β-adrenergic receptor density and adenylate cyclase activity in lead-exposed rat brain after cessation of lead exposure

To understanding the reversible or irreversible harm to the -adrenergic system in the brain of lead-exposed rats, this study sets up an animal model to estimate the change in the sympathetic nervous system of brain after lead exposure was withdrawn. We address the following topics in this study: (a) the relationship between withdrawal time of lead exposure and brain -adrenergic receptor, blood lead level, and brain lead level in lead-exposed rats after lead exposure was stopped; and (b) the relationship between lead level and -adrenergic receptor and cyclic AMP (c-AMP) in brain. Wistar rats were chronically fed with 2% lead acetate and water for 2 months. Radioligand binding was assayed by a method that fulfilled strict criteria of -adrenergic receptor using the ligand [¹²⁵I]iodocyanopindolol. The levels of lead were determined by electrothermal atomic absorption spectrometry. The c-AMP level was determined by radioimmunoassay. The results showed a close relationship between decreasing lead levels and increasing numbers of brain -adrenergic receptors and brain adenylate cyclase activity after lead exposure was withdrawn. The effect of lead exposure on the -adrenergic system of the brain is a partly reversible condition.     

Diethylstilbestrol potentiation of the dopamine-elicited formation of adenosine 3′,5′-monophosphate in incubated male rat hypothalamus

Summary Dopamine (DA) stimulates the cAMP-generating system in the male rat hypothalamus only to a very low extent (25% above control). Diethylstilbestrol (DES), a synthetic estrogen, was found to be extremely potent (a 4- and 16-fold stimulation at 20 M and 100 M, respectively). Addition of either one to an incubation medium containing varying concentrations of the other resulted in a synergistic response. The potentiation by 20 M DES of the effect elicited by 100 M DA was the most remarkable, namely, a 3-fold stimulation of the combined response. A 4- and 7.5-fold stimulation of cAMP accumulation was observed when adenosine (100 M) or adenosine (100 M)+DA (100 M) were present in the incubation medium. Theophylline (0.5 mM), an adenosine antagonist, could effectively reduce this effect, as did adenosine deaminase (10 g/ml). Clomiphene (50 M), an estrogen antagonist, exhibited a marked decrease in DES+DA-elicited cAMP formation. Pimozide (40 M) had the ability to significantly block the stimulatory effects of DES and DA.     

Effects of chronic treatment with zimelidine and REM sleep deprivation on the regulation of raphe neuronal activity in a rat model of depression

Electrophysiological investigations on the mechanism of action of antidepressants have shown that both deprivation of rapid eye movement (REM) sleep and chronic treatment with antidepressants render serotoninergic (5-HT) neurons less sensitive to the inhibitory effect of 5-HT reuptake blockers in the rat. It was of interest to test whether the same mechanisms could be evidenced in a possible experimental model of depression. The latter consisted of rats which had been treated neonatally with clomipramine and exhibited at adult age behavioural and sleep alterations which resemble the human disorder. Recording the electrophysiological activity of 5-HT neurons in the nucleus raphe dorsalis (NRD) revealed that both chronic treatment with zimelidine and REM sleep deprivation induced a hyporeactivity of these neurons to the inhibitory effect of citalopram in normal rats. However, in rats which had been treated neonatally with clomipramine, 5-HT neurons were hyporeactive to the effect of this 5-HT reuptake blocker already under baseline conditions, and no further modification could be induced by chronic zimelidine administration or REM sleep deprivation. It can be hypothesized that adaptive phenomena at the serotoninergic NRD level are not a relevant element to explain the mechanism of action of antidepressants in the present model of depression, while they have been considered as a crucial event in normal rats.     

Pharmacological characterization of central α-adrenoceptors which mediate clonidine-induced locomotor hypoactivity in the developing rat

Summary The present experiment was designed to pharmacologically characterize receptors which mediate the clonidine-induced locomotor change in the developing rat. A subcutaneous injection of clonidine (0.78 mol/kg) produced locomotor hyperactivity in 7-day-old rats but hypoactivity in 20-day-old rats. Phenoxybenzamine (1.5 mol/kg, 5.9 mol/kg and 15 mol/kg) decreased spontaneous activity in a dosedependent manner but did not antagonize clonidineinduced hypoactivity in 20-day-old rats. By contrast, the significant reversal of the clonidine-induced hypoactivity by pretreatment with phentolamine (1.6 mol/kg and 6.3 mol/kg), yohimbine (1.3 mol/kg and 5.1 mol/kg) and piperoxan (7.4 mol/kg) was observed at such doses when the blockers did not cause and hypoactivity by themselves. It is suggested that clonidine could induce locomotor hypoactivity by activating presynaptic (₁-type) -adrenoceptors in the CNS of 20-day-old rat.     

The reversal effect of antidepressants on the escape deficit induced by inescapable shock in rats

This experiment investigated the effect of antidepressants on the escape deficit induced by inescapable shock. Following exposure te escapable shock, rats received a single injection of either tricyclic antidepressants (imipramine, desipramine), and atypical antidepressant (nomifensine), or saline. In a subsequent two-way shuttle test, treatments with these antidepressants reversed the escape deficit of the inescapable-shock groups without affecting performance of the escapable-shock groups. It is suggested that catecholamine re-uptake inhibition of the acute actions of antidepressants contributed to this reversal effect. The findings are discussed in relation to the neurochemical hypothesis of the escape deficit induced by inescapable shock and to an animal model of depression.     

Effects of κ-opioid receptor ligands on intracranial self-stimulation in rats

Rationale Elevations in cAMP response element binding protein (CREB) function within the mesolimbic system of rats reduce cocaine reward in place conditioning studies and increase immobility in the forced swim test. Each of these behavioral adaptations can be interpreted as a depressive-like effect (i.e., anhedonia, despair) that may reflect reduced activity of brain reward systems. Furthermore, each effect appears due to increases in CREB-mediated expression of dynorphin, since each is attenuated by intracranial injections of the -opioid receptor antagonist norBNI.Objectives Intracranial self-stimulation (ICSS) studies were conducted in rats to determine whether administration of a -agonist would have depressive-like effects on brain stimulation reward, and whether pretreatment with a -antagonist would attenuate any such effects. Conditions that have depressive effects in people (e.g., drug withdrawal) increase the threshold amounts of stimulation required to sustain ICSS in rats.Methods Sprague-Dawley rats with lateral hypothalamic stimulating electrodes were tested in a curve-shift variant of the ICSS procedure after systemic administration of the -agonist U-69593 alone, the novel -antagonist 5-acetamidinoethylnaltrindole (ANTI) alone, or co-administration of both drugs.Results U-69593 dose dependently increased ICSS thresholds, suggesting that activation of -receptors reduced the rewarding impact of the brain stimulation. ANTI had no effects on its own, but it attenuated increases in ICSS thresholds caused by the agonist.Conclusions These data provide further evidence that stimulation of brain -receptors may trigger certain depressive-like signs, and that antagonists may have efficacy as antidepressants without having reward-related actions of their own.     

The intrinsic effects of sarmazenil on sleep propensity and performance level of sleep-deprived subjects

The effect of two dosages of sarmazenil (RO 15-3505) on sleep propensity and performance was investigated in a double-blind, placebo-controlled paradigm. The design included three 24-h testing periods, separated by at least one 7-day rest period, commencing after 24-h of sleep deprivation. Twelve normal, healthy, adult males (mean age 27±2.8 years) were paid to participate. During the experimental periods, they came to the sleep laboratory at 2100 hours and spent the night awake under close supervision. At 0700 hours, a schedule of 7 min attempting sleep in bed, 13 min awake outside the bedroom, began. This schedule was maintained for 24 h. Repeated administrations of 1 mg and 2 mg sarmazenil significantly reduced the 24-h levels of total sleep. This was particularly evident during the period 0700–2300 hours. Sarmazenil also significantly improved reaction time and tended to increase the number of correct responses in the categories search task. Sarmazenil tended to improve reaction time in the Stroop test but this was significant only for the easy version of the test during the night.     

Chronic administration ofl-sulpiride at non-neuroleptic doses reduces the duration of immobility in experimental models of “depression-like” behavior

It has been shown that long-term administration ofl-sulpiride induces a down-regulation of receptor-associated adenylate cyclase activity in the frontal cortex of rats, an adaptive response that is typically associated with the chronic administration of antidepressants. Here we show that in two animal models of depression-like behavior (forced swim in rats and tail suspension in mice), the long-term (21 days) administration ofl-sulpiride at a non-neuroleptic dose (2 mg/kg IP twice a day) significantly decreases the duration of immobility, the effect being similar to that of desipramine (20 mg/kg IP). The same dose (2 mg/kg) ofl-sulpiride, acutely administered, has no effect at all. On the other hand, either chronic (21 days) or acute administration of neuroleptic doses ofl-sulpiride have an opposite effect, and indeed increase the duration of immobility. These results are an in vivo support to the in vitro findings suggesting that low doses ofl-sulpiride may have antidepressant-like activity.     

Conceptual framework for the etiology of alcoholism: a “kindling”/stress hypothesis

Rationale The rationale for proposing the kindling/stress hypothesis is to provide a conceptual basis for the insidious development and maintenance of alcohol abuse.Objective and results An objective of the hypothesis is to emphasize how continued alcohol abuse is linked to progressive neural adaptation. Work has shown that repeated withdrawals from chronic low levels of alcohol sensitize (kindle) anxiety-like behavior (anxiety) in rats, a finding consistent with multiple withdrawal kindling of seizure activity. Additionally, stress substitutes for initial cycles of the multiple withdrawal protocol to sensitize withdrawal-induced anxiety, which is indicative that stress is capable of facilitating neuroadaptive processes related to withdrawal. The persistence of adaptation caused by stress and multiple withdrawals is revealed by the appearance of withdrawal-induced anxiety following a future re-exposure to a single 5-day period of alcohol. This persisting adaptation also permits stress to induce anxiety during a period of abstinence—a response not observed in animals without previous exposure to alcohol. Furthermore, stress interacts with repeated withdrawals to enhance voluntary alcohol drinking. Results of other preclinical and clinical studies reported in the literature are integrated with these investigations in support of the proposed hypothesis.Conclusions The kindling/stress hypothesis is based on the premise that repeated withdrawals from cycles of chronic alcohol exposure contribute to a progressive development of persisting adaptive change that sensitizes withdrawal-induced anxiety and allows stress to evoke symptoms associated with negative affect during abstinence. Thus, these consequences of repeated withdrawals account for the evolution of major characteristics of alcoholism, which include worsened acute withdrawal symptoms and increased stress-induced negative affect during abstinence, both of which enhance the likelihood of relapse—and with relapse an inability to limit an abusive pattern of alcohol intake. The kindling/stress hypothesis provides a clear strategy for future studies to explore the advancing neural adaptation proposed to contribute to the pathogenesis of alcoholism.     

In vivo pharmacological effects of dihydro-β-erythroidine,a nicotinic antagonist,in mice

The comparative in vivo pharmacology of mecamylamine and dihydro--erythroidine (DHE) in mice was studied. Modulation of the behavioral effects (antinociception, hypomotility, motor impairment and hypothermia) of nicotine in mice by DHE and mecamylamine were carried out. After SC administration, DHE and mecamylamine were nearly equipotent in blocking nicotine's effects except for antinociception, in which mecamylamine was clearly more potent. Intrathecal injection of DHE was also effective in blocking the antinociceptive effect of nicotine. In vivo interaction of DHE with calcium and calcium channels, involved in the central actions of nicotine, showed that intrathecal administration of DHE failed to reduce the antinociception induced by diverse drugs which increase intracellular calcium such as thapsigargin, (±)-BAYK 8644 and calcium, indicating that this antagonist does not affect calcium-dependent mechanisms involved in antinociception. On the other hand, mecamylamine blocked the antinociceptive effect of the calcium modulatory drugs, suggesting that it may be acting on calcium-dependent mechanisms involved in the intracellular signaling process. We conclude that DHE, a nicotinic neuromuscular antagonist, is able to block some of the central actions of nicotine after systemic and intrathecal administration. The mechanism of blockade is different from that of mecamylamine, a classical ganglionic antagonist, and may involve a direct action of DHE on nicotine receptor.     

Effects of adenosine derivatives on cAMP accumulation and lipolysis in rat adipocytes and on adenylate cyclase in adipocyte plasma membranes

Summary N⁶-monosubstituted adenosine (Ad)-derivatives and Ad-derivatives altered in the adenine-or ribose-moiety have been compared with Ad in their effects on noradrenaline (NA)-stimulated cAMP accumulation, on lipolysis stimulated by NA or theophylline (THEO) and on adenylate cyclase (AC) activity of adipocyte plasma membranes.In isolated adipocytes about 0.01 M Ad caused a 50% inhibition of cAMP accumulation stimulated maximally by 1M NA. Depending upon the structure of substituent, the Ad-N⁶-derivatives were up to one order of magnitude either more or less active than Ad itself. 2-fluoro-Ad was nearly as active as Ad, whereas 2,5-dideoxy-Ad and 2-deoxy-Ad were practically ineffective as inhibitors of NA-stimulated cAMP accumulation. All compounds showed the same order of potency relative to Ad, when tested against lipolysis stimulated maximally by 1 mM THEO or submaximally by 0.3 M NA.In adipocyte plasma membranes a 50% inhibition of AC activity stimulated by 10M NA was observed at about 10 M Ad. None of the N⁶-substituted derivatives had any effect on either basal or NA-stimulated AC activity, whereas 2,5-dideoxy-Ad proved to be about 40 times more potent than Ad. 2-deoxy-Ad and 2-fluoro-Ad were nearly equipotent to Ad. Similar results were obtained, if AC was stimulated with 5-guanylylimidodiphosphate or NaF. Neither the N⁶-derivatives nor THEO could reverse the inhibitory effect of Ad on AC in plasma membranes.It is concluded, that different mechanisms are involved in the inhibitory effects of Ad on cAMP accumulation and lipolysis in intact cells and on AC activity of adipocyte plasma membranes.     

The effect of nicotine on the swimming speed of pre-trained rats through a water alley

Summary In sessions of ten runs each, swimming time of rats through a 4 m long water alley was measured. Four doses of nicotine (0.05; 0.1; 0.2; 0.4 mg/kg given intraperitoneally 30 minutes before testing) were tested in sessions with a braking load on the tails of the animals either in all 10 runs of a session, or in every second run, or in none of the 10 runs. Regardless of the swimming condition, nicotine produced a considerable, and at doses of 0.1 mg/kg and over, significant decrease of performance in the first two runs. From the third to the 10th run, the changes caused by nicotine were smaller and differed depending on the swimming conditions.A dose of 0.1 mg nicotine/kg improved performance in the without-load-sessions and the without-load-runs of the alternating sessions, while both 0.1 and 0.2 mg/kg improved performance of the with-load-runs of the alternating sessions. Performance in the without-load-sessions and the without-load-runs was depressed by 0.4 mg/kg and that in the with-load-sessions by 0.2 and 0.4 mg/kg.     

Radioimmunoassay of β-endorphin basal and stimulated levels in extracted rat plasma

Summary A sensitive radioimmunoassay for -endorphin is described. Antibodies against human -endorphin which exhibit a high avidity for the C-terminal of the peptide were raised in rabbits following the injection of thyroglobulin-coupled human -endorphin (h-E) as immunogen. Methionineenkephalin, - -endorphin, as well as ACTH peptides did not cause interference in the radioimmunoassay. -Lipotropin, however, showed a 50% cross-reactivity. The sensitivity of the assay is 25 pg/0.5 ml tube volume for -endorphin. -Endorphin was extracted with a high recovery from the rat plasma using silicic acid and -endorphin levels as low as 100 pg/ml could be measured.Basal levels of -endorphin-like immunoreactivity in plasma of rats were about 400 pg/ml. -Endorphin levels in adrenalectomized rats and in animals chronically treated with the cortisol synthesis blocker metyrapone were found to be markedly increased (about 7-fold). Exposure of the rats to electrically induced foot-shocks caused a similar increase of immunoreactive -endorphin in plasma. A significant increase was also seen after insulin injection.     

The influence of learning on morphine analgesia and tolerance development in rats tested on the hot plate

The influence of learning on the development of tolerance to the analgesic effect of morphine in rats was examined employing the hot plate procedure. A tested-reinforced (Tr) group and its yoked-control, a tested-non-reinforced (Tnr) group, received identical exposure to the testing procedure; the Tr group was reinforced daily for its behavior on the heated plate whereas the Tnr group was reinforced only on the last day of the experiment. Paired statistical comparisons between these two groups on the last day of the experiment revealed that: 1. premorphine control reaction times on the heated plate were significantly lower in Tr than in Tnr animals; and 2. post-morphine increases in reaction time did not differ between Tr and Tnr animals. It was concluded that whereas some learning does occur in this testing procedure, learning does not influence the behavioral tolerance to morphine which develops in this analgesiometric method. An hypothesis which accommodates this behavioral tolerance and a mechanistic scheme is offered.     

Reversal of tumor-induced immunosuppression by TGF-beta inhibitors

The immune system is responsible for the early detection and destruction of newly transformed malignant cells. Some transformed cells become immunologically invisible by passive avoidance of immune surveillance (i.e., when tumor cells are immunologically indistinguishable from normal cells). Other transformed cells actively secrete cytokines that effectively blind the immune system to the presence of abnormal antigens on the tumor cell surface. Transforming growth factor- (TGF-), which is expressed by a majority of malignant tumors, is the most potent immunosuppressor and therefore, the most likely cytokine to be responsible for the latter phenomenon. In addition to playing a key role in tumor-induced immunosuppression, TGF- stimulates angiogenesis. Interestingly, tumor cells eventually become refractory to TGF--mediated growth arrest, either due to loss of TGF- receptors or due to dysregulation in TGF- signaling pathways. Neutralization of TGF- or inhibition of its production is an effective method of cancer treatment in variety of animal models. Several agents targeting TGF- are in the early stages of development and include anti-TGF- antibodies, small molecule inhibitors of TGF-, Smad inhibitors and antisense gene therapy. Since tumors may express more than one isoform of TGF-, these new drugs should target all three TGF- isoforms produced by human tumors. The effects of therapies targeting TGF- are likely to be synergistic with cytotoxic chemotherapy and immunotherapy. Reversal of TGF--induced immunosuppression is a new and promising approach to cancer therapy, with potential applications in other diseases such as AIDS.     

Relationship between behavioral and nociceptive changes in attacked mice: effects of opiate antagonists

The relationship between analgesia and behavior during and after an aggressive encounter was investigated in saline- and opiate antagonist-treated DBA mice. A low number of bites induced an analgesia that was reversed by -chlornaltrexamine but not by naloxone, and that correlated positively with increased displays of defensive upright and immobility upon contact with the opponent. Extended attacks induced a naloxone-sensitive analgesia that was linked to a delayed occurrence of panic escape behavior. In the post-conflict phase, the degree of immobility and analgesia correlated positively in attacked mice. Naltrexone prevented this analgesia and lowered immobility. Endogenous opioids released during social conflict may induce analgesia and immobility in DBA mice.     

Vasopressin stimulates release of β-lipotropin and β-endorphin in conscious rats as measured by radioimmunoassay of unextracted plasma

Summary Using a newly developed radioimmunoassay to determine the -endorphin-like immunoreactivity (-EI) in unextracted plasma, the effect of vasopressin injections on plasma -EI was investigated in conscious rats. Arginine vasopressin caused a dose-dependent increase of plasma -EI from 34.5±7.8 fmol ml^–1 (n=6) in vehicle-treated animals to 205.0±36.1 fmol ml^–1 (n=7) after injection of the highest vasopressin dose employed (486 ng/100 g b.w.). In view of the appreciable cross-reactivity of -lipotropin (-LPH) in the radioimmunoassay used, plasma was extracted and subjected to gel chromatography on a Sephadex G-50 column. On average, about 70% of the -EI co-eluted with human -LPH and about 30% with human -endorphin in plasma extracts obtained from both control and vasopressin-treated rats. No peripheral conversion of human -LPH occurred under the experimental conditions, since after i.v. bolus injection of human -LPH 97% of the -EI comigrated with human -LPH during gel filtration. A similar blood pressure increase to that induced by the vasopressin injections, when elicited by noradrenaline or angiotensin II i.v., was not followed by an elevation of plasma -EI.These data indicate that vasopressin stimulates -lipotropin and -endorphin release into the systemic circulation in vivo.     

A three-state model of the benzodiazepine receptor explains the interactions between the benzodiazepine antagonist Ro 15-1788, benzodiazepine tranquilizers, β-carbolines,and phenobarbitone

Summary The potent benzodiazepine receptor ligands -carboline-3-carboxylic acid ethyl ester (-CCM) and the corresponding methylester (-CCM) administered i.v. depressed segmental dorsal root potentials in spinal cats, reversed the prolongation of dorsal root potentials by phenobarbitone, and abolished the depression of a motor performance task induced by phenobarbitone in mice; -CCE enhanced the low-frequency facilitation of pyramidal population spikes in the hippocampus of anaesthetized rats. These effects of -carbolines reflect a depression of GABAergic synaptic transmission and, thus, are diametrically opposed to the enhancing action of benzodiazepine tranquilizers. The specific benzodiazepine antagonist, Ro 15-1788, while not affecting dorsal root potentials, hippocampal population spikes or phenobarbitone-induced motor performance depression, abolished the effects of -CCE on the three parameters and similar effects of -CCM on the spinal cord and motor performance.A three-state model of the benzodiazepine receptor is proposed in which benzodiazepine tranquilizers act as agonists enhancing the function of the benzodiazepine receptor as a coupling unit between GABA receptor and chloride channel, -carbolines act as inverse agonists reducing this coupling function, and Ro 15-1788 represents a competitive antagonist blocking both the enhancing effect of agonists and the depressant effect of inverse agonists on GABAergic synaptic transmission.     

Retrospective study on the reliability of an “approximate LD50” determined with a small number of animals

Based on 364 LD₅₀ determinations in mice and rats after intravenous and oral administration of drugs, the reliability of an approximate LD₅₀ was retrospectively tested.The difference between approximate LD₅₀ and LD₅₀ is — independent of species and route of administration — not greater than ± 20% of the LD₅₀ in 90% of the cases.Four to five doses — uniformly distributed over the dose-mortality range can suffice in reliably determining the approximate LD₅₀.The probability is 10% that an approximate LD₅₀ and LD₅₀ are significantly different from each other.152 parallel studies on male and female animals show that the LD₅₀ or approximate LD₅₀ must not be determined for both sexes. It is sufficient to test a dose near the LD₅₀ in the opposite sex. A 50–75% reduction of expenditure in animal material is possible in most of LD₅₀ determinations.     

Effects of fixed-interval duration on the development of tolerance to decreased responding byl-nantradol

The effects of several types of antidepressants in a recently developed behavioural despair model, the tail-suspension test, are described. Drug effects on the automatically recorded duration of immobility and power of movements were measured in three strains of mice. Only in one strain (NMRI) did almost all antidepressants tested showed the expected reduction in duration of immobility. Tranquillizing drugs, but not stimulants, could be distinguished from antidepressants. The power of movements could not definitively be related to the pharmacological profile of the drugs tested. The use of the tail-suspension test as a rapid and highly predictive behavioural primary screen for antidepressant drugs is suggested.