Sabrina Cavallini

Effects of cholecystokinin tetrapeptide (CCK4) and of anxiolytic drugs on GABA outflow from the cerebral cortex of freely moving rats

The effect of cholecystokinin tetrapeptide (CCK(4)) and of different anxiolytic drugs on GABA outflow from the cerebral cortex was investigated in freely moving rats, by using the epidural cup technique. CCK(4) (3-30 microg/kg, i.p.) increased GABA outflow and induced objective signs of anxiety. These neurochemical and behavioral responses were prevented by the CCK(B) antagonist GV150013 at 0.1 microg/kg (i.p.). At higher doses (up to 30 microg/kg) this compound per se reduced GABA release and caused sedation, suggesting the presence of a CCKergic positive tonic modulation on GABA interneurons. Similarly the GABA(A) receptors modulator, diazepam (2mg/kg, i.p.) and the 5-HT(1A) agonist buspirone (3mg/kg, i.p.) reduced GABA outflow and caused the expected behavioral effects (reduced muscle tone, mild 5-HT syndrome) which were prevented by the respective, selective antagonists, flumazenil (1mg/kg, i.p.) and NAN-190 (3mg/kg, i.p.). These findings support the idea that GV150013, diazepam and buspirone inhibit GABAergic cortical activity, through the respective receptors. This neurochemical effect may represent the end-effect of various anxiolytic compounds affecting the cortical circuitry.

Direct and indirect inhibition by nociceptin/orphanin FQ on noradrenaline release from rodent cerebral cortex in vitro

The modulation exerted by nociceptin/orphanin FQ (NC) on noradrenaline (NE) release in rodent cerebral cortex slices and synaptosomes was studied. Rat, mouse and guinea‐pig cortical slices and synaptosomes were preincubated with 0.1 μM [3H]‐NE and superfused. NE release was evoked by 2 min of electrical (3 Hz) stimulation in slices and by 1 min pulse of 10 mM KCl in synaptosomes. In rat cortical slices, 0.01–3 μM NC reduced the evoked [3H]‐NE efflux (Emax−54%), with a bell‐shaped concentration‐response curve, which regained its monotonic nature in the presence of either 0.1 μM naloxone (NX) or 30 μM bicuculline. In synaptosomes, the NC effect curve was sygmoidal in shape and reached a plateau at 1 μM concentration. In the rat, both 1 μM [Phe1ψ(CH2‐NH)Gly2]NC(1‐13)NH2 and 10 μM [Nphe1]NC(1‐13)NH2 (NPhe) antagonised NC‐induced inhibition, without per se modifying [3H]‐NE efflux. The effects of 0.3–1 μM NC concentrations were partially prevented by 1 μM NX; 1 μM D‐Phe‐Cys‐Thr‐D‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP) was also an effective antagonist, but 0.1 μM norbinaltorphimine was not. In the mouse cerebral cortex, NC‐induced inhibition of NE release (pEC50 6.87, Emax−61%, in the slices) was prevented by Nphe but was NX‐insensitive. In guinea‐pig cortical slices, NC effect (pEC50 6.22, Emax−38%) was prevented by Nphe, but was NX‐insensitive. These findings demonstrate that NC inhibits NE release from rodent cerebral cortex via presynaptically located ORL1 receptors. In the rat, μ opioid and GABAA receptors are involved as well.

Effects of Chemical Ischemia on Cerebral Cortex Slices Focus on Mitogen-Activated Protein Kinase Cascade

Abstract:  A variety of harmful stimuli, among them energy depletion occurring during transient brain ischemia, are thought to unbalance protein kinase cascades, ultimately leading to neuronal damage. In superfused, electrically stimulated rat cerebral cortex slices, chemical ischemia (CI) was induced by a 5‐min treatment with the mitochondrial toxin, sodium azide (10 mM), combined with the glycolysis blocker, 2‐deoxyglucose (2 mM). Thereafter, 1 h reperfusion (REP) with normal medium followed. Western blot analysis of p21Ras, extracellular signal‐regulated protein kinases (ERK)1/2 (p44/42), phospho‐ERK1/2, mitogen‐activated protein kinase (MAPK)‐p38, phospho‐p38, stress‐activated protein kinases/c‐Jun NH2‐terminal protein kinases (SAPK/JNK), phospho‐SAPK/JNK was carried out. The level of p21Ras was increased by 40% immediately after CI, and did not return to control values following REP. Both ERK1 and ERK2 levels were reduced by CI and recovered to control values following REP; no significant change in their phosphorylation degree (phosphorylated to total level ratio, about 50% in the controls) was observed. Neither p38 levels, nor phosphorylation degree were changed following CI/REP. The activation of SAPK/JNK was significantly reduced under CI, and did not recover following REP. All CI/REP‐induced effects were prevented by the NMDA receptor antagonist MK‐801, 10 μM, suggesting the involvement of glutamate. The present findings show that although CI stimulates the p21Ras protein, MAPK levels and/or phosphorylation are reduced, possibly because of acute energy depletion. Because the activation of SAPK/JNK has been related to both apoptosis and neuroprotection, the decrease observed under CI/REP conditions may instead be related to nonapoptotic neuronal death. These results could be of interest in developing preventive treatments for ischemia/REP‐induced brain damage.

Effects of cholecystokinin tetrapeptide (CCK4) and anxiolytic drugs on the electrically evoked [3H]5-hydroxytryptamine outflow from rat cortical slices

The outflow of [(3)H]5-hydroxytryptamine ([(3)H]5-HT) from electrically stimulated rat cortical slices was measured to ascertain the modulatory role of endogenous cholecystokinin (CCK) on the amine outflow and to test the hypothesis that different anxiolytic compounds inhibit 5-HT secretion. The [(3)H]5-HT outflow evoked at 10 Hz was increased up to +30% by CCK(4) 300-1000 nM, the effect being prevented by the CCK(B) receptor antagonist GV 150013, 3 nM. The limited sensitivity to CCK(4) seemed to depend on 5-HT auto-receptor feedback because pre-treatment with 100 nM methiothepin enhanced the [(3)H]5-HT outflow and lowered the CCK(4) threshold concentration from 300 to 30 nM. In addition, pre-treatment with 1 microM bacitracin to inhibit CCK metabolism increased [(3)H]5-HT efflux. This effect was concentration-dependently counteracted by GV150013 suggesting the presence of an endogenous CCK positive modulation. GV150013 30 nM, the 5-HT(1A) partial agonist buspirone 300 nM and the GABA(A) receptor modulator diazepam 10 nM, known to have anxiolytic properties, all significantly reduced the [(3)H] amine outflow from cortical slices by about 20%. This inhibition depended on their interaction with their respective receptors, which seemed to restrain the activity of functionally interconnected glutamatergic interneurones. In fact, APV (50 microM) and NBQX (10 microM) prevented the effect of the anxiolytic compounds. Thus, anxiolytic drugs with different receptor targets can reduce 5-HT outflow by dampening the glutamatergic signal, and in turn, the secretory process of the serotonergic nerve ending.