Matrix metalloproteinases inhibition promotes the polyfunctionality of human natural killer cells in therapeutic antibody-based anti-tumour immunotherapy.

Activation of human natural killer (NK) cells is associated with the cleavage of CD16 from the cell surface, a process mediated by matrix metalloproteinases (MMPs). In this report, we examined whether inhibition of MMPs would lead to improved NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) function. Using an in-vitro ADCC assay, we tested the anti-tumour function of NK cells with three different therapeutic monoclonal antibodies (mAbs) in the presence of MMPs inhibitor GM6001 or its control. Loss of CD16 was observed when NK cells were co-cultured with tumour targets in the presence of specific anti-tumour antibodies, and was found particularly on the majority of degranulating NK responding cells. Treatment with MMPs inhibitors not only prevented CD16 down-regulation, but improved the quality of the responding cells significantly, as shown by an increase in the percentage of polyfunctional NK cells that are capable of both producing cytokines and degranulation. Furthermore, MMPs inhibition resulted in augmented and sustained CD16-mediated signalling, as shown by increased tyrosine phosphorylation of CD3ζ and other downstream signalling intermediates, which may account for the improved NK cell function. Collectively, our results provide a foundation for combining MMPs inhibitors and therapeutic mAbs in new clinical trials for cancer treatment.

PARP inhibitors enhance replication stress and cause mitotic catastrophe in MYCN-dependent neuroblastoma.

High-risk and MYCN-amplified neuroblastomas are among the most aggressive pediatric tumors. Despite intense multimodality therapies, about 50% of these patients succumb to their disease, making the search for effective therapies an absolute priority. Due to the important functions of poly (ADP-ribose) polymerases, PARP inhibitors have entered the clinical settings for cancer treatment and are being exploited in a variety of preclinical studies and clinical trials. PARP inhibitors based combination schemes have also been tested in neuroblastoma preclinical models with encouraging results. However, the expression of PARP enzymes in human neuroblastoma and the biological consequences of their inhibition remained largely unexplored. Here, we show that high PARP1 and PARP2 expression is significantly associated with high-risk neuroblastoma cases and poor survival, highlighting its previously unrecognized prognostic value for human neuroblastoma. In vitro, PARP1 and 2 are abundant in MYCN amplified and MYCN-overexpressing cells. In this context, PARP inhibitors with high 'PARP trapping' potency, such as olaparib or talazoparib, yield DNA damage and cell death preceded by intense signs of replication stress. Notwithstanding the activation of a CHK1-CDC25A replication stress response, PARP-inhibited MYCN amplified and overexpressing cells fail to sustain a prolonged checkpoint and progress through mitosis in the presence of damaged DNA, eventually undergoing mitotic catastrophe. CHK1-targeted inhibition of the replication stress checkpoint exacerbated this phenotype. These data highlight a novel route for cell death induction by PARP inhibitors and support their introduction, together with CHK1 inhibitors, in therapeutic approaches for neuroblastomas with high MYC(N) activity.

Nanoparticles-cell association predicted by protein corona fingerprints.

In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a "protein corona" layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø≈ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells.

Muscle metaboreflex contribution to sinus node regulation during static exercise: insights from spectral analysis of heart rate variability.

BACKGROUND: It is currently assumed that during static exercise, central command increases heart rate (HR) through a decrease in parasympathetic activity, whereas the muscle metaboreflex raises blood pressure (BP) only through an increase in sympathetic outflow to blood vessels, because when the metaboreflex activation is maintained during postexercise muscle ischemia, BP remains elevated while HR recovers. We tested the hypotheses that the muscle metaboreflex contributes to HR regulation during static exercise via sympathetic activation and that the arterial baroreflex is involved in the HR recovery of postexercise muscle ischemia. METHODS AND RESULTS: Eleven healthy male volunteers performed 4-minute static leg extension (SLE) at 30% of maximal voluntary contraction, followed by 4-minute arrested leg circulation (ALC). Autonomic regulation of HR was investigated by spectral analysis of HR variability (HRV), and baroreflex control of heart period was assessed by the spontaneous baroreflex method. SLE resulted in a significant increase in the low-frequency component of HRV that remained elevated during ALC. The normalized high-frequency component of HRV was reduced during SLE and returned to control levels during ALC. Baroreflex sensitivity was significantly reduced during SLE and returned to control levels during ALC when BP was kept elevated above the resting level while HR recovered. CONCLUSIONS: The muscle metaboreflex contributes to HR regulation during static exercise via a sympathetic activation. The bradycardia that occurs during postexercise muscle ischemia despite the maintained sympathetic stimulus may be explained by a baroreflex-mediated increase in parasympathetic outflow to the sinoatrial node that overpowers the metaboreflex-induced cardiac sympathetic activation.

Levels of NGF, p75NGFR and ChAT immunoreactivity in brain of adult and aged microencephalic rats.

Methylazoxymethanol (MAM)-induced microencephalic aged animals with reduced cortical mass and unmodified basal nucleus were used to study the relationship between cells that produce and cells that utilize NGF. Total cortical ChAT activity of MAM 2, 19 and 27 month old animals was reduced compared to their age-matched controls. To verify whether the reduction of enzyme activity can be ascribed to changes in or ablation of projecting neurons, we carried out immunohistochemical analysis of ChAT and low affinity NGF receptor (p75NGFR) in the basal nucleus of control and MAM-treated animals. ChAT and p75NGFR immunostaining of basal forebrain cholinergic neurons showed morphological changes in MAM animals, as revealed by cellular atrophy, reduced dendritic arborization and decreased staining intensity. In the cerebral cortex of microencephalic animals, reduced levels of NGF compared to controls were observed at all examined ages. These results suggest that MAM treatment induces long-lasting ablation of cortical NGF-synthesizing cells leading to reduced trophic support to basal forebrain cholinergic neurons, which might be responsible for the cellular atrophy observed in the basal nucleus.

Evaluation of reproducibility of spontaneous baroreflex sensitivity at rest and during laboratory tests.

OBJECTIVE: The aim of the present study was to examine the reproducibility of arterial baroreflex sensitivity (BRS) provided by the spontaneous baroreflex method at rest and during laboratory tests. METHODS: Twenty healthy volunteers were studied 24 h apart, in the same laboratory and under the same environmental conditions, at rest, during active standing, while performing mental arithmetics and during static hand-gripping. Systolic blood pressure, mean arterial pressure and pulse interval were continuously and non-invasively measured by using a Finapres device. BRS was evaluated by analysing the slopes of spontaneously occurring sequences of three or more consecutive beats in which systolic blood pressure and pulse interval of the following beat both increased or decreased, in the same direction, in a linear fashion. Individual BRS were obtained by averaging all slopes computed within a given test. RESULTS: Under each test condition BRS did not differ significantly between the two consecutive days, showing strikingly similar values. The mean group coefficients of variation (CVAR), obtained by averaging individual CVAR, between the two experimental days were 15.0, 13.9 and 19.7% for resting, standing, static hand-gripping and mental arithmetic, respectively. No relationships were found between individual CVAR and individual mean arterial pressure, pulse interval and number of baroreflex sequences under any tested condition, on both experimental days. CONCLUSIONS: These results show that the spontaneous baroreflex method provides good BRS reproducibility under various stimuli that affect the neural control of circulation differently. They also suggest that BRS variability is dependent neither on haemodynamic modifications nor on the degree of baroreflex engagement, but it seems to reflect an inherent feature of the way in which arterial baroreflex modulate the heart period.

Effect of postural changes on cardiovascular responses to static exercise in hypertensive human beings.

OBJECTIVE: In hypertensive patients, exaggerated increases in vascular resistance and arterial blood pressure have been reported on changing posture from supine to upright. In this study we tested the hypothesis that in hypertensive subjects, upright posture induces an increase in the vasoconstrictor and pressor responses to physical exercise. SUBJECTS AND METHODS: We studied 17 males with mild hypertension and 10 sex- and age-matched normotensives. Each performed three bouts of static handgrip at 30% maximum voluntary contraction for 2 min after 10 min of supine rest and, in sequence, after 10 min of sitting and 10 min of standing. Arterial pressure, heart rate and forearm vascular resistance were measured by Finapres and plethysmography, respectively. RESULTS: Exercise posture did not affect the mean arterial pressure and heart rate responses to static handgrip. No significant differences in these responses were observed between the hypertensives and the normotensives in any posture. In the hypertensives (n = 12), forearm vascular resistance did not change significantly from resting values during supine and sitting static handgrip but increased significantly during standing static handgrip. In the normotensives, forearm vascular resistance did not change significantly from resting values during static handgrip in any posture. The forearm vascular resistance response to the standing static handgrip was significantly greater in the hypertensives than the normotensives. The algebraic sum of forearm vascular resistance responses to postural change from sitting to standing plus that induced by sitting static handgrip (i.e. additive reflexes) was significantly less than the forearm vascular resistance response to the standing static handgrip (i.e. combined relexes), indicating a facilitatory interaction between exercise and orthostatic stimuli in hypertensives. In contrast, the algebraic sum of the heart rate responses to postural change from sitting to standing plus that induced by sitting static handgrip was significantly greater than the response to standing static handgrip, indicating an inhibitory interaction. CONCLUSIONS: In hypertensive patients, physiological orthostasis causes an increased vasoconstrictor response to static exercise, but this is opposed by an inhibitory influence on the heart rate response, with the result that the pressor response to static exercise is unaffected by upright posture.

On the role of neural mechanisms in the cardiocirculatory inhibitory action of alpha-human atrial natriuretic peptide in the anesthetized rabbit.

The effects induced by alpha-human 28-amino acid residue atrial natriuretic peptide (alpha-hANP) on arterial pressure, heart rate and vascular resistance, measured as hindlimb perfusion pressure (HPP), were examined in anesthetized rabbits. In particular, the involvement of the autonomic nervous system in mediating the cardiocirculatory effects of alpha-hANP was investigated. Intravenous alpha-hANP (8 micrograms/kg, bolus injection) in anesthetized rabbits caused a sustained decrease in atrial pressure, a transient decrease in HPP and no significant changes in heart rate. After sinoaortic denervation, alpha-hANP produced a greater decrease in arterial pressure and in hindlimb vascular resistance and also a consistent decrease in heart rate. Bilateral vagotomy did not significantly alter the cardiocirculatory responses to alpha-hANP in either normal or in sinoaortic denervated rabbits. Intravenous infusion of alpha-hANP (2 micrograms/kg bolus + 0.2 micrograms/kg per min) did not substantially change the baroreflex cardiocirculatory responses to loading and unloading carotid and aortic baroreceptors with bilateral carotid occlusion and phenylephrine or nitroglycerin bolus injection. In addition, alpha-hANP infusion did not modify the cardiovascular reflex responses to chemical stimulation of neural receptors (sensory endings of group III and IV somatic afferents) in the hindlimb muscles which are primarily mediated by sympathetic nerves in the anesthetized rabbit. Pharmacological blockade of the autonomic nervous system with atropine and guanethidine did not reduce the hypotensive and bradycardic effects caused by alpha-hANP in sinoaortic denervated animals. The results indicate that in anesthetized rabbits: (1) alpha-hANP can induce inhibitory cardiocirculatory responses (hypotension, bradycardia, musculocutaneous vasodilation) which are consistently offset by the sinoaortic baroreceptor system; (2) alpha-hANP does not alter the reflex control of arterial pressure and heart rate by arterial baroreceptors and muscle chemosensitive receptors; (3) activation of cardiopulmonary vagally-mediated depressor reflexes does not contribute to the inhibitory cardiovascular action of alpha-hANP; and (4) inhibitory effects on sympathetic activity do not constitute a significant component of the cardiocirculatory action of alpha-hANP.

Synthesis and dopamine receptor affinities of 2-(4-fluoro-3- hydroxyphenyl)ethylamine and N-substituted derivatives.

The synthesis of 2-(4-fluoro-3-hydroxyphenyl)ethylamine (26) and of some N,N-dialkyl derivatives (27-30) starting from 4-fluoro-3-hydroxytoluene and their in vitro binding affinities for dopamine (DA) receptor are reported. The amine 26 can be regarded as a molecular modification of DA in which the para hydroxyl group is replaced by fluorine. The new compounds 26-30 were evaluated for their affinity at D-1 and D-2 DA receptor subtypes by displacement of [3H]SCH 23390 (D-1 selective) and [3H]spiperone (D-2 selective). The amine 26 had about 2-fold less affinity for D-1 and D-2 binding sites than DA. The substitution of the amino group with ethyl, n-propyl, and 2-phenylethyl groups decreased the affinity for D-1 binding sites but greatly enhanced the effectiveness on D-2 binding sites. The N-ethyl- (28) and N-n-propyl-N-(2-phenylethyl)-2-(4-fluoro-3- hydroxyphenyl)ethylamine (30) were the most potent members of the series with high selectivity for D-2 binding sites. A similar effect was observed with isomeric N-n-propyl-N-(2-phenylethyl)-2-(3-fluoro-4-hydroxyphenyl)ethylamine (31) which was approximately 65 times more selective for D-2 sites vs D-1 sites. The introduction of a 2-phenylethyl group on the nitrogen atom induce the highest effect, perhaps as a consequence of an increased liposolubility or of binding to a complementary lipophilic site on the receptor.

Long-lasting tolerance to stimulatory effects of perinatal caffeine treatment.

Pregnant rats were treated with caffeine in their drinking water at doses of 136.3 mg/kg/day during gestation and 222.2 mg/kg/day during lactation. The resulting offspring at 60 days of age (40 days after drug withdrawal) were tested in an activity monitor cage. Spontaneous locomotor activity and that induced by caffeine (10, 30, 60 mg/kg/day) were observed. Treated rats showed apparent tolerance to caffeine-induced motility. Therefore perinatal caffeine treatment seems to induce long-lasting tolerance. This finding contrasts with those previously reported for chronic caffeine treatment in adult rats, where tolerance disappears after only 2-3 weeks following drug withdrawal.

Early postnatal chlordiazepoxide administration: permanent behavioural effects in the mature rat and possible involvement of the GABA-benzodiazepine system.

The long term behavioural and biochemical effects of chronic chlordiazepoxide treatment during the period of neuronal maturation in the rat have been investigated. The administration to lactating mothers of chlordiazepoxide at very low doses (0.22 and 2.6 mg/kg) in their drinking water affects both behavioural and biochemical parameters in offspring at 60 days of age and undrugged since weaning. A deficit in the acquisition of the conditioned avoidance response in treated rats was observed, although no significant difference in spontaneous locomotor activity between control and treated rats was found. 3H-Flunitrazepam binding sites in cerebral cortex and hippocampus were decreased by the treatment, whereas no change was detected in cerebellum. Moreover, 3H-muscimol binding sites increased in hippocampus with no changes in cerebral cortex and cerebellum. According to the different regional distribution of benzodiazepine type 1 and type 2 receptors, we suggest that type 2 receptors are selectively affected by the treatment, and that the GABAergic receptor system is also permanently altered by administration of chlordiazepoxide during early postnatal life.

Cardiorespiratory response patterns to afferent stimulation of muscle nerves in the rabbit.

The aim of this study was to test the hypothesis that stimulation of thin fiber muscle afferents is capable of matching the cardiovascular and ventilatory responses. In 46 anesthetized rabbits, the central end of the gastrocnemius nerves was electrically stimulated at 3 [low-frequency stimulation (LFS)] and 100 Hz [high-frequency stimulation (HFS)]. Intensities up to 200 times motor threshold were used. LFS induced a decrease in both mean arterial pressure (-19.9 +/- 2.9%) and systemic vascular resistance (-23.9 +/- 3.2%) an increase in cardiac output (CO) (6.4 +/- 1.7%), stroke volume (7.3 +/- 3.0%) and pulmonary ventilation (VE) (26.7 +/- 2.3%); heart rate and central venous pressure were not changed significantly. HFS induced an increase in mean arterial pressure (11.1 +/- 4.9%), CO (15.8 +/- 5.4%), stroke volume (13.4 +/- 5.4%), and VE but no significant changes in heart rate, systemic vascular resistance and central venous pressure. In both response patterns, arterial and end-tidal CO2 did not change significantly. The patterns of cardiorespiratory responses to both LFS and HFS were characterized by an increase in Co and VE without concomitant decreases in arterial and end-tidal PCO2 (isocapnic hyperpnea).

Role of muscular factors in cardiorespiratory responses to static exercise: contribution of reflex mechanisms.

We investigated the effects of muscle mass and contraction intensity on the cardiorespiratory responses to static exercise and on the contribution afforded by muscle metaboreflex and arterial baroreflex mechanisms. Ten subjects performed static handgrip at 30% maximal voluntary contraction (MVC) (SHG-30) and one-leg extension at 15% (SLE-15) and 30% (SLE-30) MVC, followed by postexercise circulatory occlusion (PECO). Mean arterial pressure (MAP) and heart rate (HR) responses were greater during SLE-30 than during SHG-30. The difference in MAP was maintained by PECO, and the part of the pressor response maintained by PECO was greater after SLE-30 than after SHG-30 (88.3 +/- 10.6 and 67.8 +/- 12.7%, respectively, P = 0. 02). There were no differences in MAP and HR responses between SHG-30 and SLE-15 trials. Baroreflex sensitivity was maintained during SHG-30 and SLE-15, whereas it was significantly reduced during SLE-30 and recovered back to the resting level during PECO. Minute ventilation and oxygen uptake increased more during SLE-30 than during both SHG-30 and SLE-15 trials. Minute ventilation remained significantly elevated above rest only during PECO following SLE-30. These data suggest that during static exercise the muscle mass and contraction intensity affect 1) the magnitude of the cardiorespiratory responses, 2) the contribution of muscle metaboreflex to the cardiorespiratory responses, and 3) the arterial baroreflex contribution to HR control.

Noxious stimuli do not determine reflex cardiorespiratory effects in anesthetized rabbits.

The main purpose of this study is to examine whether the stimulation of an exclusively pain-sensing receptive field (dental pulp) could determine cardiorespiratory effects in animals in which the cortical integration of the peripheral information is abolished by deep anesthesia. In 15 anesthetized (alpha-chloralose and urethan) rabbits, low (3-Hz)- and high-frequency (100-Hz) electrical dental pulp stimulation was performed. Because this stimulation caused dynamic and static reflex contractions of the digastric muscles leading to jaw opening jaw-opening reflex (JOR); an indirect sign of algoceptive fiber activation], experimentally induced direct dynamic and static contractions of the digastric muscle were also performed. The low- and high-frequency stimulation of the dental pulp determined cardiovascular [systolic arterial pressure (SAP): -21.7 +/- 4.6 and 10.8 +/- 4.7 mmHg, respectively] and respiratory [pulmonary ventilation (VE): 145.1 +/- 44.9 and 109.3 +/- 28.4 ml/min, respectively] reflex responses similar to those observed during experimentally induced dynamic (SAP: -17.5 +/- 4.2 mmHg; VE: 228.0 +/- 58.5 ml/min) and static (SAP: 5.8 +/- 1.5 mmHg; VE: 148.0 +/- 75.3 ml/min) muscular contractions. The elimination of digastric muscular contraction (JOR) obtained by muscular paralysis did away with the cardiovascular changes induced by dental pulp stimulation, the effectiveness of which in stimulating dental pulp receptors has been shown by recording trigeminal-evoked potentials in six additional rabbits. The main conclusion was that, in deeply anesthetized animals, an algesic stimulus is unable to determine cardiorespiratory effects, which appear to be exclusively linked to the stimulation of ergoreceptors induced by muscular contraction.

Cardiorespiratory reflexes from muscles during dynamic and static exercise in the dog.

Cardiorespiratory reflex responses during the initial phase of dynamic and static contraction of hindlimb muscles were studied in anesthetized dogs. Muscle contractions were elicited by stimulating the femoral and gastrocnemius nerves at 3 and 100 Hz with the intensity of 2.0-2.5 times the motor threshold for a 20-s period. Rhythmic contractions caused a decrease in arterial pressure (Pa) and heart rate (HR) and increased pulmonary ventilation (VE) by increasing frequency (f) without significantly changing VT. Tetanic contractions provoked an increase in Pa and HR and a hyperpnea resulting from a rise in both f and VT. Similar responses were also obtained in anesthetized dogs with carotid sinuses denervated and cervical vagi cut. The abrupt increase in VE at the start of both types of exercise was not associated with immediate significant decreases in end-tidal CO2 values. These two patterns of cardiocirculatory and respiratory responses were closely similar to those reported in anesthetized rabbits in previous studies. Both patterns of responses were reflexes initiated by activation of muscle receptors verified by interrupting the afferents from the contracting muscles. It is concluded that, during dynamic and static work, two distinct muscular reflex mechanisms might exert their drives, related to the muscular metabolic rate, on the circulatory and respiratory function.

Morphometrical and microdensitometrical studies on peptide- and tyrosine hydroxylase-like immunoreactivities in the forebrain of rats prenatally exposed to methylazoxymethanol acetate.

Methylazoxymethanol acetate (MAM Ac) injected into pregnant rats at a dose of 25 mg/kg at gestational day 15 causes microcephaly due to an atrophy of various telencephalic areas, mainly neocortex, hippocampus and basal ganglia. Previous studies demonstrated alterations in various neurochemical markers of classical transmitter systems in these regions. The present paper deals with changes in peptide and tyrosine hydroxylase (TH)-containing neurons in MAM Ac-induced microcephaly using immunocytochemistry coupled with computer-assisted morphometry and microdensitometry. No change in the number of vasoactive intestinal polypeptide (VIP)-immunoreactive neurons in the neocortex and neuropeptide Y (NPY)-immunoreactive neurons in the nucleus caudatus-putamen was found whereas cholecystokinin (CCK)-and NPY-immunoreactive neurons in the neocortex and CCK- and VIP-immunoreactive neurons in the hippocampus were decreased. The reduction of the latter peptide containing neuronal populations led to a maintained density of cells in MAM Ac-exposed rats, due to the parallel reduction of the overall mass of these regions. TH immunoreactivity was found to be unchanged in the basal ganglia, and increased in the cerebral cortex in agreement with previous reports on noradrenaline cortical system after MAM Ac exposure. The present results show a heterogenous vulnerability of different peptide immunoreactive neuronal populations to MAM Ac exposure. The sparing of VIP- and NPY-immunoreactive neurons may be due to their late development in the neocortex and striatum, respectively. The hypothesis is introduced that cortical VIP interneurons can develop independent of marked alterations in the intrinsic circuitry of the cortical region.

Treatment with methylazoxymethanol at different gestational days: physical, reflex development and spontaneous activity in the offspring.

Pregnant rats were injected with a single dose of methylazoxymethanol (MAM, 25 mg/kg) on gestational day 14, 15, 16, 17, 18 or 19 and offspring were tested for their physical development, reflex development and spontaneous activity. MAM treatment did not affect gestational and litter parameters at any of the time of administration studied. Treatment at gestational day 14 (GD14) had the most severe effect on functional neurodevelopment until weaning: righting reflex at surface, chimney test, horizontal wire test resulted altered. Administration at GD15, 16, 18, 19 did not affect the performance in these tests. Offspring treated at GD17 showed a delayed eye opening and an impaired performance in the horizontal wire test. When tested at 50 days of age on the rotarod, all the treated groups performed worse than controls with the exception of GD19 treated offspring. Administration at GD14 and GD15 resulted in increased spontaneous activity of the offspring at 21 days but not at 60 days of age. Different degrees of microencephaly were observed for all treated groups. The results indicate that alterations of physical and behavioral development induced by MAM treatment are dependent on the time of MAM administration, and specific behavioral tests are able to detect different abnormalities and differentiate among treatment groups. Some alterations observed in MAM rats undergo to adaptive changes during maturation of the CNS.

Treatment with methylazoxymethanol at different gestational days: two-way shuttle box avoidance and residential maze activity in rat offspring.

Pregnant rats were injected with a single dose of methylazoxymethanol (MAM, 25 mg/kg) on gestational days 14, 15, 16, 17, 18 or 19 which resulted in various degrees of microencephaly. Offspring were tested on a two-way shuttle box avoidance and residential maze activity at 60-90 days of age. Rats treated on gestational day 19 (GD19) were severely impaired in the acquisition of the two-way shuttle box task whereas the other groups did not show any significant difference from controls. Spontaneous activity measured for 23 hr in the residential maze was altered as total, time-course and pattern depending on the time of MAM administration: treatment on GD14 prolonged exploratory behavior, treatment on GD15 and GD16 increased nocturnal activity, treatment on GD16 and GD17 induced changes in locomotion patterns and treatment on GD18 and GD19 decreased total activity. These findings indicate that treatment with MAM results in selective deficits in the acquisition of a shuttle box avoidance and alterations of locomotion patterns in the offspring which are dependent on the time of administration.

Microencephalic rats as a model for cognitive disorders.

The administration of the antimitotic compound methylazoxymethanol (MAM) to gestating rats induces a dose-dependent atrophy of specific brain areas in the offspring. This specificity is strictly dependent upon the time of MAM administration. When given at day 15 of gestation only the cortex, hippocampus, and striatum are affected, whereas when given to rat pups at postnatal day 1, the atrophy is apparent only in the cerebellum. The microencephalic offspring of dams treated at day 15 of gestation develop normally to adulthood, without manifest signs of this profound telencephalic contraction. Behavioral abnormalities are observable when subjecting these animals to tests that involve learning. The deficit in associative behavior might have its anatomical and neurochemical counterpart in the disruption of the neuronal circuitry in the neocortex, where about 50% of interneurons are absent in layers II-IV after a dose of MAM of 25 mg/kg. Loss of intrinsic neurons occurs also in the striatum, as revealed by neurochemical and pharmacological analysis. Indeed, MAM rats show a reduced dopamine-dependent adenylate cyclase activity and a reduced motor stimulation in response to dopaminergic stimulants. MAM rats are therefore an interesting animal model of chronic brain damage induced transplacentally, which could serve for studying adaptive mechanisms of the CNS to this damage and its pharmacological manipulation.

Effects of alpha-human atrial natriuretic peptide in guinea-pig isolated heart.

The aim of the present investigation has been to ascertain whether or not atrial natriuretic peptides (ANP) can exert a direct effect on myocardial contractility. Alpha-human ANP (alpha-hANP) concentrations ranging from 1 pM to 50 nM have been used to perfuse guinea-pig isolated hearts in a non-recirculating Langendorff apparatus. A dual concentration-related effect has been induced by alpha-hANP on myocardial function. A maximal increase of +LV dP/dtmax (+56%; P < 0.001) has been observed when guinea-pig hearts were perfused with 100 pM alpha-hANP, whereas a 25% decrease (P < 0.01) occurred with 50 nM alpha-hANP. Similar effects have also been induced by alpha-hANP on the coronary flow rate (CFR). A significant CFR increase (maximal at 10 pM alpha-hANP) was induced by picomolar concentrations of alpha-hANP, whereas a progressive decrease, which was maximal (-28%; P < 0.01) at 50 nM alpha-hANP, was observed with nanomolar concentrations of the peptide. No effects have been observed on heart rate. These results suggest that ANP has direct effects on both vascular and myocardial muscle cells. Coronary vasoconstriction induced by nanomolar concentrations of ANP can contribute to the cardiodepression, whereas ANP in picomolar concentrations can induce a coronary vasodilation which is not coupled with the enhanced myocardial contractility. The latter is the likely expression of a direct effect of the peptide on myocardial function.