DSP-4, a noradrenergic neurotoxin, produces more severe biochemical and functional deficits in aged than young rats

The present study examines the effects of noradrenergic lesions (either DSP-4 i.p. or 6-hydroxydopamine (6-OHDA) into the dorsal noradrenergic bundle on biochemical (noradrenaline (NA), dopamine (DA), serotonin (5-HT) and choline acetyltransferase (ChAT) activity) and cortical EEG (quantitative EEG (qEEG) and high-voltage spindle (HVS) activity in young and aged rats. Near complete 6-OHDA NA lesions, but not partial DSP-4 NA lesions, increased HVS activity in young rats. DSP-4 and 6-OHDA lesions produced no significant changes in the 5-HT or DA levels or in the ChAT activity in young rats. In some of the aged rats, DSP-4 produced similar biochemical and HVS effects, as it induced in young rats. In the remainder of the aged rats, NA levels were greatly and 5-HT levels slightly decreased. DA levels and ChAT activity were unaltered in either set of aged rats. HVS activity was increased only in that group of aged rats with the greatly lowered NA content. These results suggest that: (1) some of the aged rats are more sensitive to DSP-4 treatment than young adult rats; and (2) NA depletions have to be complete to produce an increase in HVS activity in young and aged rats.     

Spinal cholinergic and monoamine receptors mediate the antinociceptive effect of morphine microinjected in the periaqueductal gray on the rat tail, but not the feet

The antinociceptive effects of morphine (5 μg) microinjected into the ventrolateral periaqueductal gray were determined using both the tail flick and the foot withdrawal responses to noxious radiant heating in lightly anesthetized rats. Intrathecal injection of appropriate antagonists was used to determine whether the antinociceptive effects of morphine were mediated byα₂-noradrenergic, serotonergic, opioid, or cholinergic muscarinic receptors. The increase in the foot withdrawal response latency produced by microinjection of morphine in the ventrolateral periaqueductal gray was reversed by intrathecal injection of the cholinergic muscarinic receptor antagonist atropine, but was not affected by the a2-adrenoceptor antagonist yohimbine, the serotonergic receptor antagonist methysergide, or the opioid receptor antagonist naloxone. In contrast, the increase in the tail flick response latency produced by morphine was reduced by either yohimbine, methysergide or atropine. These results indicate that microinjection of morphine in the ventrolateral periaqueductal gray inhibits nociceptive responses to noxious heating of the tail by activating descending neuronal systems that are different from those that inhibit the nociceptive responses to noxious heating of the feet. More specifically, serotonergic, muscarinic cholinergic andα₂-noradrenergic receptors appear to mediate the antinociception produced by morphine using the tail flick test. In contrast, muscarinic cholinergic, but not monoamine receptors appear to mediate the antinociceptive effects of morphine using the foot withdrawal response.     

Neonatal exposure to estradiol prevents the expression of ovarian hormone-induced luteinizing hormone and prolactin surges in adulthood but not antecedent changes in neuropeptide Y or adrenergic transmitter activity: Implications for sexual differentiation of gonadotropin secretion

Sex differences in adult patterns of mating behavior and gonadotropin secretion in rats are determined in part by the presence or absence of gonadal steroids during a perinatal critical period. For example, male rats and female rats exposed neonatally to androgen do not exhibit LH surge patterns when treated appropriately with ovarian hormones in adulthood, and there is evidence that this may be due to a failure of ovarian hormones to activate the hypothalamic neuronal systems that stimulate LH secretion in such animals. Because considerable evidence suggests that estradiol formed centrally from testosterone is responsible for the permanent defeminization of mating behavior and gonadotropin secretion, the present studies compared normal females with normal males and with females treated neonatally with estradiol on the ability of ovarian hormones to induce several important neurochemical changes antecedent to the LH surge, including changes in neuropeptide Y (NPY) and LH-releasing hormone (LHRH) concentrations in the median eminence, as well as changes in turnover rates for catecholamine transmitters in the medial basal hypothalamus and medial preoptic area. Normal ovariectomized female rats responded to sequential treatment with estradiol followed by progesterone with afternoon LH and prolactin (PRL) surges, and with sequential accumulation followed by decline in concentrations of LHRH and NPY in the median eminence prior to the LH surge. In addition, administration of progesterone increased the turnover rates of norepinephrine (NE) and epinephrine (EPI) in the arcuate-median eminence region of normal females. Gonadectomized male rats receiving the same ovarian hormone treatment failed to exhibit LH or PRL surges and displayed none of the changes in neurotransmitter turnover or peptide concentrations characteristically seen in the normal female. Unexpectedly however, when females that were treated with estradiol benzoate on days 1–3 postpartum were ovariectomized and treated with ovarian hormones in adulthood, they showed the same accumulation/decline in median eminence NPY concentrations and the same activation of NE and EPI turnover in the arcuate-median eminence region as normal females, even though they showed no LH or PRL surges or changes in median eminence LHRH concentrations. These results suggest that estradiol may not mediate all of the defeminizing actions of androgen exerted during the early neonatal period, and particularly those actions that result in a lack of responsiveness in central noradrenergic, adrenergic and NPY systems in adulthood. However, an action of neonatal estradiol may result in uncoupling of the LHRH neurosecretory system from normal excitatory neurochemical influences.     

Quantification and pharmacological characterization of dialysate levels of noradrenaline in the striatum of freely-moving rats: release from adrenergic terminals and modulation by α2-autoreceptors

Information concerning striatal levels of noradrenaline (NA) remains inconsistent. Here we have addressed this issue using a sensitive method of HPLC coupled to amperometric detection. The NA reuptake-inhibitor, reboxetine, selectively elevated levels of NA versus dopamine (DA), and NA levels were also selectively elevated by the α₂-adrenoceptor (AR) antagonist, atipamezole. The actions of atipamezole were mimicked by the preferential α_2A-AR antagonist, BRL44408, while JO-1 and prazosin, preferential antagonists at α_2C-ARs, caused less marked elevations in NA levels. In contrast to antagonists, the α₂-AR agonist, S18616, decreased NA levels and likewise suppressed those of DA. Unilateral lesions of the substantia nigra with 6-hydroxydopamine depleted DA levels without affecting those of NA. Further, the D₃/D₂ receptor agonist, quinelorane, decreased levels of DA without modifying those of NA. However, the D₃/D₂ receptor antagonists, haloperidol and raclopride, and the DA reuptake-inhibitor, GBR12935, elevated levels of both DA and NA. Levels of 5-HT (but not of NA or DA) were increased only by the 5-HT reuptake-inhibitor, citalopram. They were decreased by S18616 and prazosin, reflecting the inhibitory and excitatory influence of α₂- and α₁-ARs, respectively, upon serotonergic pathways. In conclusion, NA in the striatum is derived from adrenergic terminals. Its release is subject to tonic, inhibitory control by α₂-ARs, possibly involving both α_2A- and α_2C-AR subtypes, though their respective contribution requires clarification. A role of dopaminergic terminals in the reuptake of NA likely explains the elevation in its levels elicited by DA reuptake-inhibitors and D₃/D₂ receptor antagonists.     

Only activated but not non-activated presynaptic α2-autoreceptors interfere with neighbouring presynaptic receptor mechanisms

Summary Experiments were carried out in rabbit cerebrocortical slices in order to find out whether the attenuation by presynaptic ₂-autoreceptors of effects mediated by presynaptic opioid - and adenosine A₁-receptors requires activation of the ₂-receptors. The slices were preincubated with ³H-noradrenaline and then superfused with medium containing desipramine 1 mol/l. They were stimulated electrically either with single pulses or with trains of 32 pulses at 1 Hz.The overflow of tritium elicited by a single pulse amounted to 0.21% of the tritium content of the tissue. It was Ca²⁺-dependent and tetrodotoxin-sensitive and not changed by rauwolscine 1 mol/l or yohimbine 0.3 mol/l. Ethylketocyclazocine (EK; 0.1–10 nmol/l) and R-(–)-N⁶-phenylisopropyladenosine (PIA; 1–1,000 nmol/1) potently inhibited the overflow evoked by a single pulse, and their effects were not changed by yohimbine. — The overflow of tritium elicited by trains of 32 pulses at 1 Hz amounted to 0.92% of the tritium content of the tissue and was increased approximately fourfold by yohimbine 0.3 mol/l. EK and PIA were less potent inhibitors than in the one pulse experiments. Yohimbine greatly enhanced the effects of EK and PIA. The enhancement was even more pronounced when the Ca²⁺ concentration in the medium was reduced in order to obtain a control tritium overflow similar to that evoked by 32 pulses in the absence of yohimbine.The results demonstrate that there is no ₂-adrenergic autoinhibition when noradrenaline release is elicited by a single pulse. Under these conditions, the non-activated presynaptic ₂-adrenoceptor does not interfere with presynaptic opioid - and adenosine A₁-receptor mechanisms. It is only when the autoreceptor is activated by released noradrenaline that it attenuates neighbouring presynaptic receptor mechanisms, and this attenuation is removed by ₂-adrenoceptor antagonists.Send offprint requests to N. Limberger at the above address     

Involvement of Ca+ mobilization in the amplifying effect of serotonin on responses of rabbit isolated ear artery to exogenous noradrenaline

Summary The present study examined the involvement of Ca⁺ mobilization in the amplifying effect of serotonin on steady-state responses of rabbit isolated perfused ear artery to exogenous noradrenaline (NA; 0.001–3 ol/l). In contrast to its marked amplifying effect on responses to NA, serotonin in the subconstrictor concentration of 100 nmol/l had no effect on responses to KCl. The Ca⁺-entry blocking drug diltiazem (10 gmol/1) decreased responses to NA; in addition, the amplifying effect of serotonin on responses to NA was reduced by diltiazem. Lowering the concentration of Ca⁺ in the Krebs-Henseleit perfusion solution from 2.5 to 0.25 mmol/l also reduced both responses to NA and the amplifying effect of serotonin. Using the method of Manzini et al. (1982), separate intra- and extracellular Ca⁺-dependent responses to NA were obtained. Serotonin had no effect on intracellular Ca⁺-dependent responses to NA but enhanced extracellular Ca²⁺-dependent NA responses. These results suggest that the amplifying effect of serotonin on responses of rabbit ear artery to exogenous NA involves a selective enhancement of the component of the NA response which is dependent on extracellular Ca⁺; serotonin may increase NA-evoked entry of Ca²⁺ into the vascular smooth muscle cells through receptor-operated Ca⁺ channels. Send offprint requests to A. G. Meehan at the above address     

ACTH increases noradrenaline release in the rabbit heart

Summary The effects of ACTH on the release of noradrenaline and the increase of heart rate produced by sympathetic nerve stimulation (1 Hz) were studied in isolated perfused rabbit hearts. ACTH-(1–24) 0.1–100 nmol/l increased the stimulation-evoked overflow of noradrenaline concentration-dependently, reversibly and up to two-fold. The basal outflow of noradrenaline, the basal heart rate and the stimulation-evoked increase in heart rate were not changed. Human ACTH-(1–39) also increased the evoked overflow of noradrenaline. The effect of ACTH-(1–24) 0.3 nmol/l persisted after blockade of -adrenoceptors with propranolol and blockade of neuronal catecholamine uptake by cocaine. ACTH-(1–24) 3 nmol/l did not change the removal of noradrenaline from the perfusion fluid, when hearts were perfused with medium containing 59 nmol/l noradrenaline. The results show that ACTH increases the action potential-evoked release of noradrenaline from cardiac postganglionic sympathetic neurones, probably by activating specific presynaptic ACTH receptors. The high potency of ACTH suggests that these presynaptic receptors may be activated in vivo by circulating ACTH under certain pathophysiological conditions.Send offprint requests to B. Szabo at the above address     

Release of vesicular noradrenaline in the rat tail artery induced by cocaine

Summary The effects of cocaine on overflows of endogenous noradrenaline and DOPEG from isolated rat tail arteries were examined. 1. Both overflows increased progressively with increasing concentration of cocaine, while the (NA overflow)/(DOPEG overflow) ratio first increased and then decreased. The changes in the overflows induced by cocaine (0.1 mmol/l) appeared reversible. 2. Exposure of the tissue for 30 min to cocaine, 1 mmol/l, resulted in a significant decrease in the proportion of storage vesicles containing electron-dense cores. 3. The changes in overflows of noradrenaline and DOPEG induced by cocaine (0.1 mmol/l) were unaffected by the presence of desipramine (0.1 mol/l) or removal of extracellular Ca²⁺. The effect of cocaine on the overflow of noradrenaline was potentiated by prior inhibition of MAO with clorgyline. 4. Exposure of segments to a Ca²⁺-free, high K, low Na incubation medium was accompanied by increased overflow of noradrenaline. Cocaine (0.1 mmol/l) reduced the overflow of noradrenaline to about a half, and substantially increased the overflow of DOPEG. 5. The increase in the overflow of DOPEG from segments bathed in HEPES-buffered solutions, the pH of which ranged from 6.80 to 7.38, was approximately proportional to the calculated concentration of unprotonated (uncharged) cocaine. 6. Quantitatively similar changes in the overflows were observed when norcocaine was substituted for cocaine. Ecgonine methyl ester was much less potent than cocaine, and O-benzoyl ecgonine was ineffective. 7. The small increases in the overflow of noradrenaline observed at relatively low concentration (<30 mol/l) of cocaine can be attributed primarily to inhibition of reuptake of the released transmitter by the cocaine- and desipramine-sensitive amine carrier. The overflows of NA and DOPEG in the presence of higher concentrations of the alkaloid exhibit features compatible with the following hypothesis: (A) Cocaine is translocated across the axonal membrane mainly in the form of the unprotonated species, a large fraction of which is reprotonated upon the entry into the axon. (B) Cocaine releases noradrenaline from storage vesicles into the extravesicular space, where the bulk of the amine is converted to DOPEG. (C) Efflux of the remaining noradrenaline from the axon is not mediated by the Na⁺-dependent, cocaine- and desipramine-sensitive neuronal amine carrier. It seems to represent uncoupled efflux of the protonated form of noradrenaline.Abbreviations DOPEG 3,4-dihydroxyphenylethylene glycol - DOMA 3,4-dihydroxymandelic acid - HEPES N-(2-hydroxyethyl)piperazine-N-ethanesulfonic acid - MAO monoamine oxidase - MOPEG 3-methoxy-4-hydroxyphenylethylene glycol - NA (–)noradrenaline - pH_j pH in the extravesicular space of the axon - pH_o pH of the bathing solution - pK_a negative logarithm of the dissociation constant This study was supported by the British Columbia Heart Foundation Send of fprint requests to V. Palaty at the above address     

Inhibition of N-methyl-d-aspartate (NMDA) and l-glutamate-induced noradrenaline and acetylcholine release in the rat brain by ethanol

Summary The influence of ethanol on stimulation-evoked ³H-transmitter release was examined in slices of the rat brain cortex and corpus striatum preincubated with ³H-noradrenaline and ³H-choline, respectively. ³H-Transmitter release was stimulated by NMDA, l-glutamate, electrical impulses, reintroduction of Ca²⁺ ions (Ca²⁺-evoked release; after superfusion with Ca²⁺-free, K⁺-rich solution) or veratridine. In cortical slices preincubated with ³H-noradrenaline and superfused with Mg²⁺-free, otherwise physiologically composed salt solution, ethanol inhibited the NMDA- or l-glutamate-induced tritium overflow (IC₅₀ 45 and 37 mmol/l, respectively). In contrast, the tritium overflow in response to electrical stimulation, reintroduction of Ca²⁺ ions or veratridine was not affected by ethanol at concentrations up to 320 mmol/l; these experiments were carried out in cortical slices superfused with solution containing a physiological Mg²⁺ concentration. Ethanol also failed to inhibit Ca²⁺-evoked release in the absence of Mg2⁺ ions. In the presence of 1 mol/l veratridine, but not in its absence, NMDA induced tritium overflow even when cortical slices were superfused with salt solution containing a physiological Mg²⁺ concentration; again, ethanol inhibited this NMDA-evoked tritium overflow (IC₅₀ 73 mmol/l). In striatal slices preincubated with ³H-choline and superfused with Mg²⁺-free physiological salt solution, the NMDA-evoked tritium overflow was also, although at lower potency, inhibited by ethanol (IC₅₀ 192 mmol/l).In spite of the differences between the IC₅₀ values of ethanol determined for the inhibition of cortical noradrenaline and striatal acetylcholine release, it may be concluded that the NMDA receptor-ion channel complex is one of the sites of action underlying the ethanol-induced inhibition of neurotransmitter release. Since in the brain cortex the NMDA-induced ³H-noradrenaline release appears to be mediated by an excitatory interneurone activated by NMDA, this neuronal system may be involved in the cortical actions of ethanol.     

Uptake inhibitors do not change the effect of imidazoline α2-adrenoceptor agonists on transmitter release evoked by single pulse stimulation in mouse vas deferens

Summary The present study was undertaken to compare the presynaptic interaction of neuronal noradrenaline uptake inhibitors with imidazoline and phenylethylamine ₂adrenoceptor agonists under two different conditions: at low and high noradrenaline concentrations in the biophase.Isolated mouse vasa deferentia were stimulated with trains of 7 pulses given at 0.2 Hz and the inhibition by the ₂-adrenoceptor agonists clonidine, -methylnoradrenaline, and UK-14,304 of twitch responses was measured in the absence and in the presence of either cocaine (12 mol/l) or desipramine (40 nmol/l). The effects were determined for the first (equivalent to single pulse stimulation) and the last stimulus of each train. Both uptake inhibitors antagonized the presynaptic inhibitory effects of imidazolines (clonidine and UK-14,304) on the last twitch; the effects on the first twitch remained unchanged. In contrast, the uptake inhibitors potentiated the inhibitory effect of the phenylethylamine (-methylnoradrenaline) on both the first and the last twitches.These results support the view that the concentration of noradrenaline in the biophase plays a decisive role in the inhibition by a₂-adrenoceptor agonists of the electrically evoked release of noradrenaline. Agonists that are not substrates of neuronal uptake (i.e., clonidine, UK-14,304) become less effective when noradrenaline is present in the biophase while substrates of neuronal uptake (i. e., -methylnoradrenaline) do not. The results argue against the hypothesis that uptake inhibitors interact directly with presynaptic ₂-adrenoceptors or act at some link between uptake and receptor sites. Send offprint requests to S. Guimarães at the above address