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Biomedical subjects

R B Robinson

Publications and source records attributed to R B Robinson.

At least 19 recordsLinked to original sources
PubMed1994

An excitatory muscarinic response in neonatal rat ventricular myocytes and its modulation by sympathetic innervation.

Previously, we demonstrated that low concentrations of acetylcholine (< or = 10(-9) M) increased automaticity in neonatal but not in adult rat ventricular myocardium. In the present study, we used cultured neonatal rat ventricular myocytes grown alone or in the presence of dissociated sympathetic neurons as an experimental model to study the ontogeny of the muscarinic response. McN A343 (< or = 10(-9) M), an M1 selective agonist, increased spontaneous rate from 51 +/- 4 to 56 +/- 5 beats per minute (bpm), and this excitatory response was blocked by 10(-9) M pirenzepine, an M1 selective antagonist, but not by the M2 selective antagonist AFDX-116, nor by the alpha 1 adrenergic antagonist prazosin and the beta adrenergic antagonist propranolol (all 10(-7) M) In innervated myocytes, McN A343 also increased rate from 48 +/- 6 to 55 +/- 6 bpm. However, this effect was blocked by either 10(-9) M pirenzepine or 10(-7) M propranolol. After pretreatment with 10 ng/ml of pertussis toxin, the McN A343-induced excitatory response in non-innervated myocytes was absent, thus suggesting that this response involved a pertussis toxin-sensitive G protein dependent pathway. McN A343 failed to stimulate inositol phosphate or cAMP accumulation in non-innervated myocytes. These results demonstrate the following. (1) The muscarinic excitatory response is mediated via direct stimulation of a post-synaptic M1 receptor in non-innervated myocytes. (2) The excitatory response after innervation is related to the release of catecholamines, possibly through activation of muscarinic receptors located at the pre-synaptic sympathetic nerve terminals. (3) Sympathetic innervation prevents the functional expression of the post-synaptic myocardial M1 receptor. (4) The intracellular pathway for the post-synaptic M1 excitatory response involves a pertussis toxin-sensitive G protein, but does not depend on obvious changes in cAMP or phosphoinositide hydrolysis.

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗
PubMed1994

Tubulin binding agent CI-980 has positive inotropic and local anesthetic actions.

Tubulin binding agents inhibit tubulin polymerization by actions at specific binding sites. CI-980 acts at the colchicine-binding site, which is distinct from the vinca-alkaloid binding site. We studied the actions of CI-980 in two models: neonatal rat myocytes in tissue culture and adult canine Purkinje fibers. In the first model, experiments on cell shortening and calcium signaling (using fluo3) showed that CI-980 increased the amplitude of both cell shortening and the Ca signal. The comparison drug, vinblastine, shared the effect on Ca signaling, but not that on cell shortening. In addition, high concentrations of CI-980 decreased the beating rate of spontaneously firing cell cultures. In canine Purkinje fibers, CI-980 decreased action potential amplitude (APA), Vmax, and conduction velocity and prolonged repolarization. It also decreased automaticity and suppressed delayed afterdepolarizations (DAD). These studies suggest that CI-980 is a novel compound in that it exerts antiarrhythmic effects on the AP but is positively inotropic. Whether all these actions derive from a primary effect on tubulin or whether they reflect action on both tubulin and transsarcolemmal ion channels remains to be determined.

Action Potentials↗
PubMed1992

Immunological rejection of heart transplant: how lytic granules from cytotoxic T lymphocytes damage guinea pig ventricular myocytes.

We investigated the mechanism by which lytic granules extracted from cytotoxic T lymphocytes (CTL) damage guinea pig ventricular myocytes in order to determine whether their actions can be related to the overall immunological rejection of the transplanted heart. Granule-induced myocyte morphological changes and final destruction were preceded by shortening of action potential duration (APD) and reductions of the resting potential and the action potential amplitude. APD shortening was probably caused by a granule-induced increase in outward current (most likely non-specific). Ryanodine, which blocks Ca2+ release from the sarcoplasmic reticulum, did not interfere with the morphological and electrophysiological effects of lytic granules. Fura-2 imaging indicated that [Ca2+]i initially increased about 2-fold from 90.0 +/- 11.5 nM, while cell length decreased less than 5% from a mean value of 99.0 +/- 9.0 microns. A further increase in [Ca2+]i (greater than 10 fold) was associated with progressive contracture and destruction, suggesting that the structural damage inflicted by lytic granules is caused by [Ca2+]i overload. The results indicate that the cytocidal action of CTL-derived lytic granules may be involved in immunologically induced damage, even to the extent of rejection of the transplanted heart.

Action Potentials↗
PubMed1992

Sympathetic neurons mediate developmental change in cardiac sodium channel gating through long-term neurotransmitter action.

Innervation of nerve and muscle cells during development is often accompanied by changes in the expression and function of ion channels in the postsynaptic cell. However, the signaling pathways whereby the presynaptic nerve influences the properties of the postsynaptic cell are less well understood. Indirect evidence suggests that cardiac voltage-gated Na+ channels undergo important changes during development. Here, we compare directly single voltage-gated Na+ channel currents from neonatal and adult rat ventricular myocytes and report a negative shift in the voltage dependence of channel gating during development, leading to a significant speeding of channel activation and inactivation at a fixed membrane potential. These developmental changes can be mimicked in vitro by innervation of neonatal myocytes with sympathetic neurons. The effect of sympathetic neurons is blocked by the beta-adrenergic receptor antagonist propranolol and is mimicked by prolonged coculture of neonatal myocytes with a membrane-permeable cAMP analog. Thus presynaptic neurons can control the developmental phenotype of ion channels in a postsynaptic cell through a classic receptor-mediated neurotransmitter action that involves a defined second messenger pathway.

Adrenergic Fibers↗
PubMed1991

Chronic exposure to neuropeptide Y determines cardiac alpha 1-adrenergic responsiveness.

The onset of sympathetic innervation induces a developmental change in the cardiac alpha 1-adrenergic chronotropic response from an increase to a decrease in rate. The mechanism by which innervation induces this alteration is unknown. Neuropeptide Y (NPY), which is found abundantly in cardiac sympathetic nerve terminals, was considered as a possible mediator for this effect. Chronic conditioning by NPY in noninnervated myocyte cultures stimulated the effect of sympathetic innervation in inducing the alpha 1-inhibitory chronotropic response. Chronic conditioning by the NPY antagonist PYX-2 blocked the effect of innervation. Thus endogenous NPY may modulate alpha 1-adrenergic responsiveness during the ontogeny of cardiac sympathetic innervation.

Analysis of Variance↗
PubMed1991

Thrombin modulates phosphoinositide metabolism, cytosolic calcium, and impulse initiation in the heart.

Thrombin stimulates phosphoinositide hydrolysis and increases cytosolic calcium in several types of cells. To determine whether thrombin exerts similar stimulatory actions in the heart and whether this mechanism is linked to changes in cardiac electrical activity, the effects of thrombin on several biochemical and electrophysiological parameters were examined. In neonatal rat ventricular myocyte cultures freed of fibroblast contamination by irradiation, thrombin rapidly induced the breakdown of phosphoinositides. Formation of inositol trisphosphate was detectable within 5 seconds and was followed by the sequential accumulation of inositol bisphosphate and inositol monophosphate. The effect of thrombin to stimulate phosphoinositide hydrolysis was inhibited by hirudin, but not by propranolol, prazosin, or pretreatment with pertussis toxin. The inositol phospholipid response was unassociated with changes in intracellular cAMP levels. To determine the electrophysiological effects of thrombin, we used microelectrode techniques to study canine Purkinje fibers. Thrombin increased the beating rate of fibers depolarized using barium, but not those at normal maximal diastolic potential. In addition, thrombin prolonged the action potential duration in fibers driven at a constant cycle length. This response was inhibited by hirudin and nisoldipine, but not by propranolol, prazosin, or pretreatment with pertussis toxin. Thrombin also augmented cesium-induced early afterdepolarizations. Using the fluorescent calcium indicator fura-2, we demonstrated that thrombin increased the beating rate, diastolic calcium, and peak systolic calcium of spontaneously contracting cultured ventricular myocytes. Cytosolic calcium also increased in both rat ventricular myocytes and canine Purkinje myocytes that were electrically driven at a constant basic cycle length, indicating that thrombin modulates cellular calcium metabolism independent of its actions to enhance automaticity. Taken together, these findings demonstrate several novel biological actions of thrombin in the mammalian heart that may be functionally related. The actions of thrombin to enhance automaticity and prolong repolarization may contribute to the electrical abnormalities observed in the setting of myocardial ischemia and infarction.

Action Potentials↗
PubMed1990

Functional uncoupling of the inhibitory alpha 1-adrenergic response from a G-protein in innervated cultured cardiac cells by K+ depolarization.

Under normal physiological conditions, the adult rat heart exhibits an alpha 1-adrenergic mediated decrease in rate. The negative chromotropic effect of alpha 1-stimulation in the adult depends upon maturation of sympathetic innervation and the presence of a pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein. We have previously used a cell culture model of neonatal myocardial cells to demonstrate more directly that sympathetic innervation is an important feature of the mature response. After alpha 1-adrenergic stimulation, neonatal rat ventricular myocytes cultured with sympathetic ganglion cells [nerve-muscle (NM) co-cultures] respond predominantly by a decrease in rate, whereas pure muscle cultures show an exclusive increase in rate. Since it has been reported that the inhibitory alpha 1-response in intact tissue is lost upon depolarization, the present study was designed to determine whether the negative chronotropic response could be reversed by potassium (K+) depolarization. We also investigated whether there might be an associated reduction in the PT-sensitive G protein linked to the negative chronotropic response. Thus, the effect of high K+ depolarization on both the alpha 1-adrenergic chronotropic response and the level of the PT-sensitive G protein was examined in NM co-cultures. Extracellular high K+ acutely and reversibly converted the phenylephrine-mediated chronotropic response from negative to positive. The positive chronotropic response in high K+ was alpha 1-mediated and not secondary to catecholamine release from adrenergic neurons. Loss of the inhibitory response in high K+ was not associated with a change in the level of the PT-sensitive G protein. Thus, the presence of a PT-sensitive G protein is necessary, but not sufficient to permit the expression of the mature alpha 1-adrenergic negative chronotropic response in innervated cardiac cells in culture.

Animals↗
PubMed1990

Phospholipase C modulates automaticity of canine cardiac Purkinje fibers.

Alpha-1 adrenergic agonists increase cardiac Purkinje fiber automaticity and elevate D-myo-inositol-trisphosphate (IP3) levels. To learn about the relationship between phosphoinositide metabolism and the modulation of cardiac rhythm, we used phospholipase C to activate phosphoinositide hydrolysis in an alpha-1 receptor-independent fashion and determined whether this intervention modulated automaticity. We used standard microelectrode techniques to study automaticity in adult Purkinje fiber bundles, fluorescence microscopy to study fura-2 fluorescence in isolated Purkinje and ventricular myocytes and standard biochemical techniques to measure inositol phosphate production in ventricular myocytes. Phospholipase C increased Purkinje fiber automaticity, a process that was enhanced by 10 mM lithium (which had no effect alone) and suppressed by verapamil or ryanodine (both 10 microM). Superfusion with 12-O-tetradecanoyl-phorbol-13-acetate phorbol ester, phospholipase D and A2, as well as L-alpha-phosphatidic acid, trypsin and D-myo-inositol-1-phosphate, D-myo-inositol-1,4-bisphosphate, IP3 and D-myo-inositol-1,4,5,6-tetrakisphosphate did not affect automatic rate or transmembrane potentials. Biochemical studies of ventricular myocytes demonstrated a phospholipase C-induced increase in intracellular and extracellular IP3, D-myo-inositol-1,4-bisphosphate and D-myo-inositol-1-phosphate at 3 min, with the extracellular increase persisting thereafter. Fluorescence microscopy with fura-2 revealed that phospholipase C increased systolic-free calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Muscarinic modulation of cardiac rate at low acetylcholine concentrations.

Slowing of cardiac pacemaking induced by cholinergic input is thought to arise from the opening of potassium channels caused by muscarinic receptor stimulation. In mammalian sinoatrial node cells, however, muscarinic stimulation also inhibits the hyperpolarization-activated current (If), which is involved in the generation of pacemaker activity and its acceleration by catecholamines. Acetylcholine at nanomolar concentrations inhibits If and slows spontaneous rate, whereas 20 times higher concentrations are required to activate the acetylcholine-dependent potassium current (IK,ACh). Thus, modulation of If, rather than IK,ACh, is the mechanism underlying the muscarinic control of cardiac pacing at low (nanomolar) acetylcholine concentrations.

Acetylcholine↗
PubMed1989

Single sodium channels from canine ventricular myocytes: voltage dependence and relative rates of activation and inactivation.

1. Single sodium channel currents were recorded from canine ventricular myocytes in cell-attached patches. The relative rates of single-channel activation vs. inactivation as well as the voltage dependence of the rate of open-channel inactivation were studied. 2. Ensemble-averaged sodium currents showed relatively normal activation and inactivation kinetics, although the mid-point of the steady-state inactivation (h infinity) curve was shifted by 20-30 mV in the hyperpolarizing direction. This shift was due to the bath solution, which contained isotonic KCl to depolarize the cell to 0 mV. 3. Steady-state activation showed less of a voltage shift. The threshold for eliciting channel opening was around -70 mV and the mid-point of activation occurred near -50 mV. 4. The decline of the ensemble-averaged sodium current during a maintained depolarization was fitted by a single exponential function characterizing the apparent time constant of inactivation (tau h). The apparent rate of inactivation was voltage dependent, with tau h decreasing e-fold for a 15.4 mV depolarization. 5. The relative contributions of the rates of single-channel activation and inactivation in determining the time course of current decay (tau h) were examined using the approach of Aldrich, Corey & Stevens (1983). Mean channel open time (tau o) showed significant voltage dependence, increasing from 0.5 ms at -70 mV to around 0.8 ms at -40 mV. At -70 mV tau h was much greater than tau o, while at -40 mV the two time constants were similar. 6. The degree to which the kinetics of single-channel activation contribute to tau h was studied using the first latency distribution. The first latency function was fitted by two exponentials. The slow component was voltage dependent, decreasing from 19 ms at -70 mV to 0.5 ms at -40 mV. The fast component (0.1-0.5 ms) was not well resolved. 7. Comparing the first latency distribution with the time course of the ensemble-averaged sodium current at -40 mV showed that activation is nearly complete by the time of peak inward sodium current. However, at -70 mV, activation overlaps significantly with the apparent time course of inactivation of the ensemble-averaged current. 8. Using the methods of Aldrich et al. (1983) we also measured the apparent rate of open-channel closing (a) and open-channel inactivation (b). Both rates were voltage dependent, with a showing an e-fold decrease for an 11 mV depolarization and b showing an e-fold increase for a 30 mV depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1989

Developmental changes in guanine nucleotide regulatory proteins in the rat myocardial alpha 1-adrenergic receptor complex.

During development, the cardiac alpha 1-adrenergic chronotropic response changes from positive in the neonate to negative in the adult. The negative chronotropic effect of alpha 1-adrenergic stimulation in the adult depends on maturation of sympathetic innervation and the presence of a pertussis toxin (PT)-sensitive guanine nucleotide-binding (G) protein. To examine the possibility of a developmental change in coupling of a PT-sensitive G protein to the alpha 1-adrenergic receptor, radioligand binding experiments with the iodinated alpha 1-selective radioligand [125I]-I-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone ([ 125I]-IBE 2254) were performed on membranes prepared from control and PT-treated neonatal and adult rat hearts. Scatchard analysis showed fewer alpha 1-adrenergic receptors in the adult than in the neonate (168 +/- 10 fmol/mg protein in the neonate vs. 124 +/- 13 fmol/mg protein in the adult), but similar affinities (equilibrium dissociation constant 124 +/- 29 pM in the neonate vs. 140 +/- 34 pM in the adult). PT treatment did not alter the results. In both the neonate and adult, 5'-guanylylimidodiphosphate [Gpp(NH)p, 500 microM] shifted the l-epinephrine competition curve to the right and increased the slope factor toward unity. PT had no effect on the l-epinephrine competition curve in the neonate. However, in the adult PT itself caused a partial shift in the agonist competition curve, reducing but not eliminating the effect of Gpp(NH)p. Consistent with the results from the binding experiments, PT did not have any effect on the alpha 1-adrenergic-mediated positive chronotropic response in the neonate, whereas in the adult the alpha 1-adrenergic-mediated negative chronotropic response was completely converted to a positive one after PT treatment. These results indicate the presence of a PT-insensitive G protein in the neonatal and adult rat heart and the acquisition of a PT-sensitive G protein linked to the negative chronotropic response during development.

Adenosine Diphosphate Ribose↗
PubMed1989

At physiologic albumin/oleate concentrations oleate uptake by isolated hepatocytes, cardiac myocytes, and adipocytes is a saturable function of the unbound oleate concentration. Uptake kinetics are consistent with the conventional theory.

To reexamine the role of albumin in cellular uptake of long chain fatty acids, we measured [3H]oleate uptake by isolated hepatocytes, adipocytes, and cardiac myocytes from incubations containing oleate/albumin complexes at molar ratios from 0.01:1 to 2:1. For each ratio the uptake was studied over a wide range of albumin concentrations. In all three cell types and at any given oleate/albumin ratio, the uptake appeared saturable with increasing concentrations of oleate:albumin complexes despite the fact that the unbound oleate concentration for each molar ratio is essentially constant. However, the "Km" but not the "Vmax" of these pseudosaturation curves was influenced by substrate availability. At low albumin concentrations, uptake velocities did not correlate with unbound oleate concentrations. However, observed and expected uptake velocities coincided at albumin concentrations approaching physiologic levels and were a saturable function of the oleate/albumin ratios and the consequent unbound oleate concentrations employed. Hence, under the experimental conditions employed in this study using a variety of suspended cell types, oleate uptake kinetics were consistent with the conventional theory at physiologic concentrations of albumin.

Adipose Tissue↗
PubMed1989

Alpha-1 adrenergic stimulation of 1,4,5-inositol trisphosphate formation in ventricular myocytes.

We demonstrated previously that alpha-1 adrenergic catecholamines modulate cardiac automaticity in a manner that is dependent upon the function of a pertussis toxin sensitive guanine nucleotide binding protein (G protein). Furthermore, we demonstrated that alpha-1 adrenergic receptor stimulation promotes the accumulation of inositol monophosphate (IP1). In the present study we used high-pressure liquid chromatography to resolve individual inositol phosphate isomers formed in norepinephrine-stimulated cultured rat ventricular myocytes. Norepinephrine stimulated a rapid, transient increase in 1,4,5-inositol trisphosphate (1,4,5-IP3) which was followed by slower, sustained increases in 1,3,4-IP3, inositol bisphosphate (IP2) and IP1. IP1 was composed of two major isomers with retention times characteristic of 1-IP1 and 4-IP1. 4-IP1 was the predominant IP1 isomer formed during stimulation with norepinephrine suggesting that the polyphosphoinositides rather than phosphatidylinositol are the principal targets of norepinephrine-stimulated phospholipase C activity in the heart. This was confirmed in studies performed on myocyte membranes which demonstrated proportionately greater IP2 and IP3 (relative to IP1) accumulation in response to norepinephrine. G protein regulation of alpha-1 adrenergic-dependent inositol phospholipid hydrolysis also was examined. In myocyte membranes, guanosine-5'-0-(3-thiotriphosphate) induced the accumulation of IP2 and IP3 and was required for the stimulatory effect of norepinephrine. This response was not impaired after pretreatment with pertussis toxin. These results indicate that the myocyte alpha-1 adrenergic receptor is coupled to a polyphosphoinositide-specific phospholipase C by a pertussis toxin insensitive G protein and suggest that under certain conditions IP3 may serve an important role in alpha-1 adrenergic modulation of cardiac function.

Animals↗
PubMed1988

Sympathetic neural and alpha-adrenergic modulation of arrhythmias.

alpha 1-Adrenergic stimulation of the neonatal heart may induce either an increase or a decrease in ventricular automaticity, with the latter response predominating as age increases. We used isolated tissues from the hearts of neonatal and adult dogs and rats, as well as rat myocytes in tissue culture alone or in coculture with sympathetic nerves, to study the role of sympathetic innervation in modulating the alpha-adrenergic response. In the absence of sympathetic innervation, alpha-adrenergic stimulation uniformly increases automaticity. As the myocyte is innervated, an increased quantity of a GTP regulatory protein is detectable. That this protein is an essential transducer of alpha-adrenergic inhibition of automaticity is evidenced by the conversion of the alpha response from excitatory to inhibitory as the protein develops. ADP-ribosylation of the protein with pertussis toxin causes the alpha response to revert to excitation in both adult canine hearts and innervated myocytes in tissue culture. Hence, we have evidence for sympathetic modulation of cardiac rhythm via a regulatory protein whose function depends on normal neuronal development. Abnormal development of innervation may predispose to arrhythmogenesis via persistence of a primitive response to alpha stimulation.

Animals↗
PubMed1988

Oleate uptake by cardiac myocytes is carrier mediated and involves a 40-kD plasma membrane fatty acid binding protein similar to that in liver, adipose tissue, and gut.

Uptake of [3H]oleate by canine or rat cardiac myocytes is saturable, displays the countertransport phenomenon, and is inhibited by phloretin and trypsin. Cardiac myocytes contain a basic (pI approximately 9.1) 40-kD plasma membrane fatty acid binding protein (FABPPM) analogous to those recently isolated from liver, adipose tissue, and gut, unrelated to the 12-14-kD cytosolic FABP in these same tissues. An antibody to rat liver FABPPM selectively inhibits specific uptake of [3H]oleate by rat heart myocytes at 37 degrees C, but has no influence on nonspecific [3H]oleate uptake at 4 degrees C or on specific uptake of [3H]glucose. Uptake of long-chain free fatty acids by cardiac muscle cells, liver, and adipose tissue and absorption by gut epithelial cells is a facilitated process mediated by identical or closely related plasma membrane FABPs.

Adipose Tissue↗
PubMed1988

The positive chronotropic effect of acetylcholine has muscarinic and nicotinic components in the neonatal rat heart.

Acetylcholine increases ventricular automaticity in neonatal but not adult canine Purkinje fibers. In this study, we used a rat model to investigate the mechanism for the increased automaticity, and used surface electrodes to record spontaneous rates from the ventricular septa of three different age groups: 1 to 2 days old (neonates), 6 to 9 days old (1 week old) and adults. Acetylcholine, 10(-12) and 10(-11) M, induced a significant increase in automaticity from a control of 103 +/- 6.5 beats per min to 117 +/- 9.0 and 118 +/- 10.8 beats per min, respectively, in the neonates (P less than .05). The increase was attenuated by atropine, 2 x 10(-6) M (P = .05), and eliminated by propranolol, 2 x 10(-7) M, or hexamethonium, 5 x 10(-6) M (P less than .05). In 1-week-old rats, acetylcholine, 10(-12) M, induced a lesser increase in automaticity from a control of 106 +/- 13.0 to 113 +/- 14.0 beats per min (P less than .05). The increase was blocked by atropine, 2 x 10(-6) M, propranolol, 2 x 10(-7) M, and by hexamethonium, 5 x 10(-6) M (all P less than .05). In adults, acetylcholine did not increase automaticity. Among the neonatal septa, 82% showed increased automaticity with acetylcholine alone, 78% showed increased automaticity in the presence of atropine and 13% showed increased automaticity in the presence of either propranolol or hexamethonium, suggesting a largely nicotinic mediated and catecholamine dependent component. In 1-week-old septa, 75% showed increased automaticity with acetylcholine alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1987

An inexpensive dual-excitation apparatus for fluorescence microscopy.

Ion sensitive indicator molecules can be employed in conjunction with fluorescence microscopy in single cells to measure rapid changes in the intracellular concentration of several ionic species. A number of these probes (e.g. fura-2) require the capability of measuring emission intensity at two excitation wavelengths to quantitate properly intracellular ion concentration. We have developed a simple dual-excitation apparatus for use in such applications. The apparatus switches the excitation filter within 150 ms. This economical apparatus is well suited in situations where the ionic concentration of interest is changing relatively slowly. Moreover, by synchronizing the device's action with an external stimulus, rapid and reproducible ionic changes in excitable tissue also can be measured.

Animals↗