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Initially introduced in therapy as immunosuppressants, the selective inhibitors of mTORC1 have been approved for the treatment of solid tumors. Novel non-selective inhibitors of mTOR are currently under preclinical and clinical developments in oncology, attempting to overcome some limitations associated with selective inhibitors, such as the development of tumor resistance. Looking at the possible clinical exploitation in the treatment of glioblastoma multiforme, in this study we used the human glioblastoma cell lines U87MG, T98G and microglia (CHME-5) to compare the effects of a non-selective mTOR inhibitor, sapanisertib, with those of rapamycin in a large array of experimental paradigms, including (i) the expression of factors involved in the mTOR signaling cascade, (ii) cell viability and mortality, (iii) cell migration and autophagy, and (iv) the profile of activation in tumor-associated microglia. We could distinguish between effects of the two compounds that were overlapping or similar, although with differences in potency and or/time-course, and effects that were diverging or even opposite. Among the latter, especially relevant is the difference in the profile of microglia activation, with rapamycin being an overall inhibitor of microglia activation, whereas sapanisertib was found to induce an M2-profile, which is usually associated with poor clinical outcomes.

CHF6467 is a mutated form of human recombinant nerve growth factor (NGF). The mutation selectively disrupts the binding of NGF to its p75NTR receptor while maintaining the affinity toward TrkA receptor. Because of such different profile of receptor interaction, CHF6467 maintains unaltered the neurotrophic and neuroprotective properties of wild-type NGF but shows reduced algogenic activity.In this study, we investigated the effects of CHF6467 on mortality, proliferation, cell-damage and migration in three human glioblastoma cell lines (U87MG, T98G, LN18), and in the rat astrocytoma C6 cells. Both CHF6467 and wild-type NGF, given in the range 1-50 ng/ml, did not modify cell proliferation, metabolism and migration, as well as the number of live/dead cells.The present

The immune checkpoint cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is an inhibitory regulator of T-cell mediated responses that has been investigated as target of monoclonal antibodies (mAbs) for cancer immunotherapy. The anti-CTLA-4 mAb ipilimumab represents the first immune checkpoint inhibitor that significantly improved overall survival in patients with unresectable/metastatic melanoma. The subsequent approved indications (often in the first-line setting) for melanoma and other advanced/metastatic solid tumors always require ipilimumab combination with nivolumab, an anti-programmed cell death protein 1 (PD-1) mAb. However, the improved clinical efficacy of the mAb combination is associated with increased immune-related adverse events, which might require treatment discontinuation even in responding patients. This drawback is expected to be overcome by the recent development of anti-CTLA-4 probodies proteolitycally activated in the tumor microenvironment and bispecific molecules targeting both CTLA-4 and PD-1, whose co-expression is characteristic of tumor-infiltrating T cells. These molecules would preferentially stimulate immune responses against the tumor, reducing toxicity toward normal tissues.

Ghrelin and its endogenous antagonist liver‐expressed antimicrobial peptide‐2 (LEAP‐2) are involved in GH secretion and glucose/lipids metabolism. LEAP‐2 expression in conditions of metabolic impairment may be upregulated, usually pairing with a concomitant reduction in ghrelin secretion. Adult growth hormone deficiency (aGHD) is characterized by insulin resistance, weight gain, and increased fat mass. Therefore, the primary endpoint of this cross‐sectional observational pilot study was to compare circulating LEAP‐2 and ghrelin levels in aGHD and healthy controls. Thirty patients were included in the study. Group A included adult GHD: 15 patients, 8 females, and 7 males. Median and interquartile range age of the group was 53 (41–57) years, while BMI was 27.1 (25–35) kg/m2. Group B was formed by 15 healthy controls (10 females and 5 males). Median and interquartile range age was 47 (36–57) years, while BMI 22.9 (20.8–33.1) kg/m2. They were evaluated for serum glucose and insulin, HOMA‐index, QUICKI‐index, total/LDL/HDL cholesterol, triglycerides, IGF‐1, ghrelin, and LEAP‐2. Ghrelin levels in the aGHD group were significantly lower than in healthy controls. In contrast, LEAP‐2 showed a trend toward higher levels, although the differences were not significant. However, the LEAP‐2/Ghrelin ratio was significantly higher in aGHD. No significant correlations between ghrelin and LEAP‐2 with BMI and HOMA index were found in aGHD population. However, a significant inverse correlation (r 2 = 0.15, p = .047) between BMI and ghrelin was evidenced when considering the whole population. Taken together, these results may suggest a body adaptation to a metabolic scenario typical of aGHD. The decrease in ghrelin production could prevent further weight gain and fat mass increase, although losing its secretagogue effect.

Background Purine receptors play roles in peripheral and central sensitization and are associated with migraine headache. We investigated the possibility that ATP plays a permissive role in the activation of AMPA receptors thus inducing Glu release from nerve terminals isolated from the rat trigeminal caudal nucleus (TCN). Methods Nerve endings isolated from the rat TCN were loaded with [3H]D-aspartic acid ([3H]D-ASP), layered into thermostated superfusion chambers, and perfused continuously with physiological medium, alone or with various test drugs. Radioactivity was measured to assess [3H]D-ASP release under different experimental conditions. Results Synaptosomal [3H]D-ASP spontaneous release was stimulated by ATP and to an even greater extent by the ATP analogue benzoylbenzoylATP (BzATP). The stimulation of [3H]D-ASP basal release by the purinergic agonists was prevented by the selective P2X7 receptor antagonist A438079. AMPA had no effect on basal [3H]D-ASP release, but the release observed when synaptosomes were exposed to AMPA plus a purinoceptor agonist exceeded that observed with ATP or BzATP alone. The selective AMPA receptor antagonist NBQX blocked this “excess” release. Co-exposure to AMPA and BzATP, each at a concentration with no release-stimulating effects, evoked a significant increase in [3H]D-ASP basal release, which was prevented by exposure to a selective AMPA antagonist. Conclusions P2X7 receptors expressed on glutamatergic nerve terminals in the rat TCN can mediate Glu release directly and indirectly by facilitating the activation of presynaptic AMPA receptors. The high level of glial ATP that occurs during chronic pain states can promote widespread release of Glu as well as can increase the function of AMPA receptors. In this manner, ATP contributes to the AMPA receptor activation involved in the onset and maintenance of the central sensitization associated with chronic pain.

Voltage-gated type 7 K+ (KV7 or KCNQ) channels regulate the contractility of various smooth muscles. With this study, we aimed to assess the role of KV7 channels in the regulation of human detrusor contractility, as well as the gene and protein expression of KV7 channels in this tissue. For these purposes, the isolated organ technique, RT-qPCR, and Western blot were used, respectively. XE-991, a selective KV7 channel blocker, concentration-dependently contracted the human detrusor; mean EC50 and Emax of XE-991-induced concentration-response curve were 14.1 μM and 28.8 % of the maximal bethanechol-induced contraction, respectively. Flupirtine and retigabine, selective KV7.2–7.5 channel activators, induced concentration-dependent relaxations of bethanechol-precontracted strips, with maximal relaxations of 51.6 and 51.8 % of the precontraction, respectively. XE-991 blocked the relaxations induced by flupirtine and retigabine. All five KCNQ genes were found to be expressed in the detrusor with KCNQ4 being the most expressed among them. Different bands, having sizes similar to some of reported KV7.1, 7.4, and 7.5 channel subunit isoforms, were detected in the detrusor by Western blot with the KV7.4 band being the most intense among them. In conclusion, KV7 channels contribute to set the basal tone of the human detrusor. In addition, KV7 channel activators significantly relax the detrusor. The KV7.4 channels are probably the most important KV7 channels expressed in the human detrusor. These data suggest that selective KV7.4 channel activators might represent new pharmacological tools for inducing therapeutic relaxation of the detrusor.

Voltage-gated type 7 K

In the present study, by means of genetic, biochemical, morphological, and electrophysiological approaches, the role of large-conductance voltage- and Ca(2+)-dependent K(+) channels (BK channels) in the release of excitatory and non-excitatory neurotransmitters at hippocampal and non-hippocampal sites has been investigated. The results obtained show that the pharmacological modulation of pre-synaptic BK channels selectively regulates [(3)H]D-aspartate release from cortical and hippocampal rat synaptosomes, but it fails to influence the release of excitatory neurotransmitters from cerebellar nerve endings or that of [(3)H]GABA, [(3)H]Noradrenaline, or [(3)H]Dopamine from any of the brain regions investigated. Confocal immunofluorescence experiments in hippocampal or cerebrocortical nerve terminals revealed that the main pore-forming BK α subunit was more abundantly expressed in glutamatergic (vGLUT1(+)) versus GABAergic (GAD(65-67)(+)) nerve terminals. Double patch recordings in monosynaptically connected hippocampal neurons in culture confirmed a preferential control exerted by BK channels on glutamate over GABA release. Altogether, the present results highlight a high degree of specificity in the regulation of the release of various neurotransmitters from distinct brain regions by BK channels, supporting the concept that BK channel modulators can be used to selectively limit excessive excitatory amino acid release, a major pathogenetic mechanism in several neuropsychiatric disorders.

In this study, we demonstrate the existence on trigeminal caudal nucleus (TCN) glutamatergic terminals of α4β2 nicotinic receptors (nAChRs) capable of enhancing the terminals' spontaneous release of [(3)H]d-aspartate ([(3)H]D-Asp). In rat TCN synaptosomes, spontaneous [(3)H]D-Asp release was increased by nicotine and the nicotinic receptor agonists (±)epibatidine and RJR2403. The increase was potentiated by the positive allosteric modulator of nAChRs LY2087101, inhibited by the nicotinic antagonists mecamylamine (MEC) and dihydro-β-erythroidine hydrobromide (DHβE), and unaffected by α-bungarotoxin (α-BgTx) and methyllycaconitine (MLA). Evidence of functional interaction was observed between the α4β2 nAChRs and cyclothiazide-sensitive, alfa-amino-3-hydroxy-5-methyl-4-isoxazolone propionate (AMPA) receptors co-localized on the TCN synaptosomes. Brief pre-exposure of synaptosomes to 30 μM nicotine or 10 μM RJR2403 abolished the AMPA (100 μM) -induced potentiation of [K(+)]e-evoked [(3)H]D-Asp release, an effect that seems to be caused by nicotine-induced increases in the internalization of AMPA receptors. Indeed, the effects of nicotine-pretreatment were not seen in synaptosomes containing pre-entrapped pep2-SVKI, a peptide known to compete for the binding of GluA2 subunit to scaffolding proteins involved in AMPA endocytosis, while entrapment of pep2-SVKE, an inactive peptide used as negative control, was inefficacious. These findings show that nicotine can negatively modulate the function of AMPA receptors present on glutamatergic nerve terminals in the rat TCN. Dynamic control of AMPA receptors by the nicotinic cholinergic system has been observed under other experimental conditions, and it can contribute to the control of synaptic plasticity such as long-term depression and potentiation. Nicotine's ability to reduce the functionality of presynaptic AMPA receptors could contribute to its analgesic effects by diminishing glutamatergic transmission from the primary afferent terminals that convey nociceptive input to TCN.

We examined the effects of the endocannabinoide-anandamide (AEA), the synthetic cannabinoid, WIN55,212-2, and the active phorbol ester, 4-beta-phorbol 12-myristate 13-acetate (4-beta-PMA), on the release of [(3)H]d-Aspartate ([(3)H]d-ASP) from rat hippocampal synaptosomes. Release was evoked with three different stimuli: (1) KCl-induced membrane depolarization, which activates voltage-dependent Ca(2+) channels and causes limited neurotransmitter exocytosis, presumably from ready-releasable vesicles docked in the active zone; (2) exposure to the Ca(2+) ionophore-A23187, which causes more extensive transmitter release, presumably from intracellular reserve vesicles; and (3) K(+) channel blockade by 4-aminopyridine (4-AP), which generates repetitive depolarization that stimulates release from both ready-releasable and reserve vesicles. AEA produced concentration-dependent inhibition of [(3)H]d-ASP release stimulated with 15 mM KCl (E(max)=47.4+/-2.8; EC(50)=0.8 microM) but potentiated the release induced by 4-AP (1mM) (+22.0+/-1.3% at 1 microM) and by A23187 (1 microM) (+98.0+/-5.9% at 1 microM). AEA's enhancement of the [(3)H]d-ASP release induced by the Ca(2+) ionophore was mimicked by 4-beta-PMA, which is known to activate protein kinase C (PKC), and the increases produced by both compounds were completely reversed by synaptosome treatment with staurosporine (1 microM), a potent PKC blocker. In contrast, WIN55,212-2 inhibited the release of [(3)H]d-ASP evoked by KCl (E(max)=47.1+/-2.8; EC(50)=0.9 microM) and that produced by 4-AP (-26.0+/-1.5% at 1 microM) and had no significant effect of the release induced by Ca(2+) ionophore treatment. AEA thus appears to exert a dual effect on hippocampal glutamatergic nerve terminals. It inhibits release from ready-releasable vesicles and potentiates the release observed during high-frequency stimulation, which also involves the reserve vesicles. The latter effect is mediated by PKC. These findings reveal novel effects of AEA on glutamatergic nerve terminals and demonstrate that the effects of endogenous and synthetic cannabinoids are not always identical.

Cannabinoids (CB) can act as retrograde synaptic mediators of depolarization-induced suppression of inhibition or excitation in hippocampus. This mechanism may underlie the impairment of some cognitive processes produced by these compounds, including short-term memory formation in the hippocampus. In this study, we investigated several compounds known to interact with CB receptors, evaluating their effects on K +-evoked release of [ 3H]d-aspartate ([ 3H]d-ASP) and [ 3H]GABA from superfused synaptosomes isolated from the rat hippocampus. [ 3H]d-ASP and [ 3H]GABA release were inhibited to different degrees by the synthetic cannabinoids WIN 55,212-2; CP 55,940, and arachidonyl-2′- chloroethylamide/N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA), as well as by the endocannabinoids, anandamide (AEA), and 2-arachidonoylglycerol (2-AG). Both types of release were also inhibited by capsaicin. The inhibition produced by each of the cannabinoid compounds and capsaicin was unaffected by capsazepine or by the CB1-receptor antagonists AM-251 and SR141716A. The mechanism underlying AEA- and synthetic CB-induced inhibition of the release of [ 3H]GABA and [ 3H]d-ASP from rat hippocampal synaptosomes might not involve activation of presynaptic CB1 receptors.

Prenatal exposure to diazepam (DZ), a positive allosteric modulator of the γ-aminobutyric acidA (GABAA) receptor complex, exerts profound effects that become more evident during puberty and in many cases are sex-specific, suggesting that such exposure interferes with the activity of steroid hormones. Apart from their well known effects on the genome, the reduced metabolites of many steroid hormones also interact directly with membrane receptors, including those for N-methyl- d -aspartate (NMDA). In this study, we compared the effects of several neurosteroids on NMDA receptors from normal rats and those exposed in utero to DZ (1.25 mg/kg per day) from the 14th through the 20th day of gestation. In superfused rat hippocampal synaptosomes, activation of the NMDA receptor stimulates the basal release of [ 3 H ]noradrenaline ([ 3 H ]NA), which was used in our study as an index of receptor function. [ 3 H ]NA release was evoked in a concentration-dependent manner by NMDA (100 μM) plus glycine (GLY). The maximal increase (68.23±3.86%) with respect to basal release was achieved with a GLY concentration of 10 μM, and the EC50 for GLY was 0.1 μM. Release stimulated by 100 μM NMDA+0.1 μM GLY was not modified by any of the neurosteroids tested, with the exception of pregnenolone sulfate (PREG-S), which produced a 78.57±3.94% reduction in release at the maximal concentration used (0.3 μM). In synaptosomes from animals exposed in utero to DZ, the inhibitory effect of PREG-S was reduced by 46.55±2.33%. Given the important roles played by NMDA receptors in physiological and pathological processes within the central nervous system (CNS), characterization of NMDA receptor modulation is an important objective. The fact that this modulation can be altered by exposure in utero to DZ indicates that the behavioral abnormalities observed in exposed animals might be partially attributed to an altered sensitivity of NMDA receptors to the modulatory effects of neurosteroids.

The pH fluctuations have been often interpreted as an insufficient regulation or as a consequence of the onset of pathological events, such as ischemia, in which a significant decrease in pH levels occurs. Neurotransmitter release appears to be affected by pH drop significantly. In this study, we investigated the effect of an extracellular and an intracellular acidification on tritiated dopamine release ([3H]-DA release), from superfused rat hypothalamic synaptosomes. When compared to basal release, extracellular acidification, due to a reduction in the external pH of the nominally carbonic-free superfusion media, provoked a significant increase in [3H]-DA release that showed a sensitiveness to calcium omission. Intraterminal acidification, obtained blocking the Na(+)/H(+) exchanger by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and 5-(N,N-dimethyl)-amiloride (DMA), induced a significant increase in [3H]-DA outflow which occurred in a calcium-dependent manner (80% inhibition in absence of calcium from superfusion media). To further promote an intraterminal acidification through a H(+) inner accumulation, the proton ionophore nigericin was used. At every dose employed (10 microM), this compound induced a significant increase in [3H]-DA outflow, compared to basal release. Nigericin-evoked [3H]-DA release showed a 50% decrease when calcium was omitted from superfusion media. When BAPTA-AM, a chelator of intracellular calcium, was added, nigericin-evoked [3H]-DA was completely abolished. These data indicate that [3H]-DA release can be induced by extracellular acidification due to a lowering of external pH and by an intraterminal acidification due to an internal proton accumulation. The mechanism that can trigger this exocytotic process appears to depend on calcium presence, and in particular, on an increased intraterminal calcium availability.

Central GABAergic and serotoninergic systems interact with one another and are implicated in controlling different behaviours. A gentle early long-lasting handling can prevent the deficits in locomotion and exploration in open field (O.F.) in 3-month-old male rats prenatally exposed to diazepam (DZ). Purpose of this study was to extend the research to older handled rats prenatally exposed to DZ and to assess the activity of 5-HT1A receptors (Rs), evaluating the performance in O.F. at 3 and 18 months of age following 8-OH-DPAT administration. A single daily s.c. injection of DZ (1.5 mg/kg) from gestation day 14 to gestation day 20 induced in aged, but not in young rats, a decrease in total distance travelled (TDT) and in rearing frequency (RF) and an increase of transitions from the periphery to the centre of the arena (CNT) and in the time spent in the centre of the arena (CAT), compared to controls. 8-OH-DPAT (0.150 mg/kg s.c.), given 1 h before testing, increased TDT and decreased RF, CNT and CAT in both vehicle- and DZ-exposed young rats. In aged rats prenatally exposed to DZ, 8-OH-DPAT induced an increase in TDT and a slight decrease in RF, CNT and CAT. These findings indicate that the effects of handling and of 8-OH-DPAT in prenatally DZ-exposed rats are age-dependent and suggest that O.F. test can represent a valid tool to identify the changes in 5-HT1A Rs activity following drug treatment.

In rats, exposure to diazepam (DZ) during the last week of gestation is associated with behavioral alterations (in some cases sexually dimorphic) that appear when the animals reach adulthood. This study was conducted to evaluate the effects of prenatal DZ exposure on the function of the γ-aminobutyric (GABA)A receptor complex. The method used – perfusion of rat hippocampal nerve terminals labeled with [3H]noradrenaline (NA) – allowed us to evaluate the effects of DZ on a specific native GABAA receptor subtype which is located on hippocampal noradrenergic nerve endings and mediates the release of NA. Muscimol stimulated synaptosomal release of [3H]NA in a concentration-dependent manner; maximal stimulation (50%) was achieved with a concentration of 30 µM, and the ED50 was 1.7 µM. The effect of muscimol was potentiated by the positive allosteric modulators DZ and 3α-pregnan-5α-ol-20-one (3α,5α-P; allopregnanolone), which displayed similar maximal effects and affinities. In the presence of DZ (0.1 µM), muscimol stimulated the release of [3H]NA with an ED50 of 0.5 µM; in the presence of 3α,5α-P (0.1 µM), the ED50 of muscimol was 0.3 µM. Prenatal DZ exposure did not modify the concentration-effect curve for muscimol, but it did abolish the potentiating effects of DZ and 3α,5α-P. These findings demonstrate that prenatal exposure to DZ produces functional modifications of the GABAA receptor subtype we investigated. This effect may be related to the relative contributions of the various protein subunits that compose the GABAA receptor complex. Exposure to DZ while the GABAA receptors are developing might influence the expression of these subunits, giving rise to a receptor that can be activated by muscimol but is not susceptible to allosteric modulation by DZ or 3α,5α-P.

Large conductance, calcium-activated potassium channels [big potassium (BK) channel] consist of a tetramer of pore-forming α-subunit and distinct accessory β-subunits (β1-4) that modify the channel's properties. In this study, we analyzed the effects of BK channel activators and blockers on glutamate and γ-aminobutyric acid (GABA) release from synaptosomes isolated from the cerebral cortices or trigeminal caudal nuclei (TCN) of rats. Real-time polymerase chain reaction was used to characterize BK channel α and β(1-4) subunit expression in the cortex and in the trigeminal ganglia (TG), whose neurons project primary terminal afferents into the TCN. Immunocytochemistry was used to localize these subunits on cortical and TCN synaptosomes. The BK channels regulating [(3)H]D-aspartate release from primary afferent nerve terminals projecting into the TCN displayed limited sensitivity to iberiotoxin, whereas those expressed on cortical synaptosomes were highly sensitive to this toxin. BK channels did not appear to be present on GABAergic nerve terminals from the TCN since [(3)H]-γ-aminobutyric acid release in this model was unaffected by BK channel activators or blockers. Gene expression studies revealed expression levels of the α subunit in the TG that were only 31.2 ± 2.1% of those found in cortical tissues. The β4 subunit was the accessory subunit expressed most abundantly in both the cortex and TG. Levels of β1 and β2 were low in both these areas although β2 expression in the TG was higher than that found in the cortex. Immunocytochemistry experiments showed that co-localization of α and β4 subunits (the accessory subunit most abundantly expressed in both brain areas) was more common in TCN synaptosomes than in cortical synaptosomes. On the basis of these findings, it is reasonable to hypothesize that BK channels expressed on glutamatergic terminals in the TCN and cortex have distinct pharmacological profiles, which probably reflect different α and β subunit combinations. Channels in the cortex seem to be composed mainly of α subunits and to a lesser degree by α and β4 subunits, whereas in the TG the α + β4 combination seems to prevail (although α and/or α + β2 channels cannot be excluded). In light of the BK channels' selective control of excitatory transmission and their pharmacological diversity, their effects on primary glutamatergic afferents projecting to TCN represent a potential target for drug therapy of migraines and other types of orofacial pain.

Environmental stressors can substantially affect the adaptive response of rats to novelty in a sexually dimorphic manner. Gender-related differences are also observed in neurochemical and behavioural patterns of adult rats following prenatal exposure to diazepam (DZ). In the present study the behavioural reactivity to novelty is investigated in open field (OF) and in acoustic startle reflex (ASR) tests, in non handled (NH), short-lasting handled (SLH) and long-lasting handled (LLH) adult male and female rats prenatally exposed to DZ. A single daily s.c. injection of DZ (1.5 mg/kg) over gestation days 14-20 decreases GABA/BDZ receptor function in both sexes, as shown by the decreased electrographic hippocampal response to DZ and the increased response to picrotoxin, after intra-locus coeruleus injection of the two compounds. In OF NH DZ-exposed males display a lower total distance travelled (TDT), a higher rearing frequency (RF) and a greater number of transitions in the centre of the arena (CNT) compared to NH rats prenatally exposed to vehicle. Conversely, NH DZ-exposed females show slight changes in TDT and RF and a greater reduction in CNT and in the amount of time spent in the centre of the arena (CAT). These effects are associated with an increase in the peak amplitude of the ASR in both sexes. Short-lasting handling slightly influences DZ-evoked effects in animals of both sexes. In DZ-exposed males long-lasting handling attenuates the reduction in TDT and the enhancement in RF, prevents the increase in CNT and reduces the peak amplitude of ASR. In DZ-exposed females, long-lasting handling increases TDT and RF, induces a lower avoidance of the centre of the arena, and does not modify the peak amplitude of ASR, when compared to controls. These findings indicate that prenatal exposure to DZ differently affects behavioural reactivity in adult male and female rats, and suggest that a long-lasting handling is able to attenuate some behavioural deficits induced by prenatal DZ exposure.

The effects of the formamidine pesticides amitraz and chlordimeform on the alpha(2)-adrenergic receptor subtype that mediates the release of [(3)H]noradrenaline by synaptosomes from rat hypothalami were studied. We initially characterized the presynaptic autoreceptor on noradrenergic nerve endings using selective antagonists. Yohimbine (a nonselective alpha(2) antagonist) and BRL 44408 (selective for subtypes alpha(2A)/alpha(2D)) diminished the inhibitory effect of xylazine on K(+)-evoked release of [(3)H]noradrenaline; the K(B) values were 481 and 154 nM, respectively. In contrast, prazosin (a selective alpha(2B)/alpha(2C) antagonist) did not modify the inhibitory effect of xylazine. These results indicate that the release of noradrenaline by noradrenergic nerve endings in the rat hypothalamus is regulated by alpha(2D)-adrenoceptors, a species variation of the human alpha(2A) subtype. We then assessed the effects of the two pesticides on the K(+)-evoked release of [(3)H]noradrenaline. Amitraz reduced release in a dose-dependent manner; the effect observed at the maximal concentration tested (10 microM) was 13.0 +/- 2.0% and it was reversed by yohimbine. Amitraz also diminished the inhibitory effects of the alpha(2)-adrenergic agonists clonidine and xylazine. Chlordimeform displayed no effects, possibly because the true active compound of this insecticide is its demethylated metabolite. Based on these findings we conclude that the formamidine pesticides act as partial agonists of presynaptic alpha(2D)-adrenergic receptors in the rat hypothalamus. This interaction may be responsible for the in vivo alterations in catecholaminergic regulation of cyclic variations in gonadotropin-releasing hormone (GnRH) secretion, which can have grave functional repercussions on the reproductive system of mammals exposed to these xenobiotics.

Interaction between different transmitter receptor systems is an emerging feature of neurotransmission at central synapses. G protein-coupled receptors' ability to form dimers or larger hetero-oligomers probably serves to facilitate the integration of diverse signals within the cell. We found that, in nerve terminals isolated from the cerebral cortices of rats, co-application of the GABAB agonist, baclofen, and of the non-selective mGlu agonist, L-CCG-I, potentiates the basal and depolarization-evoked release of [(3)H]GABA via a mechanism that involves mobilization of intracellular Ca(2+) ions. The effect of L-CCG-I + baclofen was abolished by the phospholipase C inhibitor U73122, reduced by Xestospongin C (an IP3 receptor blocker), and blocked by 2-APB, an IP3 receptor antagonist. Pretreatment of the synaptosomes with the lipid-soluble Ca(2+) chelator BAPTA-AM also inhibited the effects of L-CCG-I + baclofen. Subtype-selective non-competitive group I mGlu receptor antagonists, MPEP and CPCCOEt, had no effect on the release enhancement produced by baclofen + L-CCG-I. The enhancement was reversed by the GABAB receptor antagonist, CGP54626, and by the group I/group II mGlu receptor antagonist (R,S)-MCPG. The GABA release-enhancing effects of L-CCG-I + baclofen in our model might reflect the presence on cortical nerve endings of GABAB/group I mGlu receptor heteromers with pharmacological properties distinct from those of the component receptors. Activation of these heteromeric receptors might modify the function of the GABAB receptor in such a way that it facilitates GABAergic transmission, an effect that might be useful under conditions of excessive glutamatergic activity.

Voltage-dependent type 7 K + (K V 7 or KCNQ) channels modulate the excitability of neurons and muscle cells. The aims of the present study were to investigate the motor effects of K V 7 channel modulators and the expression of K V 7 channels in the human taenia coli . The effects of K V 7 channel modulators on the muscle tone of human taenia coli strips were investigated under nonadrenergic non-nitrergic conditions by organ bath studies. Gene expression and tissue localisation of channels were studied by real-time PCR and immunohistochemistry, respectively. Under basal conditions, the K V 7 channel blocker XE-991 induced concentration-dependent contractions, with mean EC 50 and E max of 18.7 μM and 30.5% respectively of the maximal bethanechol-induced contraction, respectively. The K V 7 channel activators retigabine and flupirtine concentration-dependently relaxed the taenia coli , with mean EC 50 s of 19.2 μM and 29.9 μM, respectively. Retigabine also relaxed bethanechol-precontracted strips, with maximal relaxations of 79.2% of the bethanecol-induced precontraction. The motor effects induced by the K V 7 channel modulators were not affected by tetrodotoxin or ω-conotoxin GVIA. XE-991 greatly reduced retigabine- and flupirtine-induced relaxations. Transcripts encoded by all KCNQ genes were detected in the taenia coli , with KCNQ4 showing the highest expression levels. K V 7.4 channels were clearly visualised by immunohistochemistry in colonic epithelium, circular muscle layer and taenia coli . K V 7 channels appear to contribute to the resting muscle tone of the human taenia coli . In addition, K V 7 channel activators significantly relax the taenia coli . Thus, they could be useful therapeutic relaxant agents for colonic motor disorders.

Background Gamma-aminobutyric acid (GABA) and glutamate (GLU) are involved in nociceptive signals processing in the trigeminal system. In this study, we investigated the influence of excitatory transmission on GABA release in nerve terminals isolated from the rat trigeminal caudal nucleus (TCN). Methods We utilize biochemical (superfused synaptosomes loaded with [3H]GABA) and morphological (immunofluorescence experiments with specific antibody) techniques. Results Our results show that GLU potentiates the release of [3H]GABA evoked by 9, 15 and 30 mM [K+]e; 15 mM [K+]e-evoked [3H]GABA release was also reinforced by domoate and kainate (KA), two naturally occurring GLU-receptor agonists. The enhancement of 15 mM [K+]e-evoked [3H]GABA release produced by 100 μM KA was abolished by NBQX, a mixed AMPA/KA receptor antagonist, but was not affected by GYKI52466, a selective AMPA receptor antagonist. ATPA, a selective agonist for KA receptors containing the GLUK1 subunit, had no effect on depolarization-induced [3H]GABA release, and UBP310, which selectively antagonizes these same receptors, failed to reverse the KA-induced potentiation of 15 mM [K+]e-evoked [3H]GABA release. The KA-induced potentiation was also unaffected by concanavalin A (10 μM), a positive allosteric modulator of GLUK1- and GLUK2-containing KA receptors. Immunofluorescence experiments revealed that GABAergic nerve terminals in the TCN differentially expressed GLUK subunits, with GLUK2/3-positive terminals being twice more abundant than GLUK1-containing synaptosomes. Conclusions These findings indicate that pre-synaptic KA receptors facilitating GABA release from TCN nerve terminals mainly express GLUK2/GLUK3 subunits, supporting the notion that different types of KA receptors are involved in the various stages of pain transmission.

Understanding the molecular mechanisms underlying voltage-dependent gating in voltage-gated ion channels (VGICs) has been a major effort over the last decades. In recent years, changes in the gating process have emerged as common denominators for several genetically determined channelopathies affecting heart rhythm (arrhythmias), neuronal excitability (epilepsy, pain), or skeletal muscle contraction (periodic paralysis). Moreover, gating changes appear as the main molecular mechanism by which several natural toxins from a variety of species affect ion channel function. In this work, we describe the pathophysiological and pharmacological relevance of the gating process in voltage-gated K(+) channels encoded by the K(v)7 gene family. After reviewing the current knowledge on the molecular mechanisms and on the structural models of voltage-dependent gating in VGICs, we describe the physiological relevance of these channels, with particular emphasis on those formed by K(v)7.2-K(v)7.5 subunits having a well-established role in controlling neuronal excitability in humans. In fact, genetically determined alterations in K(v)7.2 and K(v)7.3 genes are responsible for benign familial neonatal convulsions, a rare seizure disorder affecting newborns, and the pharmacological activation of K(v)7.2/3 channels can exert antiepileptic activity in humans. Both mutation-triggered channel dysfunction and drug-induced channel activation can occur by impeding or facilitating, respectively, channel sensitivity to membrane voltage and can affect overlapping molecular sites within the voltage-sensing domain of these channels. Thus, understanding the molecular steps involved in voltage-sensing in K(v)7 channels will allow to better define the pathogenesis of rare human epilepsy, and to design innovative pharmacological strategies for the treatment of epilepsies and, possibly, other human diseases characterized by neuronal hyperexcitability.

Graphical abstract, Voltage-dependent type 7 K+ (KV7) channels play important physiological roles in neurons and muscle cells. The aims of the present study were to investigate the motor effects of KV7 channel modulators in the rat gastric fundus and the expression of KV7 channels in this tissue. Muscle tone and electrical field stimulation (EFS)-evoked relaxations of precontracted longitudinal muscle strips of the rat gastric fundus were investigated under nonadrenergic noncholinergic conditions by organ bath studies. Gene expression was studied by real-time PCR and tissue localization of channels was investigated by immunohistochemistry. The KV7 channel blocker XE-991 induced concentration-dependent contractions, with mean pD2 and Emax of 5.4 and 48% of the maximal U46619-induced contraction, respectively. The KV7 channel activators retigabine and flupirtine concentration-dependently relaxed U46619-precontracted strips, with pD2s of 4.7 and 4.4 and Emax of 93% and 91% of the maximal relaxation induced by papaverine, respectively. XE-991 concentration-dependently inhibited retigabine-induced relaxation with a pIC50 of 6.2. XE-991 and DMP-543, another KV7 channel blocker, increased by 13–25% or reduced by 11–21% the relaxations evoked by low- or high-frequency EFS, respectively. XE-991 also reduced the relaxation induced by vasoactive intestinal polypeptide (VIP) by 33% of controls. Transcripts encoded by all KV7 genes were detected in the fundus, with 7.4 and 7.5 showing the highest expression levels. KV7.4 and 7.5 channels were visualized by confocal immunofluorescence in both circular and longitudinal muscle layers. In conclusion, in the rat proximal stomach, KV7 channels appear to contribute to the resting muscle tone and to VIP- and high-frequency EFS-induced relaxation. KV7 channel activators could be useful relaxant agents of the gastric smooth muscle.

We analyzed the effects of extracellular pH reductions on the release of [(3)H]D: -aspartate ([(3)H]D: -ASP) and [(3)H]noradrenaline ([(3)H]NA) from cerebrocortical synaptosomes isolated from rats. Synaptosomes were superfused with standard medium at a physiologic pH of 7.4 and with acidified medium with a pH of 6.00, 5.50, or 5.0. Medium acidification produced pH-dependent stimulation of [(3)H]D: -ASP release. The increase amounted to 202 +/- 12.6% when the pH was reduced to 5.5. The [(3)H]D: -ASP release evoked by low pH (5.50) was still observed in the absence of Ca(2+) ions, but it was abolished by DL: -threo-beta-benzyloxyaspartate (DL: -TBOA) (100 microM), which inhibits neuronal glutamate/aspartate transport. Exposure to 5-(N,N-hexamethylene)-amiloride (EIPA) (30-100 microM), a selective inhibitor of Na(+)/H(+) exchange, caused concentration-dependent stimulation of [(3)H]D: -ASP release; the increase observed with EIPA 30 microM was 160 +/- 12%. The EIPA-induced release was not dependent on the presence of Ca(2+) ions in the medium, but it was abolished when synaptosomes were pretreated with 100 microM DL: -TBOA. Reduction of the extracellular pH (5.50-5.0) also stimulated the release of [(3)H]NA from rat cortical synaptosomes. Exposure to medium with a pH of 5.50 increased basal release by 136 +/- 9.5%. The release-stimulating effect of this medium was calcium-independent and abolished by 3 muM desipramine (DMI). [(3)H]NA release was also stimulated by EIPA. The increase induced by a concentration of 30 muM amounted to 136 +/- 9.50%, and this effect was calcium-independent and abolished by pretreatment with DMI (3 muM). These findings suggest that reduction of the extracellular pH stimulates release of [(3)H]D-ASP and [(3)H]NA by reversing neurotransmitter transport in the nerve terminal. This reversal might be activated by increased cytosolic concentrations of the transmitters caused by reduction of the pH gradient between the cytoplasm and the synaptic vesicles that take up the transmitters. This hypothesis is confirmed by the results of experiments conducted with EIPA. Selective blockade of Na(+)/H(+) exchange with this compound induces accumulation of H(+) in the nerve terminals and intracellular acidification, which leads to calcium-independent, transporter-mediated release of [(3)H]D: -ASP and [(3)H]NA.

Glutamate (GLU) plays a key role in the transmission and modulation of sensory input to the trigeminal caudal nuclei (TCN). In the present study, we investigated the regulation of previously taken-up [3H] d -aspartate ([3H] d -ASP) release from nerve terminals isolated from rat caudal brainstem, in particular from the zone containing the TCN. TCN neurons can be considered integrative relay neurons linking peripheral and central pain mechanisms. Understanding the mechanisms that control the release of GLU in this area could lead to more effective treatment of migraines and other types of pain associated with the trigeminal nerve. In isolated rat caudal brainstem synaptosomes, exposure to AMPA dose-dependently potentiated [K+]e-stimulated release of [3H] d -ASP (maximum increase: 218 ± 13.08%; EC50: 1.60 ± 0.08 μM). This effect was inhibited by selective AMPA-receptor antagonists (competitive [NBQX] and non-competitive [GYKI52466]) but not by the kainate receptor subunit antagonists NS102 and ACET. AMPA-evoked responses were significantly enhanced by preventing AMPA receptor desensitization with cyclothiazide (10 μM). Basal release of [3H] d -ASP was stimulated by millimolar concentrations of ATP (maximum increase: 197.80 ± 11.85%; EC50: 545 ± 3.15 μM) and by the selective P2X7-receptor agonist benzoylbenzoyl-ATP. ATP also potentiated the release of [3H] d -ASP induced by depolarization. Its effect on basal [3H] d -ASP release was inhibited by the selective P2X7-receptor antagonist A-438079 and by the non-selective antagonist PPADS, but it was only partially suppressed by the ionotropic purinergic receptor antagonist TNP-ATP. Our findings demonstrate that glutamatergic nerve terminals in rat caudal brainstem express AMPA receptors that can facilitate [3H] d -ASP during terminal depolarization and P2X7 receptors that can also enhance this release under basal conditions.

In this study, the functional consequences of the pharmacological modulation of the M-current (I(KM)) on cytoplasmic Ca(2+) intracellular Ca(2+)concentration ([Ca(2+)](i)) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. K(v)7.2 immunoreactivity was identified in pre-synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K(+)](e), the I(KM) activator retigabine (RT, 10 microM) inhibited [(3)H]D-aspartate ([(3)H]D-Asp) release and caused membrane hyperpolarization; both these effects were prevented by the I(KM) blocker XE-991 (20 microM). The I(KM) activators RT (0.1-30 microM), flupirtine (10 microM) and BMS-204352 (10 microM) inhibited 20 mM [K(+)](e)-induced synaptosomal [Ca(2+)](i) increases; XE-991 (20 microM) abolished RT-induced inhibition of depolarization-triggered [Ca(2+)](i) transients. The P/Q-type voltage-sensitive Ca(2+)channel (VSCC) blocker omega-agatoxin IVA prevented RT-induced inhibition of depolarization-induced [Ca(2+)](i) increase and [(3)H]D-Asp release, whereas the N-type blocker omega-conotoxin GVIA failed to do so. Finally, 10 microM RT did not modify the increase of [Ca(2+)](i) and the resulting enhancement of [(3)H]D-Asp release induced by [Ca(2+)](i) mobilization from intracellular stores, or by store-operated Ca(2+)channel activation. Collectively, the present data reveal that the pharmacological activation of I(KM) regulates depolarization-induced [(3)H]D-Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca(2+)](i) occurring through P/Q-type VSCCs.

The Kv7 potassium channel family encompasses five members (from Kv7.1 to Kv7.5) having distinct expression pattern and functional role. Although Kv7.1 is prevalently expressed in the cardiac muscle, Kv7.2, Kv7.3, Kv7.4, and Kv7.5 are expressed in neural tissue. Mutations in Kv7.2 and/or Kv7.3 genes are responsible for an autosomal-dominant epilepsy of the newborn defined as benign familial neonatal seizures (BFNS), whereas defects in the Kv7.4 gene have been found in families affected by a rare form of nonsyndromic autosomal-dominant hearing loss (DFNA2). Compounds acting as direct activators of neuronal channels formed by Kv7 subunits have been approved for clinical use as analgesics or are in advanced stages of clinical evaluation as anticonvulsants; in addition to these indications, solid preclinical studies reveal their potential usefulness in other diseases characterized by neuronal hyperexcitability. In the present work, we will summarize the available evidence providing proof-of-principles that neuronal Kv7 channels are highly attractive pharmacological targets, review the molecular basis of their peculiar pharmacological sensitivity, introduce some newly synthesized I(KM) openers showing improved pharmacokinetic or pharmacodynamic properties compared to older congeners, and discuss the potential novel therapeutic application of neuronal Kv7 channels in diseases additional to epilepsy.

KCNQ2 and KCNQ3 subunits encode for the muscarinic-regulated current (I(KM)), a sub-threshold voltage-dependent K+ current regulating neuronal excitability. In this study, we have investigated the involvement of I(KM) in dopamine (DA) release from rat striatal synaptosomes evoked by elevated extracellular K+ concentrations ([K+]e) and by muscarinic receptor activation. [3H]dopamine ([3H]DA) release triggered by 9 mmol/L [K+]e was inhibited by the I(KM) activator retigabine (0.01-30 micromol/L; Emax = 54.80 +/- 3.85%; IC50 = 0.50 +/- 0.36 micromol/L). The I(KM) blockers tetraethylammonium (0.1-3 mmol/L) and XE-991 (0.1-30 micromol/L) enhanced K+-evoked [3H]DA release and prevented retigabine-induced inhibition of depolarization-evoked [3H]DA release. Retigabine-induced inhibition of K+-evoked [3H]DA release was also abolished by synaptosomal entrapment of blocking anti-KCNQ2 polyclonal antibodies, an effect prevented by antibody pre-absorption with the KCNQ2 immunizing peptide. Furthermore, the cholinergic agonist oxotremorine (OXO) (1-300 micromol/L) potentiated 9 mmol/L [K+]e-evoked [3H]DA release (Emax = 155 +/- 9.50%; EC50 = 25 +/- 1.80 micromol/L). OXO (100 micromol/L)-induced [3H]DA release enhancement was competitively inhibited by pirenzepine (1-10 nmol/L) and abolished by the M3-preferring antagonist 4-diphenylacetoxy N-methylpiperidine methiodide (1 micromol/L), but was unaffected by the M1-selective antagonist MT-7 (10-100 nmol/L) or by Pertussis toxin (1.5-3 microg/mL), which uncouples M2- and M4-mediated responses. Finally, OXO-induced potentiation of depolarization-induced [3H]DA release was not additive to that produced by XE-991 (10 micromol/L), was unaffected by retigabine (10 micromol/L), and was abolished by synaptosomal entrapment of anti-KCNQ2 antibodies. Collectively, these findings indicate that, in rat striatal nerve endings, I(KM) channels containing KCNQ2 subunits regulate depolarization-induced DA release and that I(KM) suppression is involved in the reinforcement of depolarization-induced DA release triggered by the activation of pre-synaptic muscarinic heteroreceptors.

The endogenous cannabinoid system is involved in the regulation of a number of physiologic effects in both the central and peripheral nervous systems. Its role in the control of neuronal cell proliferation has attracted major attention because of its potential implications for new therapeutic strategies. In the present study, we demonstrated that treatment of cultured cerebellar granule cells with the synthetic cannabinoid WIN55,212-2, causes cell-body and nuclear shrinkage, which are hallmarks of neuronal apoptosis, as well as concentration-dependent decrease in cell viability. Staining with the fluorescent nuclear dye, Hoechst 33258, revealed apoptosis in 27.1% and 58.5% of cells exposed to 1 and 10 microM of WIN55,212-2, respectively (P < 0.01 and P < 0.001 vs. control respectively) after 36 hr. After 24 hr of exposure to WIN55,212-2, mRNA levels for the anti-apoptotic gene bcl-xL were reduced to 45.6% of those found in control (P < 0.01). These effects were completely reverted when cells were exposed to the synthetic cannabinoid in the presence of the specific CB1-receptor antagonist, SR141716A (1 microM). Moreover, the pro-apoptotic effect of 10 microM WIN55,212-2 could be reduced by the addition to the incubation medium of a cell-permeant inhibitor of caspase-1 (50 nM). Finally, WIN55,212-2 significantly increased caspase-1 activity after 24 hr. These findings show that the activation of CB1 receptors on cerebellar granule cells induces apoptotic cell death, which is associated with downregulation of the anti-apoptotic gene, bcl-xL, and at least in part, activation of caspase-1.

Perinatal manipulations can lead to neurobehavioural changes in the progeny. In this study we investigated, in adolescent male rat offspring, the consequences of a single, intense prenatal stress induced by a 120 min-maternal immobilization at gestational day 16, and of a daily, brief maternal separation from postnatal day 2 until 21, on: unconditioned fear/anxiety-like behaviour in open field and in elevated plus-maze; learning performance in the "Can test", a non-aversive spatial and tactile/visual task; corticosterone plasma levels under basal and stress-induced conditions. Our results indicate that both prenatal stress and maternal separation procedures decrease emotionality and enhance learning performance. Maternal separation potentiates prenatal stress-induced effects in enhancing learning performance. Both basal and stress-induced corticosterone plasma levels are reduced following prenatal stress, maternal separation and the combination of two procedures. These findings suggest that a single, intense prenatal stress can enhance the adaptive stress-related responses in the progeny, probably due to the involvement of maternal factors. The synergistic effect of prenatal stress and maternal separation on learning performance may be due to a further damping of hypothalamic-pituitary-adrenal axis response in the progeny that better cope with the task administered.

In the rat, prenatal exposure to diazepam (DZ) induces a permanent reduction in GABA/BZ receptor (R) function and behavioural abnormalities. Environmental modifications during early stages of life can influence brain development and induce neurobiological and behavioural changes throughout adulthood. Indeed, a subtle, periodic, postnatal manipulation increases GABA/BZ R activity and produces facilitatory effects on neuroendocrine and behavioural responses. We here investigated the impact of prenatal treatment with DZ on learning performance in adult 3- and 8-month-old male rats and the influence of a brief, periodic maternal separation on the effects exerted by prenatal DZ exposure. Learning performance was examined employing a non-aversive spatial, visual and/or tactile task, the "Can test". Behavioural reactivity, emotional state and fear/anxiety-driven behaviour were also examined using open field (OF), acoustic startle reflex (ASR) and elevated plus-maze (EPM) tests. A single daily injection of DZ (1.5 mg/kg, s.c.), over gestational days (GD) 14-20, induced, in an age-independent manner, a severe deficit in learning performance, a decrease in locomotor and explorative activity and an increase in peak amplitude in the ASR. Furthermore, anxiety-driven behaviour in EPM was disrupted. Daily maternal separation for 15 min over postnatal days 2-21 exerted opposite effects in all the paradigms examined. Prenatally DZ-exposed maternal separated rats, in contrast to respective non-separated rats, showed an improvement in learning performance, a decrease in emotionality and a normalization of the exploratory behaviour in EPM. These results suggest that a greater maternal care, induced by separation, can serve as a source for the developing brain to enhance neuronal plasticity and to prevent the behavioural abnormalities induced by prenatal DZ exposure. © 2005 Elsevier B.V. All rights reserved.

KCNQ subunits encode for the M current (IKM), a neuron-specific voltage-dependent K+current with a well established role in the control of neuronal excitability. In this study, by means of a combined biochemical, pharmacological, and electrophysiological approach, the role of presynapticIKMin the release of previously taken up tritiated norepineprine (NE), GABA, andd-aspartate (d-ASP) from hippocampal nerve terminals (synaptosomes) has been evaluated. Retigabine (RT) (0.01-30 μm), a specific activator ofIKM, inhibited [3H]NE, [3H]d-ASP, and [3H]GABA release evoked by 9 mmextracellular K+([K+]e). RT-induced inhibition of [3H]NE release was prevented by synaptosomal entrapment of polyclonal antibodies directed against KCNQ2 subunits, an effect that was abolished by antibody preabsorption with the KCNQ2 immunizing peptide; antibodies against KCNQ3 subunits were ineffective. Flupirtine (FP), a structural analog of RT, also inhibited 9 mm[K+]e-induced [3H]NE release, although its maximal inhibition was lower than that of RT. Electrophysiological studies in KCNQ2-transfected Chinese hamster ovary cells revealed that RT and FP (10 μm) caused a -19 and -9 mV hyperpolarizing shift, respectively, in the voltage dependence of activation of KCNQ2 K+channels. In the same cells, the cognition enhancer 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991) (10 μm) blocked KCNQ2 channels and prevented their activation by RT (1-10 μm). Finally, both XE-991 (10-100 μm) and tetraethylammonium ions (100 μm) abolished the inhibitory effect of RT (1 μm) on [3H]NE release. These findings provide novel evidence for a major regulatory role of KCNQ2 K+channel subunits in neurotransmitter release from rat hippocampal nerve endings.

A gentle long-lasting handling produces persistent neurochemical and behavioural changes and attenuates the impairment in the behavioural reactivity to novelty induced by the prenatal exposure to diazepam (DZ) in adult male rat progeny. This study investigated the consequences of a late prenatal treatment with three GABA/BDZ R agonists (DZ) alprazolam (ALP) and zolpidem (ZOLP)), on different stress-related behavioural patterns, in non-handled (NH), short-lasting handled (SLH) and long-lasting handled (LLH) adult male rats exposed to forced swim test (FST), acoustic startle reflex (ASR) and Vogel test (VT). The effects on motor activity were evaluated in the open field and in the Skinner box. The seizure sensitivity to picrotoxin (PTX) was investigated as an index of the functional state of GABA/BDZ Rs. A single daily s.c. injection of DZ (1.25–2.50 mg/kg) and ALP (0.125–0.250 mg/kg) over gestational days 14–20 induced a decrease in immobility time in the FST in NH rats, no change in SLH rats and an increase in LLH rats; DZ induced an increase in the peak amplitude of the ASR in NH rats, no change in SLH rats and a reduction in LLH rats; ALP was ineffective in all groups. DZ and ALP reduced the number of punished licks in the VT in NH, SLH and LLH rats while the unpunished licks were not modified. DZ decreased locomotion and the lever pressing responses while ALP increased them. DZ and ALP increased the seizure sensitivity to PTX (2.5–4.0 mg/kg i.p.). These findings indicate a convergence on anxiety-related behaviours in the effects of prenatal exposure to DZ and ALP and a differentiation on motor activity. Long-lasting handling was able to overcompensate the increased behavioural stress reactivity induced by the prenatal exposure to DZ and ALP.

Rat brain hypothalami were exposed to various depolarizing stimuli and vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) release was measured by means of a radioimmunoassay (RIA) procedure. Under conditions of noradrenergic blockade, exposure to high K(+) (40-100 mM) produced dose-dependent increases in the VIP-LI release in a Ca(2+)-dependent manner. Exposure to veratridine (3-100 microM) also induced concentration-dependent increases in VIP-LI release, an effect that was Ca(2+)-dependent and tetrodotoxin (TTX)-sensitive. Specific ligands for the L, N, and P/Q-type voltage-operated Ca(2+) channels (VOCCs) were used to determine which channel subtypes were involved in the K(+)-evoked VIP-LI release. The L-type VOCC ligand, nifedipine (10 microM), had no effect on release. In contrast, the N-type VOCC blocker, omega-conotoxin GVIA (omega-CgTx GVIA) (0.1-100 nM), markedly reduced the K(+)-evoked response, with maximal inhibition of approximately 60+/-8%. omega-Agatoxin IVA (omega-Aga IVA) (1-50 nM), which binds P-type and, at high doses, also Q-type VOCCs, produced dose-dependent inhibition of up to 25+/-3%, while the maximal inhibition observed with the non-selective VOCCs ligand, omega-conotoxin MVIIC (omega-CmTx MVIIC) (1 nM-3 microM), amounted to 85+/-8%. These findings indicate that N and P-type Ca(2+) channels play predominant roles in the high K(+)-evoked release of VIP-LI from the rat hypothalamus.

This study was conducted to investigate mechanisms regulating the release of [(3)H]D-aspartate (or endogenous glutamate) in the rat spinal cord. Presynaptic modulation of glutamate release was studied in superfused synaptosomes depolarized with 20 mM KCl. Calcium-channel antagonists, omega-conotoxin GVIA (omega-CgTx GVIA; N-type), nifedipine (L-type), and omega-conotoxin MVIIC (omega-CmTx MVIIC; P/Q type), were used to characterize the voltage-operated Ca(2+) channels (VOCCs) involved in this release. Nifedipine had no significant effect on the K(+)-evoked release of [(3)H]D-aspartate, but the omega-conotoxins GVIA and MVIIC produced dose-dependent inhibitory effects that were additive. The most substantial reduction (54.30% +/- 4.40%) was seen with omega-CgTx GVIA, indicating that N-type channels play a major role in the release of glutamate in this tissue. We investigated the effects of neuropeptide Y (NPY), NPY(13-36), and [Leu(31)][Pro(34)]NPY on Ca(2+)-dependent, K(+)-evoked [(3)H]D-aspartate release. NPY and NPY(13-36) equipotently inhibited the release of glutamate in a concentration-dependent manner. The half-maximal response was observed at about 12 nM; maximal inhibition of 44.22% +/- 4.60% was achieved with 0.3 microM. The selective GABA(B) agonist (-)baclofen inhibited K(+)-evoked [(3)H]D-aspartate release from superfused spinal cord synaptosomes by 50.00% +/- 4.80% at 10 microM. When NPY(13-36) and (-)baclofen were used together at maximal doses, their release-inhibiting effects were not additive. In addition, neither of the agonists was able to enhance the inhibition produced by pretreating the synaptosomes with the selective inhibitor of N-type VOCCs omega-CgTx GVIA. These results are consistent with the hypothesis that presynaptic Y(2)-like and GABA(B) receptors regulate glutamate release by blocking Ca(2+) currents through N-type VOCCs. Characterization of the receptors that can inhibit the release of glutamate may provide useful information for treatment of conditions characterized by excessive glutamatergic transmission in the spinal cord.

The contribution of the different Ca(2+)-channel subtypes to the K(+)-evoked [(3)H]noradrenaline release from rat cerebral cortex synaptosomes has been investigated. In the same experimental model, it was also verified whether the calcium-mediated neurotransmitter release is influenced by IgGs purified from sera of seven patients affected by sporadic amyotrophic lateral sclerosis. Synaptosome treatment with 3.0 microM nifedipine or 2.0 microM calciseptine, which block L-type channels, slightly decreased [(3)H]noradrenaline release, the reduction being 7 and 13% of the control values, respectively. The blockade of N-type Ca(2+)-channels with omega-conotoxin-GVIA (0.001-1.0 microM) induced a concentration-dependent reduction of the neurotransmitter release, with maximum effect of 34%. omega-Agatoxin-IVA failed to significantly affect the studied release, which was instead markedly reduced by omega-conotoxin-MVIIC. After the blockade of N-type channels with maximal concentrations of omega-conotoxin-GVIA, 3.0 microM omega-conotoxin-MVIIC reduced the release by 58%. Synaptosome treatment with amyotrophic lateral sclerosis IgGs enhanced the K(+)-evoked [(3)H]noradrenaline release, which was mostly mediated by P/Q- and N-type Ca(2+)-channels. The increase induced by pathologic IgGs (0.2 mg/ml) ranged from 11 to 62% for the different patients, and it was concentration-dependent. The basal release was instead unaffected by IgG treatment. The results of the present study suggest that the K(+)-evoked [(3)H]noradrenaline release from brain cortex synaptosomes is mainly mediated by activation of P/Q- and N-type Ca(2+)-channels. Autoantibodies present in the sera of patients affected by sporadic amyotrophic lateral sclerosis may interact with these channels by producing an increased calcium influx, with consequent enhancement of the neurotransmitter release. Preliminary results of the present study have been published in abstract form (Martire et al., 1997, Pharmacol. Res. 35:9).

Rat hypothalamic synaptosomes were exposed via superfusion to various stimuli and the release of neuropeptide Y-like immunoreactivity (NPY-LI) was measured by means of radioimmunoassay procedures. High KCl (15-50 mM) concentration dependently evoked NPY-LI release; the evoked overflow reached a plateau at 30 mM KCl and was abolished in the absence of Ca2+ ions. Furthermore, a remarkable NPY-LI overflow was obtained when extracellular Na+ ions were removed. Low external Na(+)-evoked NPY-LI release was independent of the presence of Ca2+ ions from the superfusion medium. It is well known that the reduction of external Na+ ions activates the release of several neurotransmitters through an inversion of the uptake-carrier working direction; but such mechanisms, involving Na(+)-dependent uptake, have never been described for neuropeptides. The alteration of the extracellular Na+ concentration is able to modify the concentration of the intracellular Ca2+ and H+ ions. In fact, the concentrations of these two ions are regulated through Na(+)-dependent exchange mechanisms across the membrane. Amiloride, blocking the Na+/H+ exchanger, was able to maintain low Na(+)-evoked NPY-LI release, underlying that the blockade of the exchanger preserves the H+ accumulation induced by the reduction of the external Na+ ions. NPY-LI release could also be stimulated by nigericine, a proton ionophore, showing that the intracellular acidification is responsible for NPY-LI release. Intracellular acidification may stimulate Ca2+ ion release from intracellular stores, as has been shown by other workers. Large dense-core vesicles containing the peptide appear to be more sensitive to local intracellular Ca2+ release compared with extracellular Ca2+ ion entry through voltage-dependent channels.

Presynaptic receptors may reduce transmitter release with different mechanisms. Both the alpha 2-agonist, clonidine and the Y2-agonist, neuropeptide Y fragment 13-36 (NPY 13-36), induce a concentration-dependent inhibition of the 4-aminopyridine (4-AP)-evoked [3H]noradrenaline ([3H]NA) release from hypothalamic synaptosomes. Changes in alpha 2- and Y2-modulation of noradrenaline (NA) release were observed by lowering the calcium influx with the use of omega-conotoxin (omega-CgTx), a calcium-channel blocking agent. In these experimental conditions, clonidine was less active, whereas NPY 13-36 preserved its efficacy. It therefore seems possible that presynaptic alpha 2-adrenoceptors can primarily inhibit NA release by reducing calcium influx via voltage-sensitive calcium channels (VSCC), while Y2-receptors may inhibit the intracellular release process with a mechanism independent of the calcium entry.

The effects of the Y2 receptor agonist neuropeptide Y NPY-(13-36) on the depolarization-evoked release of [3H]noradrenaline (NA) from synaptosomal preparations of the medulla oblongata, the hypothalamus, the hippocampal formation and the parieto-occipital cortex of the male rat were studied. NPY-(13-36) (0.1-100 nM) caused a concentration-related inhibition of the depolarization-induced release of [3H]NA in all areas studied, except the parieto-occipital cortex. The results indicate that NPY Y2 receptors are present on NA terminals in all areas studied, except the parieto-occipital cortex and inhibit depolarization-evoked [3H]NA release.

Neuropeptide Y (NPY), first isolated in 1982, is widely distributed among the neurons of the central and peripheral nervous systems, often in close association with catecholamines. Because of its wide distribution and concentrations in selected areas of the brain, NPY is considered a putative neurotransmitter with several possible physiological effects including modulation of blood pressure, food intake and pituitary hormone release at a central level. Peripherally, the peptide seems to be involved, via direct and indirect mechanisms, in noradrenaline (NA)-mediated vasoconstriction. The ability of NPY to interact with the catecholamine transmission line may underly a possible modulatory influence of NPY on catecholamine receptor characteristics. We recently observed interaction between alpha-2 adrenergic receptors and those for NPY at the presynaptic level. Additional data from our studies in spontaneously hypertensive rats suggest that impairment of these interactions may contribute to the hypertension in this strain.

In the present study, the emetic effect of the anticancer drug cisplatin, and the protective effects of 5-HT 3 receptor antagonists against cisplatin emesis were investigated in the pigeon. The experimental set-up involved the i.v. administration of drugs and subsequent observation of the percentage of vomiting animals and the number of emetic episodes per vomiting animal. It was observed that cisplatin induced dose-dependent emesis in the pigeon. 5-HT 3 receptor antagonists afforded partial protection against cisplatin emesis, although some of them i.e. indole, indole-Iike derivatives and zacopride, displayed intrinsic emetic activity. A serotonergic mechanism appears to be involved in both cisplatin- and 5-HT 3 receptor antagonist-induced emesis, since pretreatment with an inhibitor of 5-HT synthesis, para-chlorophenylalanine (pCPA), prevented vomiting induced by either cisplatin or 5-HT 3 receptor antagonists. It is concluded that the intrinsic emetic effects of 5-HT 3 receptor antagonists provide pharmacological evidence of species differences in the properties of 5-HT 3 receptors.

In the present study, the emetic effect of the anticancer drug cisplatin, and protective effects of 5-HT3 receptor antagonists against cisplatin emesis were investigated in the pigeon. The experimental setting involved the i.v. administration of drugs and subsequent observation of the percentage of vomiting animals and number of emetic episodes per vomiting animal over a period of 5 h. In some experiments, the 5-HT and 5-HIAA content in tissues was estimated by the HPLC technique. It was observed that cisplatin (2.5-10 mg/kg) is able to induce dose-dependent emesis in the pigeon. 5-HT3 receptor antagonists (500 micrograms/kg) afford partial protection against cisplatin emesis, although some of them, i.e. indolic derivatives and zacopride, display intrinsic emetic activity at doses of 50-500 micrograms/kg. A serotonergic mechanism appears to be involved in both cisplatin- and 5-HT3 receptor antagonist-induced emesis, since pretreatment with an inhibitor of 5-HT synthesis, para-chlorophenylalanine (300 mg/kg x 3 days), is able to hamper vomiting induced by either cisplatin or 5-HT3 receptor antagonists. It is concluded that the intrinsic emetic effects of 5-HT3 receptor antagonists in the pigeon provide pharmacological evidence of species differences in the properties of 5 HT3 receptors.

During recent years we have obtained evidence for the existence of galanin (GAL)/5-hydroxytryptamine (5-HT) interactions at the pre-and postsynaptic level in the rat central nervous system (CNS). The main basis of these interactions appear to be bidirectional intramembrane interactions between GAL and 5-HT1A receptors (Fuxe et al., 1988a, 1990) which represents a way to increase plasticity of 5-HT neurotransmission, since the 5-HT1A receptor appears to be selectively modulated by GAL (Fuxe et al, 1988a). Recent work by Hedlund et al. (1990) indicates that in fact the 5-HT1A receptor subtype is regulated by an intramembrane feedback loop involving the high affinity GAL receptor.

The effects of galanin (GAL) have been evaluated on the depolarization evoked release of [3H]5-HT (serotonin or 5-hydroxytryptamine) from rat hypothalamic synaptosomal preparations, using low concentrations of potassium (15 mM). In the same preparation effects of GAL were also evaluated on [3H]5-HT uptake, using kinetic analysis to determine effects on Vmax values and Km values. GAL concentrations of 0.1-10 nM caused a concentration-related increase of the depolarization-evoked release of [3H]5-HT without influencing [3H]5-HT uptake. The results indicate the existence of high affinity GAL receptors on the hypothalamic 5-HT nerve terminals, exerting a facilitatory influence on depolarization-evoked [3H]5-HT release.

The NPY neurons play an important role in information handling in the CNS by their ability to interact in both wiring and volume transmission at the network, local circuit and synaptic level. The importance of NPY/alpha 2 receptor-receptor interactions in cardiovascular, neuroendocrine and vigilance control is emphasized. Alterations in these receptor-receptor interactions take place in the spontaneously hypertensive rats as well as in the ischemic brain, which may have profound consequences for the information handling and contribute to the functional alterations found in these pathophysiological states. Finally, in the aging brain there appears to exist a marked reduction in NPY transmission line, which may affect higher brain functions, such as learning and memory retrieval. The most impressive result is, however, the indications of a role for NPY in volume transmission, where NPY appears to produce syndromic actions via its conversion into biologically active fragments, which may have preferential actions at Y2 NPY receptors. These syndromic pathways may be altered in the spontaneously hypertensive rat and may be controlled by gonadal steroids and glucocorticoids. Glucocorticoid receptors have been demonstrated in all arcuate NPY neurons and all NA/NPY and A/NPY costoring neurons.