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Biomedical subjects

P M Iuvone

Publications and source records attributed to P M Iuvone.

At least 19 recordsLinked to original sources
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

Melatonin biosynthesis in photoreceptor-enriched chick retinal cell cultures: role of cyclic AMP in the K(+)-evoked, Ca(2+)-dependent induction of serotonin N-acetyltransferase activity.

The roles of cyclic AMP and calcium in the regulation of serotonin N-acetyltransferase (NAT) activity were studied in low density monolayer cultures of chick retinal photoreceptors and neurons. Photoreceptor-enriched retinal cell cultures were prepared from embryonic day 6 retinas and cultured for 6 days. NAT activity in these cultures could be induced by treatment with cyclic AMP protagonists, 8Br-cyclic AMP, forskolin, and 3-isobutyl-1-methylxanthine (IBMX), or by treatment with depolarizing concentrations of extracellular K+. The stimulatory effect of K+, which involves Ca2+ influx through dihydropyridine-sensitive channels, was mediated at least in part by cyclic AMP, as indicated by the following observations. Depolarizing concentrations of K+ stimulated the formation of cyclic AMP, and the stimulatory effects of K+ on both cyclic AMP formation and on NAT activity were synergistically potentiated by the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). MDL 12,330A, a putative adenylate cyclase inhibitor, inhibited K(+)-evoked cyclic AMP accumulation and induction of NAT activity over the identical concentration range. In contrast, MDL 12,300A failed to inhibit the induction of NAT elicited by 8Br-cyclic AMP. H-89, an inhibitor of cyclic AMP-dependent protein kinase, antagonized the induction of NAT activity by either forskolin or K+ with equal potency for both stimuli. These results suggest that cyclic AMP plays an essential role in the induction of NAT activity that occurs as a consequence of membrane depolarization. Cyclic AMP and Ca2+ may also interact at a step distal to adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1995

5-Methoxytryptamine inhibits cyclic AMP accumulation in cultured retinal neurons through activation of a pertussis toxin-sensitive site distinct from the 2-[125I]iodomelatonin binding site.

Melatonin and 5-methoxytryptamine inhibited forskolin-stimulated cyclic AMP formation in cultured neural cells prepared from embryonic chick retina. Both methoxyindoles exhibited similar potency and efficacy, with EC50 values of 0.8 nM for melatonin and 7.2 nM for 5-methoxytryptamine. Inhibition of cyclic AMP formation by 5-methoxytryptamine or melatonin was prevented by pretreatment with pertussis toxin. Pretreatment of cultures with 5-methoxytryptamine for 24 h reduced the subsequent inhibitory cyclic AMP response to 5-methoxytryptamine but not that to 2-iodomelatonin. Putative melatonin receptors on cultured retinal cells were labeled with 2-[125I]iodomelatonin. Melatonin displaced specific 2-[125I]iodomelatonin with a Ki value (0.8 nM) similar to the EC50 for inhibition of cyclic AMP formation. In contrast, 5-methoxytryptamine only inhibited 2-[125I]iodomelatonin binding at very high concentrations (Ki = 650 nM). Pretreating cultured cells for 24 h with 2-iodomelatonin or melatonin, but not with 5-methoxytryptamine, reduced subsequent 2-[125I]iodomelatonin binding. Thus, 5-methoxytryptamine appears to inhibit forskolin-stimulated cyclic AMP formation at a site distinct from the 2-iodomelatonin binding site.

5-Methoxytryptamine↗
PubMed1995

Melatonin biosynthesis in cultured chick retinal photoreceptor cells: calcium and cyclic AMP protect serotonin N-acetyltransferase from inactivation in cycloheximide-treated cells.

The aim of the present study was to examine the roles of membrane depolarization, calcium influx, and cyclic AMP synthesis in regulating the stability and inactivation of serotonin N-acetyltransferase activity (NAT) in cultured chick photoreceptor cells. NAT activity was induced by pretreating cells for 6 h with 1 microM forskolin. Cycloheximide was subsequently added, and the rate of loss of enzyme activity (inactivation) was determined. After induction, in the presence of cycloheximide, NAT activity declined with a half-life of approximately 30 min. The rate of inactivation was greatly reduced when depolarizing concentrations of K+, forskolin, 8-bromoadenosine 3',5'-cyclic monophosphate, or 3-isobutyl-1-methylxanthine were added together with cycloheximide. The apparent increase in NAT stability caused by K+ was abolished by addition of EGTA or nifedipine and potentiated by Bay K 8644, indicating the involvement of Ca2+ influx through dihydropyridine-sensitive channels. MDL-12330A, and inhibitor of K(+)-stimulated cyclic AMP formation, blocked the effect of depolarizing concentrations of K+. This result suggests that the effect of Ca2+ influx on the stability of NAT is at least partially mediated by increased levels of cyclic AMP. Thus, depolarization-evoked Ca2+ influx and cyclic AMP formation have two roles in the regulation of NAT activity in chick photoreceptor cells. First, they stimulate the de novo synthesis of NAT or a regulatory protein required for NAT activity. Second, they increase the half-life of the enzyme, presumably by regulating the turnover of existing enzyme molecules.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Functional interaction of melatonin receptors and D1 dopamine receptors in cultured chick retinal neurons.

The possible interaction of melatonin receptors and D1 dopamine receptors was investigated in neural cells prepared from embryonic day 8 chick retinas and cultured for 6 d. Dopamine stimulated cAMP accumulation in cultured retinal cells. This effect of dopamine was antagonized by addition of dopamine receptor antagonists (haloperidol and SCH23390) or melatonin receptor agonists (melatonin, 2-iodomelatonin, and 6-chloromelatonin). The inhibition of dopamine-stimulated cAMP accumulation by melatonin was concentration dependent, with half-maximal inhibition at approximately 160 pM. Melatonin inhibited the effect of dopamine at all dopamine concentrations, suppressing the maximal response to the neurotransmitter by approximately 70%. Melatonin also inhibited the stimulation of cAMP accumulation by SKF 82958, a selective D1 dopamine receptor agonist. Pretreatment of cultures with pertussis toxin had no significant effect on dopamine-stimulated cAMP accumulation, but inhibited the response to melatonin. In contrast to its effect on cAMP accumulation, melatonin had no effect on dopamine-stimulated inositol phosphate accumulation. These results suggest that melatonin receptors are coupled to dopamine receptor-regulated adenylate cyclase via an inhibitory G protein, and demonstrate another mechanism, in addition to inhibition of dopamine release, through which melatonin can modulate dopaminergic neurotransmission.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Alterations in light-evoked dopamine metabolism in dystrophic retinas of mutant rds mice.

In dystrophic retinas of rds mice, which are devoid of photoreceptor outer segments, high steady state levels of dopamine were found in dark and light periods. These levels were similar to those observed in normal, BALB/c mouse retinas. Major differences were determined, however, between dopamine turnover in normal and dystrophic retinas. While substantial light-evoked elevation of dopamine synthesis and utilization was observed in normal retinas, dopamine synthesis and metabolism in rds retinas was very low and response to light was depressed. Retinal dopamine metabolism was already depressed in 2 week old rds mice, prior to the onset of photoreceptor cell death, relative to that in age-matched BALB/c mice. At 1 month of age, robust light/dark differences in retinal dopamine metabolism were observed in BALB/c mice, while no significant effect of light was seen in rds mice. The limited ability of the dopaminergic system in rds retinas to respond to light may be due to the absence of normal outer segments. Interestingly, in old rds retinas, although most photoreceptor cells had degenerated, a small but significant light-evoked increase in dopamine metabolism was measured. The presence of relatively high steady state levels of dopamine in rds retinas, despite the reduced dopamine synthetic activity, is maintained by a compensatory reduction in dopamine utilization. Thus, although a considerable amount of dopamine is present in the rds retina, it might not be available to exert its biological functions.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1994

Inhibition of endogenous dopamine release in amphibian retina by L-2-amino-4-phosphonobutyric acid (L-AP4) and trans-2-aminocyclopentane-1,3-dicarboxylate (ACPD).

The metabotropic glutamate receptor agonists 2-amino-4-phosphonobutyric acid (AP4) and trans-2-aminocyclopentane-1,3-dicarboxylate (ACPD) blocked light-stimulated dopamine release from Xenopus laevis retina. ACPD suppressed release in darkness but AP4 did not. AP4 blocked release stimulated in darkness by picrotoxin, a GABA-A receptor antagonist. The data suggest that regulation of dopamine release in Xenopus retina involves subpopulations of metabotropic glutamate receptors.

Aminobutyrates↗
PubMed1994

Melatonin receptor-mediated inhibition of cyclic AMP accumulation in chick retinal cell cultures.

Melatonin receptors were characterized in cultured neurons and photoreceptors prepared from chick embryo retina. Cultured cells contained high-affinity 2-[125I]iodomelatonin binding sites (KD = 41.6 pM), similar to those in intact retina. The effects of melatonin and related indoles on cyclic AMP accumulation were examined. Melatonin (10(-7) M) had no effect on basal or K(+)-stimulated cyclic AMP accumulation, but inhibited forskolin-stimulated cyclic AMP accumulation by approximately 50%. Melatonin inhibited forskolin-stimulated cyclic AMP accumulation in the presence or absence of the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, suggesting an effect on cyclic AMP synthesis rather than degradation. Half-maximal inhibition was observed at 5.9 x 10(-10) M melatonin. The relative order of potency among melatonin analogues was 2-iodomelatonin > melatonin approximately 6-chloromelatonin > or = 6-hydroxymelatonin > N-acetylserotonin approximately 5-methoxytryptophol > serotonin. The EC50 value for inhibition of cyclic AMP accumulation by 2-iodomelatonin (36.7 pM) was comparable to the KD value for binding of the radioligand, suggesting that the binding sites represent functional receptors. The inhibitory effect of melatonin was antagonized by the putative melatonin antagonists luzindole, N-acetyltryptamine, and N-(2,4-dinitrophenyl)-5-methoxytryptamine, with estimated KB values of 0.12, 0.17, and 1 microM, respectively. At a concentration of 10 microM, N-(2,4-dinitrophenyl)-5-methoxytryptamine significantly inhibited forskolin-stimulated cyclic AMP accumulation when added alone; at 30 microM, luzindole and N-acetyltryptamine also had significant inhibitory effects. The inhibitory effect of melatonin was blocked by pretreatment with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1994

Photomechanical movements of cultured embryonic photoreceptors: regulation by exogenous neuromodulators and by a regulable source of endogenous dopamine.

In the retina of nonmammalian vertebrates, light regulates photoreceptor morphology by causing rod photoreceptor elongation and cone photoreceptor contraction. The opposite photomechanical movements occur in the dark, and proceed with a circadian rhythm in many species in vivo. Using dissociated cultures of embryonic chick retina cells, we have recently demonstrated that photoreceptor cells that differentiate in vitro acquire the capacity of responding to light/dark cycles with photomechanical movements (Stenkamp and Adler, 1993). Here we report that the putative neuromodulators melatonin and dopamine can mimic the effects of darkness and light, respectively, on in vitro photomechanical movement. Pharmacological studies showed that dopamine appears to function by means of a D2-type receptor negatively coupled to adenylate cyclase. The effects of light on the cultured photoreceptors were inhibited by dopamine D2 receptor antagonists, and were attenuated by the dopaminergic neurotoxin 6-hydroxydopamine and by the dopamine synthesis inhibitor alpha-methyl-p-tyrosine. The possible existence of an endogenous source of dopamine in the cultures was also suggested by the presence of tyrosine hydroxylase-like immunoreactivity, and of an Na(+)-dependent mechanism for the accumulation of 3H-dopamine, which was predominantly associated with nonphotoreceptor cells. Additionally, 3H-dopamine release occurred in vitro through a Ca(2+)-dependent mechanism, as well as through reverse function of a nomifensine-sensitive dopamine transporter. Both of these putative release mechanisms appeared to be regulated by light and by melatonin, suggesting a mechanism whereby the putative dopaminergic cells may interact with other cells present in the cultures. These studies suggest that complex paracrine neuromodulatory mechanisms can differentiate in low-density embryonic cell culture, that dopaminergic activities exist in vitro, and that they are important for mediating photomechanical movements.

Animals↗
PubMed1992

Melatonin synthesis in chicken retina: effect of kainic acid-induced lesions on the diurnal rhythm and D2-dopamine receptor-mediated regulation of serotonin N-acetyltransferase activity.

The effect of kainic acid (KA)-induced lesions of retinal neurons on regulation of serotonin N-acetyltransferase (NAT) activity in chicken retina was investigated. Although NAT activity was higher in KA-lesioned retinas than in controls, the pattern of diurnal variation of enzyme activity throughout 36 h of constant darkness was similar for both tissues. Quinpirole, a selective D2-dopamine receptor agonist, inhibited the nocturnal increase of NAT activity in both control and KA-treated retinas. Quinpirole was significantly more potent in KA-treated retinas than in controls; the ED50 value for quinpirole was 3 times lower in KA-treated retinas than in control tissues. KA treatment markedly reduced retinal levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC). We conclude that: (1) NAT activity in retina is localized primarily to KA-insensitive cells, presumably photoreceptors; (2) KA-sensitive inner retinal neurons are not essential to the maintenance of the circadian rhythm of NAT activity; and (3) KA-induced lesions of retinal cells result in supersensitivity of D2-dopamine receptors regulating NAT activity in a mechanism that involves adaptive changes following a decline in retinal dopamine neurotransmission.

Animals↗
PubMed1991

Circadian rhythm of tryptophan hydroxylase activity in chicken retina.

1. Retinal tryptophan hydroxylase activity in chickens (1-4 weeks old and embryos) was estimated by determination of levels of 5-hydroxytryptophan (5HTP) in retinas at defined intervals after inhibition of aromatic L-amino acid decarboxylase with m-hydroxybenzylhydrazine (NSD1015). 2. The relationship of tryptophan hydroxylase activity to photoperiod was explored. In chickens maintained on a 12-hr light: 12-hr dark cycle, a diurnal cycle in tryptophan hydroxylase activity was observed. Activity during middark phase was 4.4 times that seen in midlight phase. Cyclic changes in tryptophan hydroxylase activity persisted in constant darkness with a period of approximately 1 day, indicating regulation of the enzyme by a circadian oscillator. The phase of the tryptophan hydroxylase rhythm was found to be determined by the phase of the light/dark cycle. The relationship of the tryptophan hydroxylase rhythm to the light/dark cycle mirrors previously described rhythms of melatonin synthesis and serotonin N-acetyltransferase (NAT) activity in the retina. 3. Light exposure for 1 hr during dark phase suppressed NAT activity by 82%, while tryptophan hydroxylase activity was suppressed by only 30%. 4. Based on the differential responses of retinal NAT activity and tryptophan hydroxylase activity to acute light exposure during dark phase, it was predicted that exposure to light during dark phase would divert serotonin in the retina from melatonin biosynthesis to oxidation by MAO. In support of this, levels of 5-hydroxyindole acetic acid (5HIAA) in retina were found to be elevated approximately two-fold in chickens exposed to 30 min of light during dark phase. In pargyline-treated chickens, 2 hr of light exposure during dark phase was found to increase retinal serotonin levels by 64% over pargyline-treated controls. 5. Cyclic changes in tryptophan hydroxylase activity and NAT activity persisted for 2-3 days in constant light. Tryptophan hydroxylase activity at mid-night gradually decreased on successive days in constant light; on the first day of constant light, tryptophan hydroxylase activity at mid-night was 70% of activity seen during middark phase of the normal light/dark cycle and decreased further on subsequent days. In contrast, on each of 3 days of constant light, NAT activity at mid-night was approximately 15% of normal middark phase activity. 6. Cycloheximide completely inhibited the nocturnal increase in tryptophan hydroxylase activity when given immediately before light offset. The nocturnal increase in NAT activity was inhibited in a similar fashion. 7. Like the development of the NAT rhythm, cyclic changes of tryptophan hydroxylase activity in the retinas of chickens began on or immediately before the day of hatching. hatching.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1991

K(+)-evoked depolarization stimulates cyclic AMP accumulation in photoreceptor-enriched retinal cell cultures: role of calcium influx through dihydropyridine-sensitive calcium channels.

The effect of membrane depolarization on cyclic AMP synthesis was studied in glia-free, low-density, monolayer cultures of chick retinal photoreceptors and neurons. In photoreceptor-enriched cultures prepared from embryonic day 6 retinas and cultured for 6 days, elevated K+ concentrations increased the intracellular concentration of cyclic AMP and stimulated the conversion of [3H]adenine to [3H]cyclic AMP. The K(+)-evoked increase of cyclic AMP accumulation was blocked by omitting CaCl2 from the incubation medium, indicating a requirement for extracellular Ca2+. Stimulation of cyclic AMP accumulation was also inhibited by nifedipine, methoxyverapamil, Cd2+, Co2+, and Mg2+, and was enhanced by the dihydropyridine Ca2+ channel agonist Bay K 8644. The enhancement of K(+)-evoked cyclic AMP accumulation by Bay K 8644 was antagonized by nifedipine. Thus, Ca2+ influx through dihydropyridine-sensitive channel is required for depolarization-evoked stimulation of cyclic AMP accumulation in photoreceptor-enriched cultures.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Effects of apomorphine, a dopamine receptor agonist, on ocular refraction and axial elongation in a primate model of myopia.

The authors examined the effect of local administration of a dopamine receptor agonist on visual deprivation-induced excessive ocular growth and myopia. Eight rhesus monkeys were monocularly deprived of vision from birth with opaque contact lenses. Four of the monkeys received drops of 1% apomorphine HCl 2-3 times/day in the occluded eye; the four control monkeys received vehicle only. Axial lengths were determined by A-scan ultrasonography at birth and at 5-7 months of age. The authors assessed the axial elongation by comparing the postnatal growth in the axial dimension of the occluded eyes with the postnatal growth in nonoccluded eyes. In three of the four control monkeys, occlusion increased axial growth by an average of 1.3 mm. In contrast, they found that growth of the occluded and nonoccluded eyes of the apomorphine-treated monkeys was equivalent, except in one monkey whose nonoccluded eye did not develop normally and was anomalously small. At 6.5-9.5 months of age, three of four controls had myopic refractive errors (-3 to -7 diopters) in the occluded eyes; three of four of the apomorphine-treated monkeys had hyperopic refractive errors (+1-(+)3 diopters) in their occluded eyes. The occluded eye of the fourth monkey was only -0.5 diopters myopic. The findings suggest that apomorphine administration retards excessive axial elongation and the concomitant development of myopia associated with visual deprivation in primates.

Administration, Topical↗
PubMed1990

Arylalkylamine (serotonin) N-acetyltransferase assay using high-performance liquid chromatography with fluorescence or electrochemical detection of N-acetyltryptamine.

A sensitive, rapid, and economical method has been developed for determination of serotonin N-acetyltransferase activity from a variety of enzyme sources. The assay is based upon separation and detection of N-acetyltryptamine formed from tryptamine and acetyl coenzyme A, by means of high-performance liquid chromatography with either electrochemical or fluorometric detection. The limit of sensitivity with both detection methods is less than 20 pmol of N-acetyltryptamine formed per sample. A method for synthesis of N-acetyltryptamine, used as an external standard in the assay, is described.

Acetyltransferases↗
PubMed1990

Postnatal control of ocular growth: dopaminergic mechanisms.

A vision-dependent feedback mechanism contributes to the regulation of postnatal eye growth and refraction; this mechanism is located at least in part in the retina. In chicks and rhesus monkeys, visual deprivation leads to ocular enlargement and a myopic refractive error, and it also reduces the retinal concentration of dopamine. In neonatal chicks, local application of the dopamine agonist apomorphine limits the excessive axial elongation that is associated with visual deprivation. Both D1 and D2 dopamine receptor mechanisms may participate. Remarkably, apomorphine is geometrically selective, not limiting the exaggerated equatorial growth that occurs during visual deprivation. Preliminary results in neonatal rhesus monkeys indicate that apomorphine eye drops also suppress exaggerated axial growth and myopic refractive error during visual deprivation; equatorial diameters were not measured. Our observations are consistent with a retinal hypothesis for emmetropization and suggest that retinal dopamine participates in a mechanism linking ocular growth control to vision. In addition, we conclude that axial and equatorial dimensions are independently regulated in the chick.

Animals↗
PubMed1990

Depletion of catecholamines in the brain of rats differentially affects stimulation of locomotor activity by caffeine, D-amphetamine, and methylphenidate.

The purpose of this study was to assess the role of catecholamines in brain, in the stimulation of locomotor activity, induced by caffeine, as compared to the psychomotor stimulants D-amphetamine and methylphenidate. Adult male rats were pretreated with either (1) 2.5 mg/kg (i.p.) reserpine, 24 hr prior to testing of locomotor activity, (2) 50 mg/kg (i.p.) alpha-methyl-para-tyrosine (AMPT) 6 hr and 2 hr prior to testing of locomotor activity, (3) 200 micrograms/rat (i.c.v.) 6-hydroxydopamine (6-OHDA), or 25 mg/kg (i.p.) desmethylimipramine (DMI) and 200 micrograms/rat 6-OHDA (i.c.v.), 6-8 weeks prior to testing. Each treatment group had a matched control group. Levels of catecholamines in the forebrain were determined in each of the treatment and corresponding control groups. All rats were tested with doses of caffeine, D-amphetamine and methylphenidate (excluding the 6-OHDA-treated animals), administered in random order intraperitoneally 35 min before locomotor activity was measured for 30 min. Pretreatment with either reserpine or AMPT attenuated the stimulation of locomotor activity induced by caffeine and D-amphetamine but not that induced by methylphenidate. The dose-response curve for amphetamine was shifted downward and to the right by reserpine but was flattened by AMPT. The dose-response curve for caffeine was displaced downward in a similar manner by both reserpine and AMPT. Treatment with 6-OHDA or DMI + 6-OHDA produced the expected changes in the content of catecholamines in brain, but failed to modify dose-response curves for caffeine or amphetamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗