Richard L. Delanoy

Behavioral responses to intracerebroventricularly administered neurohypophyseal peptides in mice

Lysine vasopressin (LVP), arginine vasopressin, oxytocin, and arginine vasotocin administered intraventricularly (icv) to mice all provoked a dose-dependent behavioral response in the range 0.1 – 1.0 μg. This response included a pronounced hyperactivity, extensive foraging, increased grooming, and at higher doses, stereotyped scratching, squeaking, and occasional barrel rolling. The four hormones were all approximately equipotent. Desglycinamide lysine vasopressin and [desaminocys1, D-Arg8] vasopressin produced some of the characteristic behaviors, but were much less potent. While pretreatment of the animals with reserpine (5 mg/kg ip), haloperidol (0.5 mg/kg ip), or physostigmine (0.5 mg/kg ip) sedated the animals and attenuated the locomotion and grooming, these drugs did not substantially alter the characteristic behavioral responses to LVP. Pretreatment with α-methyl-p-tyrosine (400 mg/kg ip), p-chlorophenylalanine (320 mg/kg ip), 6-hydroxydopamine (100 μg icv), ergotamine (0.5 μg icv), ethoxolamide (52 ng icv), diphenhydramine (20 μg icv), prostaglondin F2α (2 μg icv), or naloxone (1 mg/kg ip) did not alter the LVP-induced behaviors. None of these drugs or d-amphetamine (0.5 to 20 mg/kg ip) or nicotine (0.1 or 1 μg icv) mimicked the behavioral effects of the hormones.

Footshock treatment activates catecholamine synthesis in slices of mouse brain regions.

Synthesis of catecholamines was measured in slices of frontal cortex, hypothalamus, striatum, hippocampus and brainstem by the accumulation of [3H]norepinephrine (NE) and [3H]dopamine (DA) following incubation with [3H]tyrosine. Following acute footshock (60 shocks, 0.3 mA 30 min), consistent increases in [3H]DA accumulation were seen in frontal cortex slices, but no significant effect was seen in striatal slices. The accumulation of [3H]NE was not altered consistently in frontal cortex, hypothalamus, hippocampus or brainstem. Brain slices from mice adrenalectomized 24-48 h before footshock showed similar results. However, in hypophysectomized mice, footshock did not increase the [3H]DA accumulation in slices of frontal cortex. Administration of dexamethasone before footshock prevented the footshock-induced increase in frontal cortex [3H]DA accumulation, but footshock then significantly increased [3H]NE accumulation in the hypothalamus and brainstem. Chronic footshock (5 days) had little effect on frontal cortex [3H]catecholamine accumulation but produced a consistent elevation of [3H]NE accumulation in slices from the hypothalamus. In confirmation that the slice data reflected in vivo metabolism, both dihydroxyphenylacetic acid and homovanillic acid were significantly elevated in the frontal cortex but not the striatum of mice receiving acute footshock. Since previous studies have shown that ACTH administered intracerebroventricularly also accelerated [3H]DA accumulation in frontal cortex slices, these results are consistent with the involvement of ACTH in the effects of footshock on frontal cortex DA. The effects of chronic footshock are consistent with the activation of hypothalamic tyrosine hydroxylase by corticosterone.

Peptides and the conversion of [3H]tyrosine to catecholamines: Effects of ACTH-analogs, melanocyte-stimulating hormones and lysine-vasopressin

ACTH4–10, [D-Phe7]ACTH4–10, ACTH1–24, α-MSH and lysine-vasopressin (LVP) were tested for their effects on the cerebral content and metabolism of dopamine (DA) and norepinephrine (NE). CD-1 mice were injected daily for 3 days with equimolar concentrations of long-acting preparations of the peptides. Twenty-four hours after the third peptide treatment, mice were injected with [3H]tyrosine and the specific activities of tyrosine, DA and NE were measured 10 min later. ACTH1–24, ACTH4–10 or [D-Phe7]ACTH4–10 stimulated the conversion of [3H]tyrosine to DA but did not alter the cerebral dopamine content, suggesting that the turnover of dopamine was increased. α-MSH, β-MSH or LVP did not show this effect. Since ACTH4–10 has low steroidogenic activity it appears that the effect on [3H]DA metabolism was a direct effect of the peptide and not mediated by increased corticosterone secretion. None of the peptides had any detectable effect on the metabolism of [3H]NE. ACTH1–24 but no other peptide decreased the cerebral concentration of NE. In mice adrenalectomized one day before the first peptide injection, ACTH4–10 did not alter the specific activity of DA or NE.

Mouse brain deoxyglucose uptake after footshock, ACTH analogs, α-MSH, corticosterone or lysine vasopressin

The cerebral uptake of subcutaneously injected [3H]2-deoxy-D-glucose (2DG) in 16 brain regions was examined following 30 noncontingent random footshocks or the acute injection of saline, ACTH1-24 (0.5 microgram/g), ACTH/MSH4-10 (0.25 microgram/g), [D-Phe7]ACTH4-10 (0.25 microgram/g), [Met4SO2,D-Lys8,Phe9]ACTH4-9 (0.01 microgram/g), ALPHA-MSH (0.5 microgram/g), corticosterone (2.5 microgram/g) or lysine vasopressin (0.05 microgram/g). Footshock selectively decreased 2DG uptake in parietal cortex and brain stem, and increased that in the hypothalamus. Whole brain 2DG uptake was decreased by injection of saline or most of the hormones relative to uninjected animals, but this effect was probably peripheral since plasma glucose content was increased by the injections. The only regionally specific effect of the hormones was an increased 2DG uptake in olfactory bulb by saline, ACTH/MSH4-10 And corticosterone relative to uninjected animals. Since alpha-MSH had been reported previously to decrease blood flow (measured by antipyrene uptake) in all brain regions except occipital cortex [5,6], we directly compared antipyrene uptake with 2DG uptake in the same animals using a double-isotope procedure. The results revealed an increase in 2DG uptake relative to antipyrene in cortical regions relative to subcortical regions, contradicting earlier assumptions [19].

ACTH1–24 and lysine vasopressin selectively activate dopamine synthesis in frontal cortex

The accumulation of [3H] catecholamines from [3H] tyrosine in frontal cortical, septal, striatal and hippocampal slices was examined following intracerebroventricular (i.c.v.) injections of ACTH 1-24, lysine vasopressin (LVP) and saline. Both ACTH 1-24 and LVP (1 microgram) selectively increased the accumulation of [3H] dopamine (DA) in frontal cortical slices, but did not affect that of [3H] norepinephrine (NE). LVP but not ACTH 1-24 also inhibited the accumulation of [3H] DA in striatal slices. ACTH 1-24 did not alter the accumulation of [3H] NE in hippocampal slices, nor did LVP alter the accumulation of either catecholamine (CA) in septal slices. In vitro incubations with ACTH analogs of LVP failed to alter the rate of accumulation of [3H] CAs in striatal, substantia nigral and frontal cortical slices, except for an inhibitory effect at high doses. This effect is believed to be an artifact of precursor dilution caused by release of tyrosine following degradation of the peptides. Neither peptide modified the increased [3H] CA accumulation stimulated by 26 mM K+, nor did ACTH 1-24 modify the inhibition of [3H] CA accumulation caused by 3 X 10 -6 M haloperidol or 3 X 10 -7 M apomorphine. Selective activation of the mesocortical DA system has also been reported ot occur in response to footshock, suggesting the possibility that endogenous ACTH and/or LVP might mediate the stress-induced activation of mesocortical DA synthesis. Alternatively, i.c.v. injections of these peptides may themselves be stressful and thus indirectly elicit the response.

Catecholamine metabolism in brain slices: Determination of relevant precursor pool and the effects of elevated K+

Catecholamine synthesis from [3H]tyrosine was studied in slices of striatum, cerebellum and substantia nigra of mice. If low concentrations of tyrosine (less than 5.5 microM) were added to the incubation medium, the slices released significant amounts of tyrosine into the medium during the incubation. Kinetic analysis of the same experiments indicated that medium tyrosine and not tissue tyrosine was the appropriate precursor for both dopamine (DA) synthesis and protein synthesis in striatal slice. Concentrations of medium tyrosine of 8.25 microM or greater were sufficient to prevent changes of medium tyrosine during incubation and thus maintained a constant specific activity of precursor. Increasing concentrations of medium K+ increased both the accumulation of [3H]DA and its release from striatal slices. However, accumulation was stimulated at a concentration of K+ (14 mM) that had no significant stimulatory effect on release, suggesting that the stimulatory effects of K+ on synthesis and release are mediated by separate processes. Release of 14CO2 from [1-14C]tyrosine closely paralleled the accumulation of [3H]DA from [3H]tyrosine. Release of preloaded [14C]DA closely paralleled that of [3H]DA synthesized from [3H]tyrosine, suggesting a common functional pool. The principal DA catabolite produced was dihydroxyphenylacetic acid (DOPAC). The appearance of labeled DOPAC in the media was greatly enhanced by K+ stimulation.

Neurohypophyseal hormones and behavior: Effects of intracerebroventricularly injected hormone analogs in mice

Abstract Neurohypophyseal hormones evoke spontaneous behavioral changes in mice. This study compares the potency of four naturally occuring neurohypophyseal hormones and of ten analogs with amino acid residue replacements selected in such a manner as to cover each residue position of the hormones with the exception of the cystine residue. Peptides were administered intraventricularly and the sum of foraging, scratching and squeaking, recorded at 30 second intervals during a 30 min session, was measured as a function of peptide dose. The most potent group of peptides is represented by the neurohypophyseal hormones as well as the five analogs [Hly 8 ] vasopressin, [Δ 3 -Pro 7 ]AVP, [Thi 3 ]LVP, [Abu 4 ]AVP and [Abu 4 ]LVP. [Leu 4 ]LVP showed significant activity but was far less potent than the natural hormones. None of the remaining analogs enhanced activity with an increase in peptide dose. This group included both peptides with C-terminal modifications and those in which the tyrosine (position 2) or the asparagine residue (position 5) of the hormones were substituted by alanine. The neurohypophyseal hormone-induced behavioral results of this study reveal a structure-function relationship, which is in its most important conclusions, identical to the conformation-activity model proposed for endocrine activities of neurohypophyseal peptides.

Chemical stimulation of feeding behavior in the pinfish, Lagodon rhomboides: A new approach to an old problem

Abstract o 1. A new bioassay procedure is described for studying chemically-induced feeding behavior in the pinfish, Lagodon rhomboides. The procedure involves the introduction of stimulant through a small perforated rubber bulb and an automated recording of the number of times the fish strike the bulb. The quantitative nature of the procedure and the relative reliability of alternative testing regimes are discussed. 2. Assays of extracts prepared in a similar manner from seven animals (four phyla) revealed that considerable differences in potency existed. The most potent extract (shrimp) was approximately 3 times as effective as that of crab and greater than 10 times as effective as extracts of clam, oyster, whelk, mullet or sea urchin. 3. The major stimulants in five extracts were found to be substances of less than ca. 10,000 molecular weight.

Effects of haloperidol and apomorphine on catecholamine metabolism in brain slices. Reserpine-like effects of haloperidol.

The accumulation, release and catabolism of [3H]dopamine (DA) and [3H]norepinephrine (NE) synthesized from [3H]tyrosine were measured in mouse striatal and substantia nigral slices. Apomorphine inhibited both [3H]NE and [3H]DA accumulation (IC50 less than 10(-6) M), presumably by acting on a presynaptic receptor. Haloperidol (10-8M) caused a small, but significant increase in [3H]DA accumulation from [3H]tyrosine in the presence of 26 mM K+, possible reflecting blockade of presynaptic receptors activated by release DA. However, at higher concentrations (10(-6) to 10(-5) M), haloperidol inhibited [3H]DA and [3H]NE accumulation. Reserpine also potently inhibited catecholamine synthesis; chlorpromazine had only a weak effect, and fluphenazine was ineffective. Both haloperidol (10(-5) M) and reserpine (10(-7) M), but not chlorpromazine and fluphenazine, markedly increased the formation of labeled dihydroxyphenylacetic acid (DOPAC) and increased the spontaneous release of labeled DA from striatal slices preloaded with [3H]tyrosine or [14C]DA. These data suggest that haloperidol has some direct effects on DA metabolism that are unrelated to DA-receptor blockade. Because the effects of haloperidol are apparently independent of DA release, haloperidol may elevate cytoplasmic DA by altering its vesicular storage. This would, in turn, increase the spontaneous release of labeled DA by diffusion, the oxidation of DA to DOPAC by monoamine oxidase, and the end-product inhibition of tyrosine hydroxylase.