+)-free medium. It increased in size and duration when the membrane was depolarized and decreased with hyperpolarization. These properties are consistent with the mediation of this potential via NMDA receptors. 7. These results indicate that NMDA receptors contribute to transmission at the synapse between the PP and the granule cell. This was confirmed by demonstrating that APV caused a small reduction in the size of the untreated EPSP and a shortening of the recovery phase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enhanced neurogenesis after transient global ischemia in the dentate gyrus of the rat

The dentate gyrus is one of the few areas of the mammalian brain where new neurons are continuously produced in adulthood. Certain insults such as epileptic seizures and ischemia are known to enhance the rate of neuronal production. We analyzed this phenomenon using the temporary occlusion of the two carotid arteries combined with arterial hypotension as a method to induce ischemia in rats. We measured the rate of cell production and their state of differentiation with a mitotic indicator, bromodeoxyuridine (BrdU), in combination with the immunohistochemical detection of neuronal markers. One week after the ischemic episode, the cell production in dentate gyrus was increased two- to threefold more than the basal level seen in control animals. Two weeks after ischemia, over 60% of these cells became young neurons as determined by colabeling with BrdU and a cytoplasmic protein (CRMP-4) involved in axonal guidance during development. Five weeks after the ischemia, over 60% of new neurons expressed calbindin, a calcium-binding protein normally expressed in mature granule neurons. In addition to more cells being generated, a greater proportion of all new cells remained in the differentiated but not fully mature state during the 2- to 5-week period after ischemia. The maturation rate of neurons as determined by the calbindin labeling and by the rate of migration from a proliferative zone into the granule cell layer was not changed when examined 5 weeks after ischemia. The results support the hypothesis that survival of dentate gyrus after ischemia is linked with enhanced neurogenesis. Additional physiological stimulation after ischemia may be exploited to stimulate maturation of new neurons and to offer new therapeutic strategies for promoting recovery of neuronal circuitry in the injured brain.     

Stability of synaptic density and spine volume in dentate gyrus of aged rats

The number of synapses per unit volume and per granule cell and the size of dendritic spines were studied in the dentate gyrus of Sprague-Dawley rats 6, 24, and 30 months of age. Neither synaptic density nor mean spine volume showed any age-related trends. An increase in granule cell packing density at 24 months and concomitant stability of the height of the granule cell layer is consistent with the idea that postnatal generation of granule cells may continue late into life. Possible explanations for the discrepancies in the literature regarding synaptic loss in this area include differences in morphometric techniques, age of animals used, regional differences within dentate gyrus, and sampling variability. Generalized synapse loss in the senescent rodent brain remains to be established.     

Ultrastructural evidence for bouton proliferation in the partially deafferented dentate gyrus of the adult rat

Summary A quantitative morphological study of the changes in the dentate gyrus molecular layer in response to the removal of perforant path afferents was made utilizing electron microscopic techniques. Alterations in 1. the population of remaining afferents, 2. glial cells, and 3. granule cell dendrites are reported. The major observation was an increase in intact bouton density in the region of denervation which began at 5 days post-lesion and continued through 11 days post-lesion, the longest post-lesion survival time studied.     

PKA and PKC enhance excitatory synaptic transmission in human dentate gyrus

cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) are two major modulators of synaptic transmission in the CNS but little is known about how they affect synaptic transmission in the human CNS. In this study, we used forskolin, a PKA activator, and phorbol ester, a PKC activator, to examine the effects of these kinases on synaptic transmission in granule cells of the dentate gyrus in human hippocampal slices using whole-cell recording methods. We found that both forskolin and phorbol ester increased the frequency of spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) but left the amplitude unaffected. Inactive forskolin and phorbol ester had no effect on sEPSCs in human dentate granule cells. Prior application of forskolin occluded the effects of phorbol ester on mEPSC frequency. Tetanic stimulation applied to the perforant path induced short-term depression in dentate gyrus granule cells. Both forskolin and phorbol ester significantly enhanced this short-term depression. Taken together, these results demonstrate that PKA and PKC are involved in up-regulation of excitatory synaptic transmission in human dentate granule cells, primarily by presynaptic mechanisms. In addition, the occlusion experiments suggest that the two kinases may share a common signal pathway.     

Synapses in the granule cell layer of the rat dentate gyrus: serial-sectioning study

Synaptic contacts on the granule cell somata as well as on their axon initial segments in the dentate gyrus of one juvenile 5-week-old rat and one adult 12-week-old rat were analyzed in an electron microscopic serial-sectioning study. In the dentate gyrus of the juvenile rat, somata of 17 granule cells were nearly completely reconstructed from a series of 183 serial sections, and the axon initial segments of 15 of these granule cells were traced in various lengths. On the other hand, in the dentate gyrus of the adult rat, somata of 31 granule cells were almost completely reconstructed from a series of 238 serial sections, and the axon initial segments of 23 of these granule cells were traced in various lengths. Both symmetrical and asymmetrical synapses were observed on the somata, whereas almost all synapses on the axon initial segments were of symmetrical type and asymmetrical synapses on the axon initial segments were rather exceptional. Although we confirmed two conclusions from previous random-section studies to some extent, that is, the superficial-to-deep gradient of synaptic densities on granule cell somata and the presence of a substantial number of asymmetrical synapses on granule cell somata (about 23% of total somatic synapses), the present serial-sectioning study clearly revealed that granule cells vary greatly with regard to the number of synapses on their somata (15–186 in a 5-week-old rat and 9–144 in a 12-week-old rat) and axon initial segments. The granule cells also differed in the proportion of somatic asymmetrical synapses to total synapses they received (0–44% in a 5-week-old rat and 0–60% in a 12-week-old rat). The results of the present study indicated that, when a relatively small number of granule cells are analyzed, one should take the heterogeneity of synaptic contacts on granule cells in number and type into consideration.     

Sprouting of remaining substance P-immunoreactive fibers in the monkey dentate gyrus following denervation from its substance P-containing hypothalamic afférents

This study analyzed the response of intrinsic substance P-immunoreactive fibers in the monkey dentate gyrus to disruption of the supramammillo-hippocampal projection. This projection normally forms a thin plexus of large, substance P-immunoreac tive terminals in the innermost portion of the dentate molecular layer and establishes exclusively asymmetric synapses with dendritic shafts and spines of dentate neurons. Conversely, substance P-containing terminals have never been observed in synaptic contact with granule cell bodies. Ten days after ipsilateral fimbria-fornix transection, the prominent band of large immunostained axons in the inner molecular layer of the ipsilateral fascia dentata disappeared. Four and five weeks following transection, however, some small, substance P-containing terminals were observed in the innermost portion of the dentate molecular layer and the granule cell layer. These terminals established exclusively symmetric synapses with the somata and proximal dendritic shafts of granule cells. These results suggest that, following transection of the hypothalamo-hippocampal fiber tract, presumptive intrinsic substance P-containing axons are capable of sprouting into the granule cell layer and the former termination field of the hypothalamic fibers. The symmetric synapses established with granule cell bodies and their proximal dendrites might indicate a shift from an extrinsic excitation to an intrinsic inhibition of granule cells following disruption of substance P-containing hypothalamic afferents.     

Sprouting of remaining substance P-immunoreactive fibers in the monkey dentate gyrus following denervation from its substance P-containing hypothalamic afferents

This study analyzed the response of intrinsic substance P-immunoreactive fibers in the monkey dentate gyrus to disruption of the supramammillohippocampal projection. This projection normally forms a thin plexus of large, substance P-immunoreactive terminals in the innermost portion of the dentate molecular layer and establishes exclusively asymmetric synapses with dendritic shafts and spines of dentate neurons. Conversely, substance P-containing terminals have never been observed in synaptic contact with granule cell bodies. Ten days after ipsilateral fimbria-fornix transection, the prominent band of large immunostained axons in the inner molecular layer of the ipsilateral fascia dentata disappeared. Four and five weeks following transection, however, some small, substance P-containing terminals were observed in the innermost portion of the dentate molecular layer and the granule cell layer. These terminals established exclusively symmetric synapses with the somata and proximal dendritic shafts of granule cells. These results suggest that, following transection of the hypothalamo-hippocampal fiber tract, presumptive intrinsic substance P-containing axons are capable of sprouting into the granule cell layer and the former termination field of the hypothalamic fibers. The symmetric synapses established with granule cell bodies and their proximal dendrites might indicate a shift from an extrinsic excitation to an intrinsic inhibition of granule cells following disruption of substance P-containing hypothalamic afferents.     

Cell proliferation in the dentate gyrus of the adult rat fluctuates with the light-dark cycle

This study measured cell proliferation in the hippocampal dentate gyrus in the adult rat at different times within a 12:12h light-dark cycle. The experiments were conducted in animals living in either a complex environment or in standard lab cages. A single dose of the thymidine analog 5-Bromo-2'-deoxyuridine (BrdU) was injected 2h before animals were sacrificed either 4, 11, 16, or 23h after the beginning of the light phase of the light-dark cycle (designated ZT0). In both studies, we found a significant increase in the number of BrdU-positive cells in the subgranular cell layer (SGZ) following BrdU administration at ZT9 and sacrifice at ZT11, compared to other circadian times examined. BrdU administration at ZT9 was timed to primarily identify proliferating cells that were in the S phase of the cell cycle during the light phase. Our results suggest that cell proliferation is enhanced either by sleep or by other variables coupled to the light phase of the circadian cycle.     

Effects of changes in extracellular potassium,magnesium and calcium concentration on synaptic transmission in area CA1 and the dentate gyrus of rat hippocampal slices

The dependence of stimulus-induced synaptic potentials on changes of extracellular ionic concentrations of potassium ([K⁺]_o 3, 5, 8 mM), magnesium ([Mg²⁺]_o 2, 4, 8 mM) and calcium [Ca²⁺]_o (2 mM and continuous lowering by washing with Ca²⁺-free solutions) was investigated in area CA1 and dentate gyrus of rat hippocampal slices. Field potentials (fps), [K⁺]_o and [Ca²⁺]_o were measured with double-barreled ion selective/reference microelectrodes. Paired pulse stimulation (interval 50-ms) was applied either to the lateral perforant path or to the Schaffer collaterals. Elevation of [K⁺]_o from 5 to 8 mM and of [Mg²⁺]_o from 2 to 8 mM depressed the rise of excitatory postsynaptic potentials, as well as the amplitude of population spikes. With elevation of [K⁺]_o, the effect was stronger in the dentate gyrus, while with elevation of [Mg²⁺]_o, the reduction was more pronounced in area CA1. During washout of Ca²⁺, synaptic potentials became reduced and finally depressed. The [Ca²⁺]_o at which synaptic transmission was blocked increased with higher [Mg²⁺]_o and decreased with a change of [K⁺]_o from 3 to 5 mM, whereas with an elevation of [K⁺]_o from 5 to 8 mM, it rose in area CA1 but was reduced in dentate gyrus. All ionic changes also affected frequency habituation and potentiation in paired pulse experimentes. In dentate gyrus, frequency habituation was reversed to frequency potentiation with moderate lowering of [Ca²⁺]_o and with elevation of [Mg²⁺]_o and [K⁺]_o. In contrast, in area CA1 frequency potentiation was reduced upon elevation of [K⁺]_o.     

Morphine augments excitatory synaptic transmission in the dentate gyrus through GABAergic disinhibition

The present study investigated the effect of morphine on synaptic transmission and long-term potentiation (LTP) in the dentate gyrus using rat hippocampal slice preparations. Field excitatory postsynaptic potential (fEPSP) and population spike (PS), evoked by stimulation of the perforant path, were recorded from the dentate molecular layer and the stratum granulosum, respectively. Following application of 10 microM morphine, PS amplitude increased gradually in 10 min and was eventually potentiated by approximately 50%. The phenomenon showed a concentration-dependent manner and was completely canceled by naloxone, a mu opioid receptor antagonist. Furthermore, morphine-induced PS augmentation was not detected in disinhibited hippocampal slices, which suggests that the inhibitory input to the dentate granule cells was required for the facilitatory effect of morphine. Neither fEPSP nor tetanus-induced LTP of PS was altered by morphine application. The data support the hypothesis that mu opioid receptor activity modulates inhibitory recurrent circuits in the dentate gyrus and thereby, indirectly plays a regulatory role for hippocampal excitatory neurotransmission.     

Medial septal projections to the dentate gyrus of the rat: electrophysiological analysis of distribution and plasticity

Summary Previous electrophysiological experiments in rabbits have suggested that medial septal stimulation activates dentate granule cells and evokes an associated negative field response at the granule cell layer, without an associated dendritic response. Anatomical studies have suggested that septal input to the granule cells may be to stratum moleculare, or close to the cell layer, or may not exist at all. The present experiments confirmed in rats anaesthetised with urethane that medial septal stimulation elicits single action potentials from cells in the granule layer. The associated negative field potential was maximal in the granule cell layer and there was no sign of a separate dendritic potential. The fibres responsible for this potential travel to the dorsal hippocampus in the fornix superior rather than the fimbria, taking the same course as the fibres which contribute to the dense cholinesterase staining just above the granule cell layer. Stimulation at 100 Hz for 1 s of either medial septal, or perforant path, input to the dentate granule cell layer produced long term potentiation of the subsequent evoked field responses to the stimulated pathway. The responses to the non-stimulated pathway were unchanged. Paired pulse stimulation produced both homosynaptic and heterosynaptic potentiation. These data suggest that medial septal input synapses close to granule cell bodies and produces a negative field potential which is a combination of dendritic and population spike potentials. Medial septal input also appeared to produce direct activation of hilar neurones, some of which may be basket cells or other interneurones. The data also show that long term potentiation is specific to this input, perhaps dependent on presynaptic mechanisms. Paired pulse potentiation, at least in the heterosynaptic case appears to depend on postsynaptic mechanisms.     

Molarless condition suppresses proliferation but not differentiation rates into neurons in the rat dentate gyrus

The dentate gyrus (DG) of the hippocampal complex is one of the few areas of the rodent brain where neurogenesis continues throughout adulthood. We investigated the effects of the molarless condition on cell proliferation, rate of differentiation into neurons in the subgranular zone of the DG, and plasma corticosterone levels. The molarless condition decreased cell proliferation in the DG and increased plasma corticosterone levels. Approximately 80% of newly generated cells differentiated into neurons and the remaining 20% of the cells differentiated into astrocytes. These ratios were not significantly different between control and molarless rats. In conclusion, the rates of neurogenesis and gliogenesis in the DG are suppressed by the molarless condition, and this suppression might be associated with the increased corticosteroid levels in molarless subjects.     

Sustained increase in adult neurogenesis in the rat hippocampal dentate gyrus after transient brain ischemia

It is known that the number of newly generated neurons is increased in the young and adult rodent subventricular zone (SVZ) and dentate gyrus (DG) after transient brain ischemia. However, it remains unclear whether increase in neurogenesis in the adult DG induced by ischemic stroke is transient or sustained. We here reported that from 2 weeks to 6 months after transient middle cerebral artery occlusion (MCAO), there were more doublecortin positive (DCX+) cells in the ipsilateral compared to the sham-control and contralateral DG of the adult rat. After the S-phase marker 5-bromo-2'-deoxyuridine (BrdU) was injected 2 days after MCAO to label newly generated cells, a large number of BrdU-labeled neuroblasts differentiated into mature granular neurons. These BrdU-labeled neurons survived for at least 6 months. When BrdU was injected 6 weeks after injury, there were still more newly generated neuroblasts differentiated into mature neurons in the ipsilateral DG. Altogether, our data indicate that transient brain ischemia initiates a prolonged increase in neurogenesis and promotes the normal development of the newly generated neurons in the adult DG.     

Action of norepinephrine in the dentate gyrus. II. Iontophoretic studies

Summary The effects of iontophoretic application of 1-norepinephrine (NE) and related drugs on granule cell responses evoked by a stimulus pulse applied to the medial perforant pathway were studied in anesthetized rats. Drugs were applied and responses recorded at successive dorso-ventral positions along the dendrites and at the cell body layer. 1. Reciprocal actions of alpha and beta receptors were revealed in the cell body region. The beta agonist isoproterenol decreased the population spike while the beta antagonist sotalol increased it. In contrast, the alpha agonists phenylephrine and clonidine increased the population spike whereas the alpha antagonist prazosin decreased it. The action of the drugs was rapid, dose dependent and reversible. NE itself had no effect when applied in the granule cell layer. 2. In contrast to the failure of NE to elicit a short term response, and in confirmation of a previous report (Neuman and Harley 1983), the prolonged application of NE in the granule cell layer produced a longterm enhancement of the population spike. However, this effect was also observed after the application of d-NE. 3. NE affected granule cell responses in the middle third of the dendrites where it reduced the evoked synaptic potential (ESP, current flow produced by excitatory synaptic activity) in a dose-dependent manner. Recordings taken simultaneously in the cell body region revealed a reduction of the population spike and no change in the ESP at the cell body layer (the positive-going ESP reflecting an outward current flow from the cell). In an attempt to delineate receptor specificity, a series of alpha and beta agonists and antagonists were applied to the mid-dendritic region. All drugs reduced the ESP in a manner similar to NE. Such lack of specificity in the action of NE has been previously reported in the spinal motoneuron (Engberg et al. 1976; Marshall 1983). The function of NE in the dentate gyrus is discussed in the light of these and previous results.     

Evidence for the sprouting of entorhinal afferents into the "hippocampal zone" of the molecular layer of the dentate gyrus.

Summary Fragments of the dentate gyrus containing portions of the stratum granulosum and the overlying stratum moleculare were isolated from their commissural, associational and hypothalamic inputs in 11 and 28-day-old rats. Six weeks later the distribution of the entorhinal afferents to the isolated portion of the stratum moleculare was determined autoradiographically by the injection of ³H-proline into the medial entorhinal area, and the appearance of the molecular layer was examined in Timm's sulfide silver preparations. It is evident from our material that under these circumstances the entorhinal afferents to the dentate gyrus can extend right up to the stratum granulosum and that the normal staining pattern that characterizes the hippocampal zone of the molecular layer in Timm's preparations may disappear and be replaced, in part, by a narrow zone of dense, mossy fiber-like staining.The simplest interpretation of these findings is that fibers from the medial entorhinal area are capable of sprouting into the deafferented hippocampal zone of the dentate gyrus. However, the observation that entorhinal afferents may occupy the entire thickness of the molecular layer does not, by itself, establish this point because in every case examined the whole molecular layer was shrunken. While this calls for some caution in the interpretation of the findings, our results suggest that there is no special barrier to the growth of entorhinal fibers and that the inner part of molecular layer of the dentate gyrus is capable of receiving afferents other than those normally present.This work was supported in part by grants NS-10943 and EY-01255 from the National Institutes of Health