Medardo Hernández

Motor behavior and nigrostriatal dopaminergic activity in adult rats perinatally exposed to cannabinoids

We have recently reported several neurochemical alterations, measured at perinatal and peripubertal ages, in the maturation of nigrostriatal dopaminergic neurons following perinatal hashish exposure. In the present work, we tried to undertake whether these neurochemical changes during ontogeny: a) were accompanied by changes of motor behavior, the main neurobiological process regulated by nigrostriatal dopaminergic neurons; and b) persisted in adulthood, leading to disturbances in the expression of an adult motor activity. To this end, two different experiments were performed. In the first, we examined, by using an actimeter, the ontogeny of spontaneous locomotor activity in immature male and female rats born from mothers perinatally exposed to hashish extract. Results showed a complete absence of significant changes in locomotor activity in females, whereas males presented a constant trend to decrease, although never statistically significant, at all ages studied as a consequence of the perinatal cannabinoid exposure. In the second experiment, we evaluated neurochemical indices--dopamine (DA) and L-3,4-dihydroxyphenylacetic acid (DOPAC) contents, tyrosine hydroxylase (TH) activity, and number and affinity of D1 and D2 dopaminergic receptors in the striatum--and behavioral parameters--spontaneous locomotor activity and spontaneous and induced stereotypic behavior--both indicating nigrostriatal dopaminergic activity, in adult female and male rats perinatally exposed to hashish extract. Results were as follows. The spontaneous locomotor activity, measured in the actimeter, was not affected by perinatal hashish exposure in both adult males and females. This was also seen in an open-field test as measured by total number of sector crossings. However, when differentiated between internal and external sectors hashish-exposed males presented a higher number of external crossings than controls, which did not appear in females. Moreover, several induced stereotypic behaviors, such as self-grooming and shaking induced by water spraying, were also altered by hashish treatment in a sexually dimorphic manner, whereas the number of spontaneous rears and self-grooms, measured in the open-field test, was unchanged. Thus, the frequency of water spraying-induced self-grooming was significantly increased in both males and females perinatally exposed to hashish, although the increase was more marked in males (200.4%) than females (121.2%). In addition, the frequency of shaking was also markedly increased in males but remained unchanged in females. These behavioral effects were paralleled by modifications in striatal neurochemical parameters. Thus, there was a significant increase in the DOPAC/DA ratio, indicating increased presynaptic activity, in females perinatally exposed to hashish, but compensated by a lower density of D1 receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Motor disturbances induced by an acute dose of Δ9-tetrahydrocannabinol: Possible involvement of nigrostriatal dopaminergic alterations

Exposure to cannabinoids has been reported to affect several neurotransmitter systems and their related behaviors. The present study has been designed to further explore the effects of cannabinoids on motor behavior and test the involvement of nigrostriatal dopaminergic neurotransmission and other neurotransmitters as possible neurochemical targets for these cannabinoid effects. Male rats treated with an oral dose of delta 9-tetrahydrocannabinol (THC), the main psychoactive ingredient of cannabinoid derivatives, or vehicle were used 1 h after treatment for analyses of spontaneous motor and stereotypic activities together with neurochemical analyses of the nigrostriatal dopaminergic activity. Treatments and analyses were performed in the dark phase of photoperiod because it corresponds to the maximum behavioral expression in the rat. Neurochemical analyses were measurements of presynaptic activity--dopamine (DA) and L-3,4-dihydroxyphenylacetic acid (DOPAC) contents, tyrosine hydroxylase (TH) activity, and in vitro DA release--and postsynaptic sensitivity--number and affinity of D1 and D2 receptors--in the striatum. In addition, measurements of 5-hydroxytryptamine (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) contents were also performed to evaluate serotoninergic activity in the striatum. An oral dose of THC produced a loss of spontaneous motor activity, measured in both actimeter and open-field test, and a decrease in the frequency of several stereotypic behaviors, such as rearing and self-grooming. This decrease was correlated to a low number of D1-dopaminergic receptors in the striatum, although neither DA and DOPAC contents nor TH activity and D2 receptors were altered.(ABSTRACT TRUNCATED AT 250 WORDS)

An acute dose of δ9-tetrahydrocannabinol affects behavioral and neurochemical indices of mesolimbic dopaminergic activity

Cannabinoid consumption has been reported to affect several neurotransmitter systems and their related behaviors. The present study has been designed to examine cannabinoid effects on certain behaviors, which have been currently located in the limbic forebrain, in parallel to their effects on mesolimbic dopaminergic neurons. To this end, male rats treated with an oral dose of delta 9-tetrahydrocannabinol (THC) or vehicle were used 1 h after treatment for two different behavioral tests or neurochemical analyses of mesolimbic dopaminergic activity. Treatments, behavioral tests and sacrifice were performed in the dark phase of photoperiod because it corresponds to the maximum behavioral expression in the rat. Behavioral tests were a dark-light emergence test, which allows measurements of emotional reactivity, and a socio-sexual approach behavior test, which allows measurements of sexual motivation and also of spontaneous and stereotypic activities. Neurochemical analyses consisted of measurements of dopamine (DA) and L-3,4-dihydroxyphenylacetic acid (DOPAC) contents, tyrosine hydroxylase activity, in vitro DA release and number and affinity of D1 receptors in the limbic forebrain. Results were as follows. THC exposure markedly altered the pattern executed by the animals in both tests. Concretely, THC-exposed animals exhibited a low number of visits to an incentive female in addition to high time spent in the vicinity of an incentive male, both observed in the socio-sexual approach behavior test, and an increased emergence latency to go out of a dark compartment in the dark-light emergence test. However, the fact that THC also decreased spontaneous activity and the frequency of rearing and self-grooming behaviors, in addition to the observations of either low total number of visits to both incentive sexual areas or high escape latency to go out of a light compartment, when the animal is placed in this compartment, also suggest the possible existence of an accompanying motor deficit. These behavioral effects were accompanied by increases in DA and DOPAC contents and in D1 receptor density in the limbic forebrain and to a slight decrease in the pattern of K(+)-evoked DA release in vitro from perifused limbic fragments, with no changes in the remaining neurochemical parameters. Collectively, these results allow us to conclude that acute THC markedly altered the behavioral pattern executed by the animals in a socio-sexual approach behavior test and in a dark-light emergence test, presumably indicating loss of sexual motivation and increased emotionality, although also accompanied by motor deficiencies.(ABSTRACT TRUNCATED AT 400 WORDS)

Noradrenaline modulates smooth muscle activity of the isolated intravesical ureter of the pig through different types of adrenoceptors

1 We have studied the effects of α‐ and β‐adrenoceptor agonists and antagonists on both phasic peristaltic activity and basal tone of the isolated intravesical ureter of the pig by means of isometric techniques in vitro. 2 Spontaneous phasic activity was exhibited by 21% of pig intravesical ureter preparations manifested as rhythmic contractions with average frequency and amplitude of 2.54 ± 0.18 min−1 and 1.48 ± 0.16 g (n = 31), respectively. 3 Adrenaline, noradrenaline and phenylephrine induced concentration‐dependent increases in both phasic activity and basal tone of ureteral preparations, all three agonists being more potent in modifying ureteral phasic activity than baseline tone. B‐HT 920, B‐HT 933 and clonidine had no significant effect. 4 Phentolamine (10−9 − 10−7 m) and prazosin (3 × 10−11 − 3 × 10−8 m) significantly inhibited increases in both frequency of phasic activity and baseline tone induced by a submaximal dose of noradrenaline. Rauwolscine (10−9 − 10−7 m) affected only the tone evoked by noradrenaline and higher concentrations of this antagonist were needed to block phasic activity. 5 Pretreatment of ureteral strips with the β‐adrenoceptor antagonist, propranolol (10−6 m), significantly increased the maximum contraction evoked by noradrenaline. After incubation with phentolamine (10−6 m), noradrenaline (10−7 − 10−6 m) decreased phasic activity induced by prostaglandin F2α (10−5 m). Isoprenaline and salbutamol also abolished PGF2α‐induced phasic activity. Pafenolol (10−6 m) and butoxamine (10−6 m) blocked the inhibitory effect of noradrenaline, isoprenaline, and salbutamol on PGF2α‐induced phasic activity. 6 These results suggest that noradrenaline may modulate both phasic peristaltic activity and basal tone of pig intravesical ureter through both α‐ and β‐adrenoceptors.

Prejunctional alpha sub 2-Adrenoceptors Inhibit Nitrergic Neurotransmission in Horse Penile Resistance Arteries

PURPOSE To study the influence of alpha-adrenergic stimuli on non-adrenergic non-cholinergic (NANC) neurogenic relaxation in isolated horse penile resistance arteries. MATERIALS AND METHODS Deep intracavernous penile arteries with an internal lumen diameter of 200-500 microns., isolated from the corpus cavernosum of young horses, were mounted in microvascular myographs for isometric tension recording and electrical field stimulation (EFS) of autonomic nerve terminals. RESULTS In the presence of guanethidine (10(-5) M) and atropine (10(-7) M) tone of the arteries was raised by the thromboxane analogue, U46619. EFS (1, 4 and 32 Hz) induced frequency-dependent relaxations, which were abolished in the presence of tetrodotoxin, while NG-nitro-L-arginine (L-NOARG, 10(-4) M) abolished the relaxations to EFS at 1 Hz, and significantly reduced the relaxations at 4 Hz and 32 Hz by 82.5 +/- 10.2% and 52.9 +/- 4.7%, respectively (n = 6). EFS induced relaxations of a similar magnitude in penile arteries contracted with U46619 or the alpha 1-adrenoceptor agonist, phenylephrine, while the alpha 2-adrenoceptor agonist, BHT920 (10(-6) M), produced an inhibitory effect on the EFS-evoked relaxations which was inversely related to the stimulus frequency (1, 4 and 32 Hz). BHT920 had no effect on the relaxations induced by exogenous nitric oxide (NO), added as acidified sodium nitrite (10(-6)-10(-3) M). The inhibitory effect of BHT920 on NANC relaxations was reversed by 10(-7) M rauwolscine. CONCLUSION These results suggest that the release of a NANC neurotransmitter primarily thought to be NO is inhibited by stimulation of prejunctional alpha 2-adrenoceptors in horse penile resistance arteries.

Contribution of K+ channels and ouabain-sensitive mechanisms to the endothelium-dependent relaxations of horse penile small arteries

Penile small arteries (effective internal lumen diameter of 300–600 μm) were isolated from the horse corpus cavernosum and mounted in microvascular myographs in order to investigate the mechanisms underlying the endothelium‐dependent relaxations to acetylcholine (ACh) and bradykinin (BK). In arteries preconstricted with the thromboxane analogue U46619 (3–30 nM), ACh and BK elicited concentration‐dependent relaxations, pD2 and maximal responses being 7.71±0.09 and 91±1% (n=23), and 8.80±0.07 and 89±2% (n=24) for ACh and BK, respectively. These relaxations were abolished by mechanical endothelial cell removal, attenuated by the nitric oxide (NO) synthase (NOS) inhibitor, NG‐nitro‐L‐arginine (L‐NOARG, 100 μM) and unchanged by indomethacin (3 μM). However, raising extracellular K+ to concentrations of 20–30 mM significantly inhibited the ACh and BK relaxant responses to 63±4% (P<0.01, n=7) and to 59±4% (P<0.01, n=6), respectively. ACh‐ and BK‐elicited relaxations were abolished in arteries preconstricted with K+ in the presence of 100 μM L‐NOARG. In contrast to the inhibitor of ATP‐sensitive K+ channels, the blockers of Ca2+‐activated K+ (KCa) channels, charybdotoxin (30 nM) and apamin (0.3 μM), each induced slight but significant rightward shifts of the relaxations to ACh and BK without affecting the maximal responses. Combination of charybdotoxin and apamin did not cause further inhibition of the relaxations compared to either toxin alone. In the presence of L‐NOARG (100 μM), combined application of the two toxins resulted in the most effective inhibition of the relaxations to both ACh and BK. Thus, pD2 and maximal responses for ACh and BK were 7.65±0.08 and 98±1%, and 9.17±0.09 and 100±0%, respectively, in controls, and 5.87±0.09 (P<0.05, n=6) and 38±11% (P<0.05, n=6), and 8.09±0.14 (P<0.01, n=6) and 98±1% (n=6), respectively, after combined application of charybdotoxin plus apamin and L‐NOARG. The selective inhibitor of guanylate cyclase, 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ, 5 μM) did not alter the maximal responses to either ACh or BK, but slightly decreased the sensitivity to both agonists, δpD2 being 0.25±0.07 (P<0.05, n=6) and 0.62±0.12 (P<0.01, n=6) for ACh and BK, respectively. Combined application of ODQ and charybdotoxin plus apamin produced further inhibition of the sensitivity to both ACh (δpD2=1.39±0.09, P<0.01, n=6) and BK (1.29±0.11, P<0.01, n=6), compared to either ODQ or charybdotoxin plus apamin alone. Exogenous nitric oxide (NO) present in acidified solutions of sodium nitrite (NaNO2) and S‐nitroso‐cysteine (SNC) both concentration‐dependently relaxed penile resistance arteries, pD2 and maximal responses being 4.84±0.06 and 82±3% (n=12), and 6.72±0.07 and 85±4% (n=19), respectively. Charybdotoxin displaced to the right the dose‐relaxation curves for both NO (δpD2 0.38±0.06, P<0.01, n=6) and SNC (δpD2 0.50±0.10, P<0.01, n=5), whereas apamin only reduced sensitivity (δpD2=0.35±0.12, P<0.05, n=5) and maximum response (65±9%, P<0.05, n=6) to SNC. ODQ shifted to the right the dose‐relaxation curves to both NO and SNC. The relaxant responses to either NO or SNC were not further inhibited by a combination of ODQ and charybdotoxin or ODQ and charybdotoxin plus apamin, respectively, compared to either blocker alone. In the presence of 3 μM phentolamine, 5 μM ouabain contracted penile resistance arteries by 50±6% (n=17) of K‐PSS, but did not significantly change the relaxant responses to either ACh, BK or NO. However, in the presence of L‐NOARG ouabain reduced the ACh‐ and BK‐elicited relaxation from 94±3% to 16±5% (P<0.0001, n=6), and from 98±2% to 13±3% (P<0.0001, n=5), respectively. Combined application of ODQ and ouabain inhibited the relaxations to NO from 92±2% to 26±3% (P<0.0001, n=6). The present results demonstrate that the endothelium‐dependent relaxations of penile small arteries involve the release of NO and a non‐NO non‐prostanoid factor(s) which probably hyperpolarize(s) smooth muscle by two different mechanisms: an increased charybdotoxin and apamin‐sensitive K+ conductance and an activation of the Na+‐K+ATPase. These two mechanisms appear to be independent of guanylate cyclase stimulation, although NO itself can also activate charybdotoxin‐sensitive K+ channels and the Na+‐K+ pump through both cyclic GMP‐dependent and independent mechanisms, respectively.

Differential structural and functional changes in penile and coronary arteries from obese Zucker rats.

Erectile dysfunction frequently coexists with coronary artery disease and has been proposed as a potential marker for silent coronary artery disease in type 2 diabetes. In the present study, we comparatively assessed the structural and functional changes of both penile arteries (PAs) and coronary arteries (CAs) from a prediabetic animal model. PAs and CAs from 17- to 18-wk-old obese Zucker rats (OZRs) and from their control counterparts [lean Zucker rats (LZRs)] were mounted in microvascular myographs to evaluate vascular function, and stained arteries were subjected to morphometric analysis. Endothelial nitric oxide (NO) synthase (eNOS) protein expression was also assessed. The internal diameter was reduced and the wall-to-lumen ratio was increased in PAs from OZRs, but structure was preserved in CAs. ACh-elicited relaxations were severely impaired in PAs but not in CAs from OZRs, although eNOS expression was unaltered. Contractions to norepinephrine and 5-HT were significantly enhanced in both PAs and CAs, respectively, from OZRs. Blockade of NOS abolished endothelium-dependent relaxations in PAs and CAs and potentiated norepinephrine and 5-HT contractions in arteries from LZRs but not from OZRs. The vasodilator response to the phosphodiesterase 5 inhibitor sildenafil was reduced in both PAs and CAs from OZRs. Pretreatment with SOD reduced the enhanced vasoconstriction in both PAs and CAs from OZRs but did not restore ACh-induced relaxations in PAs. In conclusion, the present results demonstrate vascular inward remodeling in PAs and a differential impairment of endothelial relaxant responses in PAs and CAs from insulin-resistant OZRs. Enhanced superoxide production and reduced basal NO activity seem to underlie the augmented vasoconstriction in both PAs and CAs. The severity of the structural and functional abnormalities in PAs might anticipate the vascular dysfunction of the more preserved coronary vascular bed.

Time-course of the effects of ovarian steroids on the activity of limbic and striatal dopaminergic neurons in female rat brain

This paper studies the time-course of the effects of pharmacological administrations of ovarian steroids on the functional state of dopaminergic terminals in the striatum and the limbic forebrain, using the ratio between the contents of dopamine (DA) and its metabolite, L-3,4 dihydroxyphenylacetic acid (DOPAC), as an index of nerve activity. Estradiol produced an increase in the dopaminergic activity of both limbic and striatal neurons, reflected in the high DOPAC/DA ratio observed in both areas. This estrogenic effect was only observed at 4 hours, disappearing in the subsequent times studied. The effect was antagonized by progesterone in both tissues, since a single injection of this steroid to estrogen-pretreated rats restored to control values the estradiol-induced increase, suggesting the existence of negative interactions between both steroids. Furthermore, treatment with progesterone produced also a late decrease of the DOPAC/DA ratio in the striatum, which was observed only in the animals nonpretreated with estrogens.

The prenatal exposure to δ9-tetrahydrocannabinol affects the gene expression and the activity of tyrosine hydroxylase during early brain development

We have previously reported that the exposure of pregnant female rats to delta 9-tetrahydrocannabinol (THC) during the perinatal period affected the gene expression and the activity of tyrosine hydroxylase (TH) in the brain of their male offspring. Those studies were done in animals perinatally exposed to THC but tested at peripubertal and adult ages. In the present work, we explored whether these effects also appear during early fetal brain development, when TH expression plays an important role in neuronal development. To this end, TH-mRNA concentrations were measured by Northern blot analysis with a specific TH probe in the brain of fetuses at gestational days 14 and 16 which had been prenatally exposed to THC or vehicle from day 5 of gestation. In parallel, measurements of TH activity and catecholamine contents by HPLC were also done. The results obtained were as follows. The prenatal exposure to THC markedly affected the expression of the TH gene in the brain of fetuses at gestational day 14. Thus, the amounts of TH-mRNA at this age were higher (2-fold) in THC-exposed fetuses than in controls. This corresponded with a marked increase in the activity of this enzyme (3-fold) at this age. Normalization was found in both parameters at gestational day 16. In summary, the prenatal exposure to THC affected the expression of the TH gene and the activity of this enzyme in brain catecholaminergic neurons during early fetal brain development. These results support the notion that cannabinoids are able to act at the level of the gene expression of specific key proteins for brain development.

Neuronal and smooth muscle receptors involved in the PACAP- and VIP-induced relaxations of the pig urinary bladder neck

As pituitary adenylate cyclase‐activating polypeptide 38 (PACAP 38)‐ and vasoactive intestinal peptide (VIP) are widely distributed in the urinary tract, the current study investigated the receptors and mechanisms involved in relaxations induced by these peptides in the pig bladder neck.