Effect of catecholamines on excitability of single mechanoreceptors of Pacinian corpuscles

A method of continuous external perfusion was used to study the effect of adrenalin and noradrenalin on the excitability of single isolated mechanoreceptors of Pacinian corpuscles. On the addition of adrenalin and noradrenalin in concentrations of 1·10^–5–1·10^–7 g/ml to the solution the excitability of the receptors was increased by 10–15% and the amplitude of the receptor potential rose on the average by 10–20%. It is postulated that the change in excitability is due to an increase in the receptor membrane potential. The results are discussed in connection with the possible adrenergic innervation of the Pacinian corpuscles.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 6, No. 3, pp. 312–317, May–June, 1974.     

Analysis of the modulating effect of catecholamines on electrogenesis in an isolated mechanoreceptor

Perfusion of isolated Pacinian corpuslces in the cat mesentery with solutions of dibutyryl-cAMP or theophylline caused an increase in spike activity against the background of a reduction in amplitude of receptor potentials and lowering of the threshold for spike generation. The opposite effect on mechanoreceptor electrical responses was found when catecholamines were used. It was postulated on the basis of these findings that the action of catecholamines on Pacinian corpuscles is accompanied by a fall in the cAMP level in the sensory nerve terminal. Noradrenalin caused a decrease in adenylate cyclase activity in receptor homogenates. The activating effect of noradrenalin on Na⁺, K⁺-ATPase, abolished by phentolamine but unchanged by propranolol, was demonstrated. The possible role of -adrenoreceptors in the modulating effect of catecholamines on electrical activity of Pacinian corpuscles is discussed.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 14, No. 2, pp. 158–163, March–April, 1982.     

Role of non-quantum acetylcholine secretion in neural control of the membrane potential in skeletal muscle fibers of rats

Experiments on muscle fibers of the rat diaphragm (in vitro denervation) showed that their three-hour incubation in the cultural medium results in an 8-mV drop in the resting membrane potential (RMP). Addition of 5·10^–8 M carbacholine to the cultural medium, mimicing the effect of non-quantum acetylcholine, delayed depolarization of the denervated muscle. The effect of carbacholine could not be eliminated byd-tubocurarine (5·10^–6 M), a postsynaptic acetylcholine receptor blocker, and by ouabain (1·10^–4 M), and inhibitor of Na⁺, K⁺-ATPase of the membrane. At the same time, the effect could be completely eliminated by Mg²⁺ ions (5·10^–3 M), which blocked Ca²⁺ channels of the membrane, by N-nitroarginine (1·10^–4 M), which inhibited the enzyme NO-synthase, and by hemoglobin (2·10^–5 M), which inactivated the extracellular NO molecules. It is concluded that the released non-quantum acetylcholine can contribute to neural control of RMP of cross-striated muscle fibers via the Ca²⁺-dependent activation of NO synthesis in the sarcoplasm. The NO molecules can play the role of a retrograde signal indicative of the normal functioning of the neuromuscular synapse. The impairment of this link caused by a denervation-induced cessation of the non-quantum secretion can serve as a signal triggering the early changes in the muscle membrane following nerve transection.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 67–71, January–February, 1995.     

Nitric oxide control of large veins in the toad <Emphasis Type="Italic">Bufo marinus</Emphasis>

This study examined the nitric oxide (NO) control of the vascular smooth muscle of the ventral abdominal vein and vena cava of the toad, Bufo marinus, by using anatomical and physiological approaches. Nicotinamide adenine di-nucleotide phosphate-diaphorase histochemistry and immunohistochemistry using endothelial nitric oxide synthase (NOS) and neural NOS antibodies produced no evidence for endothelial NOS in the veins, but, neural NOS-immunoreactive perivascular nerves were present. Acetylcholine (10^–5 M) caused a vasodilation in both veins that was endothelium-independent, and which was blocked by the soluble guanylyl cyclase inhibitor, ODQ (10^–5 M). The NOS inhibitors, L-NNA (10^–4 M) and L-NAME (10^–4 M), did not significantly reduce the vasodilatory effect of acetylcholine in the veins; this suggested that the vasodilation was not due to NO. However, in the presence of phenoxybenzamine (10^–7–10^–8 M), L-NNA significantly reduced the vasodilatory effect of acetylcholine in the veins. This unusual response is due to phenoxybenzamine partially inactivating the muscarinic receptor pool in the veins. In addition, the neural NOS inhibitor, vinyl-L-NIO (10^–5 M), significantly reduced the acetylcholine-mediated vasodilation in the presence of phenoxybenzamine. The results show that in toad veins, nitrergic nerves rather than an endothelial NO system are involved in NO-mediated vasodilation.     

Investigation of mechanisms of synaptic transmission in ampullae of Lorenzini in the skate

The effect of magnesium ions, L-glutamate (L-GLU), and the diethyl ester of glutamic acid (DEE-GLU) on temperature and electrical sensitivity of the ampullae of Lorenzini in skates was studied by the method of perfusion of the basal membrane of electroreceptor cells and recording spike activity from single nerve fibers. Addition of 10^–4–10^–5 M L-GLU to the solution was shown to cause an increase in the spontaneous discharge frequency of receptors with low initial level of activity (8–20 spikes/sec) and a decrease in receptors with spontaneous activity of 22–42 spikes/sec. In solution with an increased magnesium ion concentration (15–50 mM) both spontaneous and evoked receptor activity was blocked, Under these conditions the addition of L-GLU to the solution caused partial recovery of spontaneous receptor activity. Reversible blocking of spontaneous and evoked receptor activity was observed in a solution containing 10^–4–10^–3 M DEE-GLU. It is suggested that L-GLU is the synaptic transmitter in the ampullae of Lorenzini of the skate.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 13, No. 3, pp. 292–298, May–June, 1981.     

Analysis of the mechanism of amplitude change in Pacinian corpuscle receptor potential in a solution with a decreased calcium ion concentration

The effect of Ca2+-free solution on the amplitude increase in the receptor potential (RP) of Pacinian corpuscles was studied using external perfusion technique. The RP amplitude increased in Ca2+-free solution. It was blocked after addition of 10-20 mM of tetraethylamonium. A temporary increase in the RP amplitude is seen in the solution with 0.2 mM of 2.4-dinitrophenol. Sensitivity of the receptor membrane to mechanical stimuli does not change in Ca2+-free solution. It is suggested that near the mechanosensitive ionic canal of Pacinian corpuscle receptor membrane the fixed negative charges which could influence the "gate" system state of the mechanosensitive canal are absent.     

Activation of muscarinic receptors in porcine airway smooth muscle elicits a transient increase in phospholipase D activity

Phospholipase D (PLD) is a phosphodiesterase that catalyses hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. In the presence of ethanol, PLD also catalyses the formation of phosphatidylethanol, which is a unique characteristic of this enzyme. Muscarinic receptor-induced changes in the activity of PLD were investigated in porcine tracheal smooth muscle by measuring the formation of [³H]phosphatidic acid ([³H]PA) and [³H]phosphatidylethanol ([³H]PEth) after labeling the muscle strips with [³H]palmitic acid. The cholinergic receptor agonist acetylcholine (Ach) significantly but transiently increased formation of both [³H]PA and [³H]PEth in a concentration-dependent manner (>105–400% vs. controls in the presence of 10^–6 to 10^–4 M Ach) when pretreated with 100 mM ethanol. The Ach receptor-mediated increase in PLD activity was inhibited by atropine (10^–6 M), indicating that activation of PLD occurred via muscarinic receptors. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) increased PLD activity that was effectively blocked by the PKC inhibitors calphostin C (10^–8 to 10^–6 M) and GFX (10^–8 to 10^–6 M). Ach-induced increases in PLD activity were also significantly, but incompletely, inhibited by both GFX and calphostin C. From the present data, we conclude that in tracheal smooth muscle, muscarinic acetylcholine receptor-induced PLD activation is transient in nature and coupled to these receptors via PKC. However, PKC activation is not solely responsible for Ach-induced activation of PLD in porcine tracheal smooth muscle.     

Has nonquantal acetylcholine secretion from motor nerve endings a role in neurotrophic control of resting membrane potential in rat muscle fibers?

The resting membrane potential of fibers of the rat diaphragm was measured by a microelectrode technique 3 h after division of the phrenic nerve and incubation in culture medium for 5 days after denervation. The membrane potential was recorded in synaptic regions of fibers close to (2–3 mm) and distant from (9–11 mm) the site of nerve division. The membrane potential of the synaptic region of the close fibers 3 h after denervation became smaller, whereas that of the synaptic region of distant fibers did not change relative to the control. Placing the muscle 3 h after denervation into medium with carbamylcholine (1·10^–8 M), cGMP (1·10^–4 M), or dibutyryl-cGMP (1·10^–6 M) led to hyperpolarization of the synaptic region of the close fibers but did not change the resting potential in the synaptic region of the distant fibers, and abolished differences between them. Five days after division of the nerve, incubation of the muscle in a solution with the above-mentioned substances did not affect the resting membrane potential. Nonquantal release of acetylcholine from motor nerve endings, assessed by the amplitude of hyperpolarization of the postsynaptic membrane, induced by application of curarine against the background of acetylcholine esterase inhibition, 3 h after denervation was identical in the synaptic region of the close and distant fibers and did not differ from the control. It is postulated that the postdenervation fall of membrane potential of rat muscle fibers is not due to disturbance of nonquantal secretion of acetylcholine from motor nerve endings.S. V. Kurashov Kazan' Medical Institute, Ministry of Health of the USSR. Translated from Neirofiziologiya, Vol. 17, No. 3, pp. 358–365, May–June, 1985.     

Possible role of voltage-sensitive potassium channels in generation of response in Pacinian corpuscles

The effects of 20 mM tetraethylammonium (TEA) and 5 mM 4-aminopyridine (4-AP) on mechanically and electrically excitable membranes of Pacinian corpuscles were investigated using the air gap technique for producing constant superfusion and recording electrical response at the receptor. The effects of TEA led to a 150% rise in the duration of receptor potential, the amplitude of which declined by 40%. No statistically significant changes in response to mechanical stimulation could be detected after applying 4-AP to the receptor membrane. The two blockers mentioned did modify the membrane of the first nodes of Ranvier, producing a 2–3-fold increase in the duration of action potentials. Computations based on the Dodge model would indicate that the observed effects may be explained by inhibition of voltage-dependent potassium channels which help to transform receptor current into spike response in the intact receptor.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 20, No. 5, pp. 623–630, September–October, 1988.     

Reception of millimeter-band electromagnetic radiation by the ampullae of Lorenzini of skates

During recording of impulse activity from single nerve fibers of electroreceptors of the ampullae of Lorenzini of skates, we studied the responses to electromagnetic radiation (EMR) at a frequency of 37–55 GHz and an intensity of 1–100 mW/cm².Exposure of the ampullar canal pore to EMR at an intensity of 1–5 mW/cm² and a distance of 1–10 mm evoked a transient increase in the frequency of low-threshold receptor activity (current threshold was 0.04–0.2 µA). An increase in EMR intensity by more than 8–10 mW/cm² produced, together with elevation of receptor activity, an inhibition due to a rise in temperature of 1–3°C in the region exposed. The phase of increase in frequency of activity was absent in high current-threshold receptors (0.3–2.0 µA) when exposed to EMR. The receptors responded to irradiation of the ampullar canal pore at a distance of 15–20 mm by an increase in discharge frequency for 20 min. Direct irradiation of the ampullae of Lorenzini induced only inhibitory responses in receptor cells regardless of their excitability.The results obtained indicate that the sensory receptors of vertebrates are sensitive to EMR. It is concluded that the excitatory effects are due to direct reception of EMR by electroreceptors, and the inhibitory effects are related to local heating of the Lorenzini ampullar pore.Neirofiziologiya/Neurophysiology, Vol. 25, No. 5, pp. 325–329, September–October, 1993.     

Water-soluble proteins of Pacinian corpuscles

The composition of water soluble proteins has been investigated in different parts of Pacinian corpuscles. The Pacinian corpuscles fluid proteins have been compared with the receptor homogenate proteins and blood serum of a cat. It is supposed that proteins of the Pacinian corpuscle fluid are produced from the blood system on the whole. The composition of the Pacinian corpuscle fluid as well as a great variety of glycoproteins in it allows to suppose that the Pacinian corpuscles fluid perform a transport function.     

cAMP, not Ca/calmodulin, regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

Inhibition of muscarinic receptor-mediated Ca27#x002B; mobilization by phorbol 12-myristate 13-acetate in chick embryo cells

Summary A muscarinic cholinergic receptor is present on undifferentiated cells of the chick embryo. Stimulation of the muscarinic receptor with muscarinic agonists triggers intracellular Ca^2#x002B; mobilization. Here, we investigate the effect of phorbol 12-myristate 13-acetate (PMA) on the muscarinic receptor-mediated Ca^2#x002B; mobilization, which is monitored in cell suspensions of chick embryos of stage 24 by chlorotetracycline fluorescence. PMA inhibits the Ca^2#x002B; mobilization in a time-dependent and concentration-dependent manner without changing the ED₅₀ of acetylcholine. The concentration of PMA that gives halfmaximal inhibition is 3.1×10^–9 M PMA.     

Decrease in excitability of LG following habituation of the crayfish escape reaction

Crayfish escapes from threatening stimuli to the abdomen by tailflipping upwards and forwards. This lateral giant (LG)-mediated escape reaction habituates readily upon repetitive sensory stimulation. Using an isolated abdominal nerve cord preparation, we have analyzed the change in LG activity by applying additional sensory stimulation after different periods following habituation to characterize the retention of LG habituation. Results show that the LG mediated response habituates more quickly, but the retention time is shorter, as repetitive sensory stimulation is applied at progressively shorter inter-stimulus time intervals. The spike response of LG recovers quickly, within several minutes after habituation, but they fail to spike when an additional stimulus is applied after specific long periods following habituation. The critical period of the delay for this decrease in excitability of LG is dependent on the inter-stimulus time interval of the initial repetitive stimulus. As the inter-stimulus interval became longer, the delay needed for decrease in excitability became shorter. Furthermore, the local injection of 10^–6 mol l^–1 octopamine into the neuropil just following habituation promotes the achievement of decrease in excitability. No effects were observed when 10^–6 mol l^–1 serotonin and tyramine were injected. These results suggested octopamine promotes decrease in excitability of LG following habituation.     

Multiplicity of chemical mechanisms of regulation of muscle contractions in <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> L.

Effects of acetylcholine, octopamine, and their antagonists, as well as of glutamic acid were studied on contractions of dorsal longitudinal muscle of the mollusc Lymnaea stagnalis L., evoked by electrical stimulation of n. cervicalis inferior. Acetylcholine and octopamine increased amplitude of contractions evoked by applications at concentrations about 10^–8 M and decreased at concentrations higher than 10^–7 M. Glutamic acid produced only inhibitory effect on the contraction amplitude, which appeared at concentrations beginning from 10^–9 M and higher. The acetylcholine antagonists atropine and d-tubocurarine also decreased amplitude of evoked contractions. Their blocking effect rose with increase of their concentrations in the range from 10^–9 M to 10^–5 M. Specificity of the effect of the studied substances was established in experiments with a combined application of the transmitters and their antagonists. The obtained results indicate multiplicity of chemical mechanisms of regulation of contractions of the dorsal longitudinal muscle in L. stagnalis.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 44–50.Original Russian Text Copyright © 2005 by Kononenko, Zhukov.     

Ionic mechanisms underlying modulating effects of serotonin on acetylcholine-induced responses in Helix pomatia neurons

The ionic mechanisms underlying modulatory effects of serotonin on acetylcholine-response in identified and nonidentifiedHelix pomatia neurons were investigated using voltage-clamping techniques at the neuronal membrane. External application of 10^–5–10^–4 M serotonin to the membrane of neurons responding to application of acetylcholine depending on Na⁺ depolarization (D_Na response) reduced membrane conductivity during response to acetylcholine without changing reversal potential of acetylcholine-induced current. Acetylcholine (10^–6–10^–4 M) administration took place 1–3 min later. Neurons with response to acetylcholine application dependent on Cl⁺ depolarization (D_Cl response) or hyperpolarization (H_Cl response) behaved similarly. Analogous effects could be produced by external application of theophylline which, together with the latency and residual effect characteristic of serotonin action points to the participation of intracellular processes associated with the cellular cyclase system in the changes produced by serotonin in acetylcholineinduced response. Serotonin brought about a shift in reversal potential and an increase in the acetylcholine-induced current in those neurons where this response was associated with changed permeability at the membrane to certain types of ions. During two-stage acetylcholine-induced response of the D_Na-H_K type, serotonin inhibited the inward current stage. Mechanisms underlying modulatory serotonin action on acetylcholine-induced response in test neurons are discussed in the light of our findings.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 57–64, January–February, 1988.     

Acetylcholine activates a chloride channel as well as glutamate and GABA

Summary In conventional two microelectrode experiments, acetylcholine had qualitatively the same effect as GABA and glutamate on membrane potential and input resistance of muscle fibres of the opener and intrinsic stomach muscles of crayfish (Austropotamobius torrentium). In patch-clamp experiments, acetylcholine occasionally elicited single channel openings in cell-attached patches on these muscles. If outside-out patches were excised and the Cl^– concentration was high on both sides of the membrane, acetylcholine at concentrations of 1 nM regularly elicited single channel currents. The amplitude of single channel currents depended strongly on the intracellular concentration of Cl^–. The reversal potential of the channel, determined after replacing intracellular K⁺ with Cs⁺, corresponded to the Nernst potential for Cl^–. The voltage dependence and the reversal potential of single channel current amplitudes elicited by ACh, glutamate and GABA were identical. The distribution of life times of openings (>1 ms) elicited by ACh and glutamate could be fitted by a single exponential with a time constant of about 2.5 ms, corresponding to the mean open time. ACh and glutamate applied to the same outside-out patch showed cross-desensitization, and thus ACh and glutamate activate the same channels. An excitatory, cationic ACh-activated channel could not be identified. Permeabilities of the chloride channel were calculated according to the Goldman-Hodgkin-Katz equation at different membrane potentials. Negative single channel current amplitudes (inward currents) could be fitted with a permeability of ₂= 3.9×10^–14 cm³s^–1. For positive currents (outward) the channel had a permeability of ₁= 1.4× 10^–14 cm³s^–1. The permeability of the channel declined from 16×10^–14 cm³s^–1 to 2.3×10^–14 cm³s^–1 if the intracellular Cl^–-concentration was raised from 6 to 257 mM. The activation elicited by acetylcholine was inhibited by extracellular Ca⁺⁺. The mean current activated by ACh was reduced by a factor of 50 if the extracellular concentration of Ca⁺⁺ was raised from 0.1 mM to the physiological concentration of 13.5 mM.     

Pharmacology of the neuronal nicotinic acetylcholine receptor of cultured Kenyon cells of the honeybee, <Emphasis Type="Italic">Apis mellifera</Emphasis>

We investigated the pharmacology of the nicotinic acetylcholine receptor of honeybee Kenyon cells, a subset of olfactory interneurons, which are crucial for olfactory learning and memory. Whole-cell currents were recorded using patch-clamp techniques. Pressure application of agonists induced inward currents in cultured Kenyon cells at holding potentials of –110 mV. Acetylcholine or carbamylcholine were full agonists, nicotine, epibatidine and cytisine were only partial agonists. Coapplications of these partial agonists with acetylcholine reduced the current amplitude. The most efficient antagonists were dihydroxy--erythroidine (EC₅₀=0.5 pmol·l^–1) and methyllycaconitine (EC₅₀=24 pmol·l^–1). The open channel blocker mecamylamine, d-tubocurarine and hexamethonium were rather weak blockers of the honeybee nicotinic response. Bath applications of the muscarinic antagonist atropine inhibited nicotinic currents dependent on concentration (EC₅₀=24.3 mol·l^–1). Muscarine, pilocarpine or oxotremorine (1 mmol·l^–1) did not induce any measurable currents. The non-cholinergic drugs strychnine, bicuculline and picrotoxin partially and reversibly blocked the acetylcholine-induced currents. Our results indicate the expression of only one nicotinic acetylcholine receptor subtype in cultured Kenyon cells. Muscarinic as well as non-cholinergic antagonists also inhibit the receptor function, distinguishing the honeybee nicotinic receptor from the typical nicotinic receptor of vertebrates and from many described insects receptors.     

The effects of transmitters on the affinity of the insoluble fraction of Ox brain for Na+ and K+

The insoluble fraction of ox-brain, which had previously been shown to have a non-linear affinity for Na⁺ and K⁺, was prepared. Acetylcholine (1×10^–8 mol/l and 1×10^–7 mol/l) reduced the affinity of the fraction for Na⁺ and K⁺ to zero, while at 1×10^–6 mol/l, the affinity for the cations was almost as high as in the absence of the transmitter; the affinities for Na⁺ and K⁺ were particularly high, when the supernatant concentrations of these ions exceeded 80–100 mM. Addition of eserine (3×10^–5 mol/l) considerably modified the response of the fraction to acetylcholine (1×10^–5 mol/l). Atropine (1×10^–8 mol/l) in the absence or presence of acetylcholine (1×10^–5, or 1×10^–4 mol/l) reduced the affinity of the fraction for Na⁺ and K⁺ to zero. Epinephrine (3×10^–10 mol/l) lowered the affinity for Na⁺ and K⁺, while ergotamine itself (1×10^–5 mol/l) reduced it to zero. The addition of both epinephrine and ergotamine at the latter concentrations restored the affinity of the fractions for Na⁺ and K⁺ to what it had been in the absence of the transmitter or antagonist, previously reported. Norepinephrine (3×10^–10 mol/l), or ouabain (1×10^–7 mol/l) reduced the affinity of the fraction for Na⁺ and K⁺ to zero. Thus, the transmitters and antagonists altered the affinity of the insoluble fraction for Na⁺ and K⁺ nonlinearity, dependent upon their concentrations, the concentrations of the cations, and the interaction of transmitter and antagonist.     

Effects of L-alanine and L-alanine peptides on the chemotaxis of tetrahymena: Evolutionary conclusions

L-alanine and its peptides (L-Ala-2–6) do not attract or repulse Tetrahymena in a 10^–8M concentration. In 10^–10M concentration there is a consistent repellent effect. Twenty four hours after L-alanine or L-alanine-peptides' pretreatment (imprinting) the progeny generation of the cells react differently to the same materials. L-Alanine, L-alanine penta- and hexapeptide in both concentrations are chemoattractant, while L-alanine tetrapeptide is repellent. L-Alanine dipeptide is inert in 10^–10M and repellent at 10^–8M concentrations, while L-alanine tripeptide is strongly repellent at 10^–10M and attractant at 10^–8M concentrations. This means, that the first encounter (imprinting) with an exogeneous amino acid or peptide is decisive to the later reaction of the protozoan cell. The chain length is important in the imprinting, however the reaction is not consistent. The experiments call the attention to the significance of imprinting in the receptor and hormone evolution.