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Biomedical subjects

W K Stell

Publications and source records attributed to W K Stell.

At least 19 recordsLinked to original sources
PubMed1999

Nitric oxide synthase-containing cells in the retina, pigmented epithelium, choroid, and sclera of the chick eye.

Nitric oxide is a nonconventional neurotransmitter that is produced as needed by the enzyme nitric oxide synthase (NOS). NOS has been detected in numerous neural structures, including distinct populations of retinal neurons in a variety of vertebrate species. The purpose of this study was to identify NOS-containing cells in the retina and extraretinal ocular tissues of hatched chicks. NOS was detected in frozen sections by using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry and antisera to neuronal NOS. In the retina, NADPH-diaphorase and NOS immunolabelling were present in four subtypes of amacrine cells, some ganglion cells, efferent fibers, efferent target cells, and neuronal processes in both plexiform layers, whereas diaphorase alone was detected in photoreceptor ellipsoids and Müller cells. In addition, NADPH-diaphorase and immunoreactive NOS were detected in axon bundles and innervation to vascular smooth muscle in the choroid, whereas stromal and endothelial cells in the choroid, scleral chondrocytes, and the retinal pigmented epithelium contained only NADPH-diaphorase. The excitotoxin quisqualate destroyed all but one subtype of NOS-immunoreactive amacrine cell and caused increased NADPH-diaphorase activity in Müller cells. We conclude that nitric oxide is produced by many different cells in the chick eye, including retinal amacrine and ganglion cells, Müller cells, retinal pigmented epithelium, and cells in the choroid, and likely has a broad range of visual and regulatory functions.

Animals↗
PubMed1999

Colchicine causes excessive ocular growth and myopia in chicks.

Colchicine has been reported to destroy ganglion cells (GCs) in the retina of hatchling chicks. We tested whether colchicine influences normal ocular growth and form-deprivation myopia, and whether it affects cells other than GCs. Colchicine greatly increased axial length, equatorial diameter, eye weight, and myopic refractive error, while reducing corneal curvature. Colchicine caused DNA fragmentation in many GCs and some amacrine cells and photoreceptors, ultimately leading to the destruction of most GCs and particular sub-sets of amacrine cells. Colchicine-induced ocular growth may result from the destruction of amacrine cells that normally suppress ocular growth, and corneal flattening may result from the destruction of GCs whose central pathway normally plays a role in shaping the cornea.

Animals↗
PubMed1999

Localization of retinoid binding proteins, retinoid receptors, and retinaldehyde dehydrogenase in the chick eye.

Retinoids have many functions in the eye, including, perhaps, the visual guidance of ocular growth. Therefore, we identified where retinoid receptors, binding proteins, and biosynthetic enzymes are located in the ocular tissues of the chick as a step toward discovering where retinoids are generated and where they act. Using antibodies to interphotoreceptor retinoid binding protein (IRBP), cellular retinol binding protein (CRBP), cellular retinoic acid binding protein (CRABP), cellular retinaldehyde binding protein (CRALBP), retinaldehyde dehydrogenase (RALDH), and retinoic acid receptors (RAR and RXR), we localized these proteins to cells in the retina, retinal pigmented epithelium, choroid and sclera of the chick eye. IRBP was detected in the photoreceptor layer and pigmented epithelium; CRBP was in the pigmented epithelium; CRABP was in amacrine and bipolar cells in the retina; CRALBP was in Müller cells, pigmented epithelium, choroid, and fibrous sclera; RALDH was in retinal amacrine cells, pigmented epithelium, and choroid; RAR was in amacrine cells, choroid, and chondrocytes and fibroblasts in the sclera; and RXR was in amacrine and ganglion cells, bipolar cell nuclei, choroid, and chondrocytes. We also found that the growth-modulating toxins colchicine and quisqualate destroyed selectively different subsets of CRABP-containing amacrine cells. We conclude that the distribution of proteins involved in retinoid metabolism is consistent with a role of retinoids not only in phototransduction, but also in maintenance of cellular phenotype and visual guidance of ocular growth.

Aldehyde Oxidoreductases↗
PubMed1999

Light- and focus-dependent expression of the transcription factor ZENK in the chick retina.

Ocular growth and refraction are regulated by visual processing in the retina. We identified candidate regulatory neurons by immunocytochemistry for immediate-early gene products, ZENK (zif268, Egr-1) and Fos, after appropriate visual stimulation. ZENK synthesis was enhanced by conditions that suppress ocular elongation (plus defocus, termination of form deprivation) and suppressed by conditions that enhance ocular elongation (minus defocus, form deprivation), particularly in glucagon-containing amacrine cells. Fos synthesis was enhanced by termination of visual deprivation, but not by defocus and not in glucagon-containing amacrine cells. We conclude that glucagon-containing amacrine cells respond differentially to the sign of defocus and may mediate lens-induced changes in ocular growth and refraction.

Adaptation, Ocular↗
PubMed1998

Cholinergic amacrine cells are not required for the progression and atropine-mediated suppression of form-deprivation myopia.

Muscarinic cholinergic pathways have been implicated in the visual control of ocular growth. However, the source(s) of acetylcholine and the tissue(s) which regulate ocular growth via muscarinic acetylcholine receptors (mAChRs) remain unknown. We sought to determine whether retinal sources of acetylcholine and mAChRs contribute to visually guided ocular growth in the chick. Cholinergic amacrine cells were ablated by intraocular injections of either ethylcholine mustard aziridinium ion (ECMA; a selective cholinotoxin) or quisqualic acid (QA; an excitotoxin that destroys many amacrine cells, including those that release acetylcholine). Disruption of cholinergic pathways was assessed immunocytochemically with antibodies to the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT) and three different isoforms of mAChR, and by biochemical assay for ChAT activity. ECMA (25 nmol) destroyed two of the four subtypes of cholinergic amacrine cells and attenuated retinal ChAT activity, but left retinal mAChR-immunoreactivity intact. QA (200 nmol) destroyed the majority of all four subtypes of cholinergic amacrine cells, and ablated most mAChR-immunoreactivity and ChAT activity in the retina. ECMA and QA had no apparent effect on mAChRs or cholinergic fibres in the choroid, only marginally reduced choroidal ChAT activity, and had little effect on ChAT activity in the anterior segment. Toxin-treated eyes remained emmetropic and responded to form-deprivation by growing excessively and becoming myopic. Furthermore, daily intravitreal injection of 40 microg atropine for 6 days into form-deprived toxin-treated eyes completely prevented ocular elongation and myopia. We conclude that neither cholinergic amacrine cells nor mAChRs in the retina are required for visual regulation of ocular growth, and that atropine may exert its growth-suppressing influence by acting upon extraretinal mAChRs, possibly in the choroid, retinal pigmented epithelium, or sclera.

Animals↗
PubMed1998

Immunocytochemical characterization of quisqualic acid- and N-methyl-D-aspartate-induced excitotoxicity in the retina of chicks.

A single, large dose of N-methyl-D-aspartate (NMDA) or quisqualic acid (QA) injected into the chick eye has been shown previously to destroy many retinal amacrine cells and to induce excessive ocular growth accompanied by myopia. The purpose of this study was to identify distinct populations of retinal cells, particularly those believed to be involved in regulating ocular growth, that are sensitive to NMDA or QA. Two pmol of NMDA or 0.2 micromol of QA were injected unilaterally into eyes of 7-day-old chicks, and retinas were prepared for observation 1, 3, or 7 days later. Retinal neurons were identified by using immunocytochemistry, and cells containing fragmented DNA were identified by 3'-nick-end labelling in frozen sections. NMDA and QA destroyed many amacrine cells, including those immunoreactive for vasoactive intestinal polypeptide, Met-enkephalin, and choline acetyltransferase, but they had little effect upon tyrosine hydroxylase-immunoreactive cells. Other cells affected by both QA and NMDA included those immunoreactive for glutamic acid decarboxylase, gamma-aminobutyric acid, parvalbumin, serotonin, and aminohydroxy methylisoxazole propionic acid (AMPA) receptor subunits GluR1 and GluR2/3. Cells largely unaffected by QA or NMDA included bipolar cells immunoreactive for protein kinase C (alpha and beta isoforms) and amacrine cells immunoreactive for glucagon. DNA fragmentation was detected maximally in many amacrine cells and in some bipolar cells 1 day after exposure to QA or NMDA. We propose that excitotoxicity caused by QA and NMDA induces apoptosis in specific populations of amacrine cells and that these actions are responsible for the ocular growth-specific effects of QA and NMDA reported elsewhere.

Animals↗
PubMed1998

Identification and localization of muscarinic acetylcholine receptors in the ocular tissues of the chick.

The purpose of this study was to characterize the distribution of muscarinic acetylcholine receptors (mAChRs) in the ocular tissues of hatched chicks. In the chick, different isoforms of these receptors have been detected in the brain, heart, and retina, and mAChRs in ocular tissues have been implicated in the pathogenesis of form-deprivation myopia. However, the precise anatomical distribution of mAChRs within the retina, retinal pigment epithelium, choroid, ciliary body, and ciliary ganglion remains unknown. We used affinity-purified, type-specific antibodies directed to three different chick mAChR subtypes (cm2, cm3, and cm4) to detect receptor immunoreactivity in sections and extracts of these ocular tissues. We found cm2, cm3, and cm4 in the retina, retinal pigment epithelium, choroid, and ciliary body. Within the retina, cm2 was expressed in numerous amacrine and ganglion cells; cm3 was expressed in many bipolar cells and small subsets of amacrine cells; and cm4 was found in most, if not all, amacrine and ganglion cells. Each mAChR was localized to distinct strata within the inner plexiform layer that cumulatively form three broad bands that closely match previously described localizations of subtype-nonspecific muscarinic ligand binding. Only cm3 was detected in the outer plexiform layer, and only cm4 was detected in the ciliary ganglion. We propose that each mAChR subtype has unique functions in each ocular tissue.

Animals↗
PubMed1997

Light-modulated release of RFamide-like neuropeptides from nervus terminalis axon terminals in the retina of goldfish.

The nervus terminalis of teleosts, a cranial nerve anatomically associated with the olfactory system, projects to visual system targets including retina and optic tectum. It is known to contain gonadotropin-releasing hormone and RFamide-like peptides, but its function remains unknown. We have probed nervus terminalis function in goldfish by measuring peptide content in retina and tectum with a radioimmunoassay for A18Famide (neuropeptide AF; bovine morphine-modulating peptide). We found that retinal peptide content increased in the dark and decreased in the light, whereas tectal peptide content decreased in the dark and increased in the light. In addition, RFamide-like peptide content in the retina was transiently decreased by severing both olfactory tracts, increased in light-adapted eyes treated with a GABAergic agonist (isoguvacine), and decreased in dark-adapted eyes treated with GABAergic antagonists (bicuculline and picrotoxin). We also found that RFamide-like peptide release could be induced in dark-adapted isolated-superfused retinas by exposure to light or a high concentration (102.5 mM) of potassium ions. We interpret the increase and decrease in peptide content as reflecting a decrease and increase, respectively, in rate of peptide release. We propose that the release and accumulation of RFamide-like peptides in axon terminals of nervus terminalis processes in the retina are modulated primarily by neurons intrinsic to the retina and regulated by light. Peptide release appears to be inhibited tonically in the dark by GABA acting through GABAA receptors; light facilitates peptide release by disinhibition due to a reduction in GABA release. In addition, we propose that electrical signals originating outside the retina can override these intrinsic release-modulating influences.

Adaptation, Ocular↗
PubMed1997

Basic fibroblast growth factor, its high- and low-affinity receptors, and their relationship to form-deprivation myopia in the chick.

Form deprivation myopia in chickens is a widely accepted model to study visually-regulated postnatal ocular growth. Recently we showed that basic fibroblast growth factor-2 provides a "stop" signal for the growing eye. To understand further its action, we have localized basic fibroblast growth factor-2 and its low- and high-affinity receptors in the chicken eye, and determined the localization of basic fibroblast growth factor receptors in the inner plexiform layer with respect to that of neurotransmitter systems known to play a role in form-deprivation myopia. By immunocytochemistry and in situ hybridization, two complementary methods, we found that nearly all cells in the retina, and scleral chondrocytes, contain basic fibroblast growth factor-2 protein and messenger RNA as well as high-affinity basic fibroblast growth factor receptor protein and messenger RNA. Immunocytochemical localization of basic fibroblast growth factor-2 binding sites (a high resolution alternative to autoradiography), combined with N-glycanase and heparitinase treatment or heparin competition, revealed additional binding sites in specific synaptic layers of the inner plexiform layer and low-affinity binding sites in the choroid and optic fibre layer. Some binding sites in the synaptic layers were found to co-stratify with neurites of dopamine-, vasoactive intestinal polypeptide- or enkephalin-containing amacrine cells, suggesting that basic fibroblast growth factor-2 could modulate synaptic transmission to or from these cells. Form deprivation did not affect the levels of basic fibroblast growth factor receptor-1 messenger RNA in retina/retinal pigment epithelium/choroid (Northern blotting), but it abolished the decrease in amount of extractable basic fibroblast growth factor normally observed in the dark (Western blotting). The results are discussed with respect to previous findings on basic fibroblast growth factor-2 and basic fibroblast growth factor receptor-1 localization in the avian and other vertebrate eyes, and their relevance to form-deprivation myopia. The widespread distribution of basic fibroblast growth factor-2 and its receptor makes it impossible to predict which cells might mediate the action of basic fibroblast growth factor-2 in form-deprivation myopia. However, the alteration in amounts of extractable retinal basic fibroblast growth factor-2 in form-deprived, dark-adapted retinas, in which basic fibroblast growth factor-2 probably serves as a "stop" signal for ocular growth, is consistent with a role for basic fibroblast growth factor-2 in the regulation of ocular growth.

Animals↗
PubMed1997

Identification and localization of an immunoreactive AMPA-type glutamate receptor subunit (GluR4) with respect to identified photoreceptor synapses in the outer plexiform layer of goldfish retina.

L-glutamate, the main excitatory synaptic transmitter in the retina, is released from photoreceptors and evokes responses in second-order retinal neurons (horizontal, bipolar cells) which utilize both ionotropic and metabotropic types of glutamate receptors. In the present study, to elucidate the functional roles of glutamate receptors in synaptic transmission, we have identified a specific ionotropic receptor subunit (GluR4) and determined its localization with respect to photoreceptor cells in the outer plexiform layer of the goldfish retina by light and pre-embedding electron-microscopical immunocytochemistry. We screened antisera to mammalian AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate)-preferring ionotropic glutamate receptors (GluR 1-4) of goldfish retina by light- and electron-microscopical immunocytochemistry. Only immunoreactive (IR) GluR4 was found in discrete clusters in the outer plexiform layer. The cones contacted in this manner were identified as long-wavelength ("red") and intermediate-wavelength ("green") cones, which were strongly immunoreactive to monoclonal antibody FRet 43 and antisera to goldfish red and green-cone opsins; and short-wavelength ("blue") cones, which were weakly immunoreactive to FRet 43 but strongly immunoreactive with antiserum to blue-cone opsin. Immunoblots of goldfish retinal homogenate with anti-GluR4 revealed a single protein at M(r) = 110 kDa. Preadsorption of GluR4 antiserum with either the immunizing rat peptide, or its goldfish homolog, reduced or abolished staining in retinal sections and blots. Therefore, we have detected and localized genuine goldfish GluR4 in the outer plexiform layer of the goldfish retina. We characterized contacts between photoreceptor cells and GluR4-IR second-order neurons in the electron microscope. IR-GluR4 was localized to invaginating central dendrites of triads in ribbon synapses of red cones, semi-invaginating dendrites in other cones and rods, and dendrites making wide-cleft basal junctions in rods and cones; the GluR4-IR structures are best identified as dendrites of OFF-bipolar cells. The results of our studies indicate that in goldfish retina GluR4-expressing neurons are postsynaptic to all types of photoreceptors and that transmission from photoreceptors to OFF-bipolars is mediated at least in part by AMPA-sensitive receptors containing GluR4 subunits.

Amino Acid Sequence↗
PubMed1997

N-methyl-D-aspartate-induced excitotoxicity causes myopia in hatched chicks.

OBJECTIVE: To characterize the effect of the amacrine cell-selective toxin N-methyl-D-aspartate (NMDA) on ocular growth in chicks. DESIGN: Single injections of NMDA in doses ranging from 20 to 2000 nmol in 20 microL of sterile saline were made into the vitreous chamber of one eye of 7-day-old white leghorn chicks (six chicks per group); the contralateral (control) eye was injected with saline. Six NMDA-treated eyes were also deprived of form vision by applying a translucent goggle 7 days after treatment, to determine whether myopia could still be induced or enhanced in NMDA-treated eyes. OUTCOME MEASURES: Axial length, equatorial diameter and refractive error, measured immediately after and 7, 14, 21, 28 and 35 days after treatment. RESULTS: NMDA-treated eyes became myopic within 7 days of treatment and remained so until at least 35 days after treatment. During this time the eyes continued to grow but could not be induced to become more myopic by depriving them of patterned images. The half-maximal effective dose of NMDA was calculated to be 670 nmol, 7 days after treatment. CONCLUSIONS: NMDA-induced excitotoxicity destroys retinal pathways by which patterned visual stimuli restrain ocular growth in the chick.

Animals↗
PubMed1996

Immunocytochemical localization of the high-affinity glutamate transporter, EAAC1, in the retina of representative vertebrate species.

The glutamate transporter, EAAC1, was localized immunocytochemically in goldfish, salamander, turtle, chicken, and rat retinas, using affinity-purified oligopeptide antibodies. Immunoreactive (IR) EAAC1 was present in the inner plexiform layer of all species, and in cell bodies of bipolar, amacrine, and ganglion cells of most species, but absent from photoreceptors and Müller's glial cells. Western blots revealed an IR-EAAC1 band at 70 kDa. Staining was abolished by preabsorption with EAAC1 peptide.

ATP-Binding Cassette Transporters↗
PubMed1996

Characterization of the RFamide-like neuropeptides in the nervus terminalis of the goldfish (Carassius auratus).

FMRFamide-immunoreactivity has been demonstrated in the CNS of many vertebrate species. We sought to further characterize this immunoreactivity in nervus terminalis retinal efferents of the goldfish using an antiserum raised against a bovine morphine modulating peptide (A18Famide). This antiserum robustly labels nervus terminalis efferents to the retina, as well as a sub-population of retinal amacrine cells. Under immunocytochemical conditions the antiserum cross-reacted with neuropeptide Y-like as well as A18Famide-like peptides, but under conditions of radioimmunoassay it was highly specific for A18Famide-like peptides. High pressure liquid chromatography, gel permeation chromatography and radioimmunoassay showed that at least two different RFamide-like peptides, approximately the same size as the bovine RFamide-like peptides, are present in the goldfish nervus terminalis.

Animals↗
PubMed1995

Localization of putative dopamine D2-like receptors in the chick retina, using in situ hybridization and immunocytochemistry.

The actions of dopamine are mediated by 5 or more receptor subtypes, any of which may be coupled by G-proteins to adenylate cyclase (D1-family: stimulatory, D2-family: inhibitory or no action). Postnatal ocular growth in the chick is a vision-dependent mechanism which involves D2-type receptors in either the retina or the retinal pigment epithelium (RPE). Although the dopaminergic amacrine cells are well described in the chick retina, only D2-receptors, but not D3- and D4-receptors have been clearly localized, and the cells that express them have not been identified. In this study we showed that immunoreactive D2/3-receptor protein is localized to the photoreceptor inner segments, outer and inner plexiform layer and ganglion cell layer, as described previously (Wagner et al., J. Comp. Neurol., 330 (1993) 1-13). D2-receptor mRNA was localized to cell bodies in all nuclear layers of the retina, whereas D4-receptor mRNA was restricted to the inner half of the retina. Immunoreactive D2-type receptors and their mRNA were observed also in the basal region of the RPE. Because of the widespread distribution of both D2- and D4-receptor mRNA in the chick retina and RPE and the lack of D3- and D4-receptor-specific antibodies, we were unable to identify which of the D2/3/4-receptor-bearing cells are involved in controlling ocular growth.

Animals↗
PubMed1995

Stimulation of dopaminergic amacrine cells by stroboscopic illumination or fibroblast growth factor (bFGF, FGF-2) injections: possible roles in prevention of form-deprivation myopia in the chick.

Form-deprivation myopia (FDM) in the chick is a popular model for studying the postnatal regulation of ocular growth. Using this model, we have shown previously that dopamine and FGF-2 can counteract the effects of form-deprivation, thereby producing emmetropia. In the present study, we tested the hypothesis that the emmetropizing effects of flickering light and intraocular injections of FGF-2 in the chick are mediated by the activity of dopaminergic retinal amacrine cells. We have assessed the rate of dopamine synthesis in the retina by measuring the accumulation of 3,4-dihydroxyphenylalanine (DOPA). We found that form-deprivation reduces the rate of dopamine synthesis in the light-adapted retina, and that the normal rate of dopamine synthesis in the light can be restored by stroboscopic illumination at frequencies around 10 Hz. By labeling cells immunocytochemically we have shown that the synthesis of c-fos, a putative transcriptional regulator of the tyrosine hydroxylase gene, is induced in dopaminergic amacrine cells by stroboscopic illumination at around 10 Hz. These observations are consistent with a critical role for dopaminergic amacrine cells in the regulation of ocular growth by intermittent illumination. We have found also that intraocular injections of FGF-2 cause emmetropization without altering levels of expression of c-fos, amounts of tyrosine hydroxylase, or rates of dopamine synthesis with respect to vehicle-injected controls. We conclude that FGF acts either in parallel to or downstream from the dopaminergic amacrine cells, rather than through them. We observed that intravitreal injection per se induces high levels of c-fos expression in both form-deprived and non-deprived retinas, and causes partial emmetropization in form-deprived eyes, while inhibiting dopamine synthesis in non-deprived retinas. It is likely, therefore, that injection stimulates the production and/or release of unknown factors whose diverse effects on ocular growth and dopamine metabolism are mediated by complex pathways. Taken together, our results are consistent with the view that the retinal circuitry that controls postnatal ocular growth in the chick involves multiple messengers and pathways.

Animals↗
PubMed1995

Substance P-, F8Famide-, and A18Famide-like immunoreactivity in the nervus terminalis and retina of the goldfish Carassius auratus.

We re-investigated the occurrence of substance P-like immunoreactivity in the retina of the goldfish Carassius auratus using antisera to substance P and other tachykinins. Most antisera labelled a previously described single class of mono-stratified amacrine cells arborizing in layer 3 of the inner plexiform layer. Preabsorption experiments showed that these amacrine cells contained at least one tachykinin-like peptide. One antiserum (INC 353) to substance P labelled not only these amacrine cells but also fibres in layer 1 of the inner plexiform layer and fibres in the optic nerve. These fibres were identified as retinopetal projections of the nervus terminalis, in part because of colocalized labelling with antisera against gonadotropin-releasing hormone and FMRFamide. Preabsorption experiments showed that the substance P-immunoreactive material in the nervus terminalis was not substance P or any other typical tachykinin. Labelling of the nervus terminalis with INC 353 was blocked by preabsorption with two bovine FMRF-amide-like peptides, F8Famide and A18Famide, which contain a substance P(4-7)-like region. Antisera to F8Famide and A18Famide strongly labelled ganglia of the nervus terminalis and retinopetal fibres. We suggest that labelling of the nervus terminalis by antisera to substance P and FMRFamide occurs because of homologies between these antigens and a non-tachykinin, endogenous peptide that is similar to F8Famide and A18Famide.

Animals↗
PubMed1995

The effect of vasoactive intestinal peptide on development of form deprivation myopia in the chick: a pharmacological and immunocytochemical study.

The role of vasoactive intestinal peptide (VIP) in the development of form deprivation myopia (FDM) was examined. Daily intravitreal injection of porcine VIP reduced, but did not eliminate FDM at a maximal daily dose of 1 x 10(-5) mol/injection. A VIP analogue reported to be relatively hydrolysis-resistant in vivo, had no effect on development of FDM at any dose tested. Two VIP antagonists completely abolished FDM. The one reported to be selective for central nervous system VIP receptors was 100 times more potent than one reported to be selective for peripheral nervous system receptors (ED50 = 2 x 10(-10) and 2 x 10(-8) mol/injection respectively). By immunofluorescence using antiserum to porcine VIP, VIP-like immunoreactivity was localized to a subset of amacrine cells (AC) and in three parallel layers in the inner plexiform layer (IPL) (10%, 40% and 70% of IPL thickness from the AC layer). Immunoreactive nerve fibres were also seen in the choroid, the ciliary body and the iris. These results suggest that VIP may play a role in both normal development of the refractive properties of the eye, and in the development of FDM.

Animals↗