Characteristics of Rod Regeneration in a Novel Zebrafish Retinal Degeneration Model Using N-Methyl-N-Nitrosourea (MNU)

Primary loss of photoreceptors caused by diseases such as retinitis pigmentosa is one of the main causes of blindness worldwide. To study such diseases, rodent models of N-methyl-N-nitrosourea (MNU)-induced retinal degeneration are widely used. As zebrafish (Danio rerio) are a popular model system for visual research that offers persistent retinal neurogenesis throughout the lifetime and retinal regeneration after severe damage, we have established a novel MNU-induced model in this species. Histology with staining for apoptosis (TUNEL), proliferation (PCNA), activated Müller glial cells (GFAP), rods (rhodopsin) and cones (zpr-1) were performed. A characteristic sequence of retinal changes was found. First, apoptosis of rod photoreceptors occurred 3 days after MNU treatment and resulted in a loss of rod cells. Consequently, proliferation started in the inner nuclear layer (INL) with a maximum at day 8, whereas in the outer nuclear layer (ONL) a maximum was observed at day 15. The proliferation in the ONL persisted to the end of the follow-up (3 months), interestingly, without ongoing rod cell death. We demonstrate that rod degeneration is a sufficient trigger for the induction of Müller glial cell activation, even if only a minimal number of rod cells undergo cell death. In conclusion, the use of MNU is a simple and feasible model for rod photoreceptor degeneration in the zebrafish that offers new insights into rod regeneration.     

Effects of prenatal cocaine exposure in the photoreceptor cells of the rat retina

Despite the increasing evidence of eye abnormalities, the effects of prenatal exposure to cocaine on the visual system are still poorly understood. This study was aimed at analyzing the qualitative and quantitative organization of the retinal photoreceptor cells (PR) and outer nuclear layer (ONL) after prenatal exposure to cocaine in the rat. Pregnant Wistar rats were given sc injections of cocaine hydrochloride (60 mg/kg body wt/d) or saline or were not manipulated; analyses were performed in the 14- and 30-d-old male offspring. Radial semithin and ultrathin sections of epon-embedded flat mounts of the retina showed displaced PR-like cells in the inner nuclear layer (INL), picnotic PR nuclei in INL, and ONL, and retinal PR rosettes and outer-segment debris in the subretinal space. The quantitative study showed an increased density of PR-like nuclei in the INL in PND14 cocaine-treated rats that were within normal values at PND30; no changes were detected in the PR mean nuclear diameter and in the packing density of PR nuclei in the ONL. These data constitute the first morphological demonstration of photoreceptor damage after prenatal cocaine-exposure probably owing to a direct action of the drug and/or to the cocaine-induced ischemia/hypoxia.     

Retinal development and the expression profiles of opsin genes during larval development in Takifugu rubripes

The light-sensitive capacity of fish larvae is determined by the structure of the retina and the opsins expressed in the retinal and nonretinal photoreceptors. In this study, the retinal structure and expression of opsin genes during the early developmental stage of Takifugu rubripes larvae were investigated. Histological examination showed that at 1 days after hatching (dah), seven layers were observed in the retina of T. rubripes larva, including the pigment epithelial layer [retinal pigment epithelium layer (RPE)], photoreceptor layer (PRos/is), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL). At 2 dah, optic fibre layer (OFL) can be observed, and all eight layers were visible in the retina. By measuring the thickness of each layer, opposing developmental trends were found in the thickness of ONL, OPL, INL, IPL, GCL and OFL. The nuclear density of ONL, INL and GCL and the ratios of ONL/INL, ONL/GCL and INL/GCL were also measured and the ratio of ONL/GCL ranged from 1.9 at 2 dah to 3.4 at 8 dah and no significant difference was observed between the different developmental stages (P > 0.05). No significant difference was observed for the INL/GCL ratio between the different developmental stages, which ranged from 1.2 at 2 dah to 2.0 at 18 dah (P > 0.05). The results of quantitative real-time polymerase chain reaction (PCR) showed that the expression of RH1, LWS, RH2-1, RH2-2, SWS2, rod opsin, opsin3 and opsin5 could be detected from 1 dah. These results suggest that the well-developed retina and early expression of the opsins of T. rubripes during the period of transition from endogenous to mixed feeding might be critical for vision-based survival skills during the early life stages after hatching.     

Regeneration of the goldfish retina after exposure to different doses of ouabain

Summary After ouabain-induced degeneration, the retina of the goldfish shows a remarkable regeneration capacity. The extent of the damage depends on the dose of ouabain used in the experiment. After intraocular injection of 7l 10^–5 M ouabain, the ganglion cells and the cells of the inner nuclear layer (INL) become necrotic except for most of the outer horizontal cells, some bipolar cells, and Müller cells. The outer nuclear layer (ONL) and the marginal growth zone at the ora serrata remain intact; the plexiform layers become spongy. The degenerated material is removed by the proliferated reactive macroglial cells and invading macrophages. The degenerated cellular elements of the retina are replaced by mitosis of neuroblasts in the marginal growth zone and of cells in the ONL.After intraocular injection of a 5-fold higher dose of ouabain (7 l 5·10^–5M), the degeneration of the retina proceeds more rapidly and completely. In this experiment, the ONL is destroyed and the receptor outer segments are phagocytosed by cells of the pigment epithelium. In contrast to the regeneration of the amphibian retina, in the goldfish cells of the pigment epithelium do not participate by metaplastic transformation in the regeneration of the retina. The only source of cellular regeneration of the retina after complete destruction of its differentiated neural elements is the marginal growth zone, which is highly resistant to ouabain. The rate of mitoses in this region is strongly increased. The derivatives of these cells spread out tangentially over the entire fundus of the eye in a concentric manner. In this regenerate, mitotic processes continue in a radial direction, resulting in thickening and layering of the new retinal formation.     

Putative stem cells and the lineage of rod photoreceptors in the mature retina of the goldfish

The retinas of teleost fish grow continuously, in part, by neuronal hyperplasia and when lesioned will regenerate. Within the differentiated retina, the growth-associated hyperplasia results in the generation of new rod photoreceptors only, whereas injury-induced neurogenesis results in the regeneration of all retinal cell types. It is believed, however, that both new rod photoreceptors and regenerated neurons originate from the same populations of intrinsic progenitors. Experiments are described here that attempt to identify in the normal retina of goldfish neuronal progenitors intrinsic to the retina, particularly those which have remained cryptic because they divide infrequently. Long-term, systemic exposure to bromodeoxyuridine (BrdU) was used to label these cells. Five populations of proliferative cells were labeled: microglia, which are briefly described but not studied further; retinal progenitors in the circumferential germinal zone (CGZ); and rod precursors in the outer nuclear layer (ONL), both of which have been well characterized previously; and two populations of slowly-dividing cells in the inner nuclear layer (INL). The majority of these cells have a fusiform morphology, whereas the remaining ones are spherical. Longitudinal BrdU labeling suggests that the fusiform cells migrate to the ONL to replenish the pool of rod precursors. A subset of the spherical cells express pax6, although none are stained with markers of differentiated amacrine or bipolar cells. It is hypothesized that these rare, pax6-expressing cells are retinal stem cells, which give rise to the pax6-negative fusiform cells. Based on these data, two models are proposed: the first describes the lineage of rod photoreceptors in goldfish; the second is a consensus model of neurogenesis in the retinas of all teleosts.     

Genetic dissection reveals two separate pathways for rod and cone regeneration in the teleost retina

Development of therapies to treat visual system dystrophies resulting from the degeneration of rod and cone photoreceptors may directly benefit from studies of animal models, such as the zebrafish, that display continuous retinal neurogenesis and the capacity for injury-induced regeneration. Previous studies of retinal regeneration in fish have been conducted on adult animals and have relied on methods that cause acute damage to both rods and cones, as well as other retinal cell types. We report here the use of a genetic approach to study progenitor cell responses to photoreceptor degeneration in the larval and adult zebrafish retina. We have compared the responses to selective rod or cone degeneration using, respectively, the XOPS-mCFP transgenic line and zebrafish with a null mutation in the pde6c gene. Notably, rod degeneration induces increased proliferation of progenitors in the outer nuclear layer (ONL) and is not associated with proliferation or reactive gliosis in the inner nuclear layer (INL). Molecular characterization of the rod progenitor cells demonstrated that they are committed to the rod photoreceptor fate while they are still mitotic. In contrast, cone degeneration induces both Müller cell proliferation and reactive gliosis, with little change in proliferation in the ONL. We found that in both lines, proliferative responses to photoreceptor degeneration can be observed as 7 days post fertilization (dpf). These two genetic models therefore offer new opportunities for investigating the molecular mechanisms of selective degeneration and regeneration of rods and cones.     

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial world. Animal models are of pivotal importance to study such diseases. In this regard the photoreceptor-specific toxin N-methyl-N-nitrosourea (MNU) has been widely used in rodents to pharmacologically induce retinal degeneration. Previously, we have established a MNU-induced retinal degeneration model in the zebrafish, another popular model system in visual research.A fascinating difference to mammals is the persistent neurogenesis in the adult zebrafish retina and its regeneration after damage. To quantify this observation we have employed visual acuity measurements in the adult zebrafish. Thereby, the optokinetic reflex was used to follow functional changes in non-anesthetized fish. This was supplemented with histology as well as immunohistochemical staining for apoptosis (TUNEL) and proliferation (PCNA) to correlate the developing morphological changes.In summary, apoptosis of photoreceptors occurs three days after MNU treatment, which is followed by a marked reduction of cells in the outer nuclear layer (ONL). Thereafter, proliferation of cells in the inner nuclear layer (INL) and ONL is observed. Herein, we reveal that not only a complete histological but also a functional regeneration occurs over a time course of 30 days. Now we illustrate the methods to quantify and follow up zebrafish retinal de- and regeneration using MNU in a video-format.     

Regeneration of dopaminergic neurons in goldfish retina.

The conditions necessary to trigger regeneration of dopaminergic neurons were investigated in the goldfish retina. Intraocular injection of 6-hydroxydopamine (6-OHDA) was used to destroy dopaminergic neurons, and neuronal regeneration was monitored by injections of the thymidine analog bromodeoxyuridine (BUdR). Regenerated dopaminergic neurons, (identified by double-labeling with anti-tyrosine hydroxylase and anti-BUdR antibodies) were found within 3 weeks after 2 injections of 0.6 mg/ml 6-OHDA (estimated intraocular concentration), but not after injection of lower doses. All retinas with regenerated dopaminergic neurons also contained other types of regenerated neurons, including cones and ganglion cells, consistent with nuclear counts which revealed non-selective cell loss (34-36%) in both the outer and inner nuclear layers after exposure to the high dose, but not lower doses of 6-OHDA. Regenerated neurons were produced by clusters of dividing neuroepithelial cells probably derived from rod precursors in the outer nuclear layer. These results demonstrate that dopaminergic neurons will not regenerate after they are selectively ablated but only as part of a developmental process that involves generation of multiple cell types.     

In Vivo Voltammetric Study of 6-Hydroxydopamine-Induced Neuronal Degradation

Freely moving rats were injected intraventricularly with 200 micrograms of 6-hydroxydopamine (6-OHDA) and examined for transport of this substance to the striatum, degradation of dopamine (DA) neurons, and functional recovery through microcomputer-controlled in vivo voltammetry. Approximately 6 min after the injection, 6-OHDA began to appear in the extracellular fluid of the contralateral striatum. It increased linearly and began to decrease exponentially with the termination of the injection. Two hours after the injection with 6-OHDA, a peak began to appear at the same potential as the peak of DA in the differential pulse voltammogram. It persisted for approximately 3 days. When the rats were injected intraperitoneally with L-3,4-dihydroxyphenylalanine (L-Dopa), the conversion of L-Dopa to DA was not found 1 week after the 6-OHDA injection, but was clearly recognized 5 months after the injection. When the rats were examined for behavioral changes arising from the L-Dopa injection, they were found to be clearly less hyperactive 5 months after the 6-OHDA injection than 1 week after.     

Safety and Efficacy of Human Wharton's Jelly-Derived Mesenchymal Stem Cells Therapy for Retinal Degeneration

Purpose

To investigate the safety and efficacy of subretinal injection of human Wharton’s Jelly-derived mesenchymal stem cells (hWJ-MSCs) on retinal structure and function in Royal College of Surgeons (RCS) rats.

Methods

RCS rats were divided into 2 groups: hWJ-MSCs treated group (n = 8) and placebo control group (n = 8). In the treatment group, hWJ-MSCs from healthy donors were injected into the subretinal space in one eye of each rat at day 21. Control group received saline injection of the same volume. Additional 3 animals were injected with nanogold-labelled stem cells for in vivo tracking of cells localisation using a micro-computed tomography (microCT). Retinal function was assessed by electroretinography (ERG) 3 days before the injection and repeated at days 15, 30 and 70 after the injection. Eyes were collected at day 70 for histology, cellular and molecular studies.

Results

No retinal tumor formation was detected by histology during the study period. MicroCT scans showed that hWJ-MSCs stayed localised in the eye with no systemic migration. Transmission electron microscopy showed that nanogold-labelled cells were located within the subretinal space. Histology showed preservation of the outer nuclear layer (ONL) in the treated group but not in the control group. However, there were no significant differences in the ERG responses between the groups. Confocal microscopy showed evidence of hWJ-MSCs expressing markers for photoreceptor, Müller cells and bipolar cells.

Conclusions

Subretinal injection of hWJ-MSCs delay the loss of the ONL in RCS rats. hWJ-MSCs appears to be safe and has potential to differentiate into retinal-like cells. The potential of this cell-based therapy for the treatment of retinal dystrophies warrants further studies.     

人胎视网膜神经肽Y免疫反应神经元的发育

本文用免疫细胞化学ＡＢＣ法,研究１５—３８周龄人胎视网膜神经肽Ｙ免疫反应（ＮｅｕｒｏｐｅｐｔｉｄｅＹｉｍｍｕｎｏｒｅｃｔｉｖｅ,ＮＰＹ－ＩＲ）神经元（以下称ＮＰＹ－ＩＲ细胞）的发育。结果表明：①胎龄１５周视网膜中央部已出现不同类型的ＮＰＹ－ＩＲ细胞：位于黄斑及其周围外核层的为ＮＰＹ－ＩＲ视锥细胞;位于内核层最内一列的为ＮＰＹ－ＩＲ无长突细胞位于节细胞层的可能为ＮＰＹ－ＩＲ移位无长突细胞或节细胞;内核层和节细胞层的ＮＰＹ－ＩＲ细胞的突起均分布在内网层的第１亚层。②胎龄２４周后,ＮＰＹ－ＩＲ视锥细胞完全消失。③随着视网膜的发育,内核层和节细胞层的ＮＰＹ－ＩＲ细胞数量增多,突起增粗增长,胞体分布由中央部扩展到周边部,其中内核层ＮＰＹ－ＩＲ细胞的密度呈现从中央部向周边部逐渐降低的分布方式,节细胞层ＮＰＹ－ＩＲ细胞则多数集中分布在视网膜的边缘和黄斑之间,形成较高密度的环状区。     

根田鼠视网膜的胚后发育

视觉对动物的生活习性尤其是取食具有重要意义。本文对根田鼠视网膜的胚后发育进行了研究,结果表明:出生3d内根田鼠视网膜分化程度较低,神经节母细胞层尚未分化,占据了视网膜层的一半以上;5日龄时,外网层开始出现;6日龄时,外网层开始清晰,外核层与内核层更加清晰;18日龄时,视网膜结构与成年根田鼠结构相似,各层结构清晰可见。测量了神经节细胞层和外核层的细胞密度以及核层厚度,结果表明:随着个体发育,外核层细胞层厚度及细胞密度不断增加;而神经节细胞层厚度及细胞密度不断减少。与褐家鼠、黑线姬鼠、大仓鼠、棕色田鼠、甘肃鼢鼠、达乌尔黄鼠、岩松鼠视网膜相比,根田鼠视网膜结构介于夜行性与昼行性鼠类之间[动物学报52(2):376-382,2006]。     

表皮生长因子及其受体和erb-B在鼠视网膜的表达

用免疫组织化学方法,对鼠视网膜的表皮生长因子(EGF)及其受体(EGF-R),和erb-B的表达,作了研究.结果表明:①EGF,EGF-R和erb-B分别在视网膜各层有不同表达;②EGF免疫反应着色不强,但有特异性地均匀分布在节细胞层(GCL)、内核层(INL)、外核层(ONL)和视网膜色素上皮层(RPE);③EGF-R免疫反应着色以GCL和INL的细胞明显,分布于细胞浆和细胞核,以细胞核更明显;④erb-B的分布类似EGF.但在ONL内出现一条较明显的着色带.对以上结果的可能意义进行了讨论.     

Central catecholamine depletion inhibits peripheral lymphocyte responsiveness in spleen and blood

Experimental and clinical evidence has demonstrated extensive communication between the CNS and the immune system. To analyse the role of central catecholamines in modulating peripheral immune functions, we injected the neurotoxin 6-hydroxydopamine (6-OHDA) i.c.v. in rats. This treatment significantly reduced brain catecholamine content 2, 4 and 7 days after injection, and in the periphery splenic catecholamine levels were reduced 4 days after treatment. Central catecholamine depletion induced an inhibition of splenic and blood lymphocyte proliferation and splenic cytokine production and expression (interleukin-2 and interferon-gamma) 7 days after injection. In addition, central treatment with 6-OHDA reduced the percentage of spleen and peripheral blood natural killer (CD161 +) cells, and T-cytotoxic (CD8 +) cells in peripheral blood. The reduction in splenocyte proliferation was not associated with a glucocorticoid alteration but was completely abolished by prior peripheral sympathectomy. These data demonstrate a crucial role of central and peripheral catecholamines in modulating immune function.     

Photoreceptor cell death, proliferation and formation of hybrid rod/S-cone photoreceptors in the degenerating STK38L mutant retina

A homozygous mutation in STK38L in dogs impairs the late phase of photoreceptor development, and is followed by photoreceptor cell death (TUNEL) and proliferation (PCNA, PHH3) events that occur independently in different cells between 7-14 weeks of age. During this period, the outer nuclear layer (ONL) cell number is unchanged. The dividing cells are of photoreceptor origin, have rod opsin labeling, and do not label with markers specific for macrophages/microglia (CD18) or Müller cells (glutamine synthetase, PAX6). Nestin labeling is absent from the ONL although it labels the peripheral retina and ciliary marginal zone equally in normals and mutants. Cell proliferation is associated with increased cyclin A1 and LATS1 mRNA expression, but CRX protein expression is unchanged. Coincident with photoreceptor proliferation is a change in the photoreceptor population. Prior to cell death the photoreceptor mosaic is composed of L/M- and S-cones, and rods. After proliferation, both cone types remain, but the majority of rods are now hybrid photoreceptors that express rod opsin and, to a lesser extent, cone S-opsin, and lack NR2E3 expression. The hybrid photoreceptors renew their outer segments diffusely, a characteristic of cones. The results indicate the capacity for terminally differentiated, albeit mutant, photoreceptors to divide with mutations in this novel retinal degeneration gene.     

Time course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafish

Constant intense light causes apoptosis of rod and cone photoreceptors in adult albino zebrafish. The photoreceptors subsequently regenerate from proliferating inner nuclear layer (INL) progenitor cells that migrate to the outer nuclear layer (ONL) and differentiate into rods and cones. To identify gene expression changes during this photoreceptor regeneration response, a microarray analysis was performed at five time points during the light treatment. The time course included an early time point during photoreceptor death (16 h), later time points during progenitor cell proliferation and migration (31, 51, and 68 h) and a 96 h time point, which likely corresponds to the initial photoreceptor differentiation. Mean expression values for each gene were calculated at each time point relative to the control (0 h light exposure) and statistical analysis by one-way ANOVA identified 4567 genes exhibiting significant changes in gene expression along the time course. The genes within this data set were clustered based on their temporal expression patterns and proposed functions. Quantitative real-time PCR validated the microarray expression profiles for selected genes, including stat3 whose expression increased markedly during the light exposure. Based on immunoblots, both total and activated Stat3 protein expression also increased during the light treatment. Immunolocalization of Stat3 on retinal tissue sections demonstrated increased expression in photoreceptors and Müller glia by 16 h of light exposure. Some of the Stat3-positive Müller cells expressed PCNA at 31 h, suggesting that Stat3 may play a role in signaling a subset of Müller cells to proliferate during the regeneration response.     

Distribution of zinc in the substantia nigra of rats treated with 6-hydroxydopamine

To study the relationship between tissue accumulation of Zinc (Zn) and neurodegeneration in the nigrostriatal dopaminergic pathway,⁶⁵Zn distribution in this pathway was examined after unilateral injection of 6-hydroxydopamine (6-OHDA) into the substantia nigra of rats. When⁶⁵ZnCl₂ was intravenously injected 4 days after treatment with 6-OHDA,⁶⁵Zn was concentrated in the ipsilateral substantia nigra 6 days after⁶⁵Zn injection. On the other hand, 19 d after treatment with 6-OHDA,⁶⁵Zn distribution in the ipsilateral substantia nigra was decreased to the level of the contralateral one. When NH₄ ⁹⁹TcO₄, which cannot go through the blood-brain barrier, was injected into rats 4 d after treatment with 6-OHDA,⁹⁹Tc was concentrated in the ipsilateral substantia nigra 30 min after⁹⁹Tc injection, but no longer detectable 6 d after injection. These results suggest that Zn is necessary for a repair process called replacement gliosis after the death of neurons and that excess Zn does not accumulate in the lesion after completion of the gliosis.     

Cellular distribution ofl-glutamate decarboxylase (GAD) andγ-aminobutyric acidA (GABAA) receptor mRNAs in the retina

1. Gamma-aminobutryic acid (GABA), a major inhibitory transmitter of the vertebrate retina, is synthesized from glutamate by L-glutamate decarboxylase (GAD) and mediates neuronal inhibition at GABAA receptors. GAD consists of two distinct molecular forms, GAD65 and GAD67, which have similar distribution patterns in the nervous system (Feldblum et al., 1990; Erlander and Tobin, 1991). GABAA receptors are composed of several distinct polypeptide subunits, of which the GABAA alpha 1 variant has a particularly extensive and widespread distribution in the nervous system. The aim of this study was to determine the cellular localization patterns of GAD and GABAA alpha 1 receptor mRNAs to define GABA- and GABAA receptor-synthesizing neurons in the rat retina. 2. GAD and GABAA alpha 1 mRNAs were localized in retinal neurons by in situ hybridization histochemistry with 35S-labeled antisense RNA probes complementary to GAD67 and GABAA alpha 1 mRNAs. 3. The majority of neurons expressing GAD67 mRNA is located in the proximal inner nuclear layer (INL) and ganglion cell layer (GCL). Occasional GAD67 mRNA-containing neurons are present in the inner plexiform layer. Labeled neurons are not found in the distal INL or in the outer nuclear layer (ONL). 4. GABAA alpha 1 mRNA is expressed by neurons distributed to all regions of the INL. Some discretely labeled cells are present in the GCL. Labeled cells are not observed in the ONL. 5. The distribution of GAD67 mRNA demonstrates that numerous amacrine cells (conventional, interstitial, and displaced) and perhaps interplexiform cells synthesize GABA. These cells are likely to employ GABA as a neurotransmitter. 6. The distribution of GABAA alpha 1 mRNA indicates that bipolar, amacrine, and perhaps ganglion cells express GABAA receptors having an alpha 1 polypeptide subunit, suggesting that GABA acts directly upon these cells.     

Differential subcellular localization of zinc in the rat retina.

In the retina, zinc is believed to be a modulator of synaptic transmission and a constituent of metalloenzymes. To determine whether the intracellular localization of zinc correlates with function, we examined the localization of endogenous zinc in the rat retina using the silver amplification method. By light microscopy, reaction products were detected in the pigment epithelial cells (PE), the inner segment of photoreceptors (IS), the outer nuclear layer (ONL) and the inner nuclear layer (INL), the outer plexiform layer (OPL) and the inner plexiform layer (IPL), and the ganglion cell layer (GC). The heaviest accumulation of precipitate was observed in PE and IS, whereas only a little precipitate was found in GC. When the intracellular zinc was chelated with diethyldithiocarbamate, a small amount of precipitate was observed only in ONL. By electron microscopy, zinc was associated with three compartments. In OPL and IPL, zinc was associated with neural processes, while in PE, IS, INL, and GC it was associated with the Golgi apparatus. In ONL, zinc was associated with the nucleus. Zinc in the neural processes is believed to act as a modulator of synaptic transmission, and zinc associated with the Golgi apparatus is assumed to catalyze metalloenzyme reactions.