Jörg Traber

Shock-induced ultrasonic vocalization in young adult rats: a model for testing putative anti-anxiety drugs

A putative animal model of anxiety based on shock-induced ultrasonic vocalization was pharmacologically validated in young adult rats. Suppression of shock-induced ultrasonic vocalization was obtained with diazepam, chlordiazepoxide, meprobamate and pentobarbital; the serotonin (5-HT)1A receptor agonists 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin], buspirone, ipsapirone, gepirone and tandospirone; the nonselective 5-HT receptor agonists TFMPP [1-(3-trifluoromethylphenyl)piperazin], mCPP [1-(3-chlorophenyl)piperazin] and DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane); the NMDA antagonists PCP (phencyclidine) and MK-801; the alpha 2-adrenoceptor antagonists idazoxane, yohimbine and 1-PP (1-pyrimidinylpiperazine); and the atypical neuroleptic clozapine. The alpha 2-adrenoceptor agonist clonidine, the 5-HT2/5-HT1C antagonist ritanserin, the 5-HT3 antagonists ondansetron and ICS-205,930, and the 5-HT reuptake inhibitor fluoxetine did not, or only partially, reduce ultrasonic vocalization. Tricyclic and tetracyclic, as well as some atypical antidepressants and a monoamineoxidase (MAO) inhibitor, showed no ultrasonic vocalization reducing effects, or reduced ultrasonic vocalization only at high doses. An opiate, an antimuscarinic, (pro)convulsants and typical neuroleptics did not reduce ultrasonic vocalization. The present findings suggest that the ultrasonic vocalization model specifically measures anxiolytic effects. Because ultrasonic vocalization responding develops within five days, remains stable for at least three months and gives highly reproducible results, the test appears suitable for rapid and repeated testing of new anxiolytics in the same animals.

Brain serotonin receptors as a target for the putative anxiolytic TVX Q 7821

In animal behavioral tests of anxiolytic efficacy, TVX Q 7821 was active and equipotent with diazepam, but did not produce muscle relaxation or anti-convulsant effects. The high affinity, specific binding of 3H-TVX Q 7821 to calf hippocampal membranes was displaced by serotonin (5-HT) but not by diazepam. Similarly, unlabeled TVX Q 7821 displaced 3H-5-HT but not 3H-flunitrazepam binding. Since ketanserin (a putative 5-HT2 ligand) was equally weak in displacing labeled 5-HT or TVX Q 7821, TVX Q 7821 may preferentially bind to 5-HT1, receptors.

Influence of opiates on the levels of adenosine 3':5'-cyclic monophosphate in neuroblastoma X glioma hybrid cells.

Abstract In neuroblastoma x glioma hybrid cells prostaglanddin E 1 (PGE 1 ) increases the level of adenosine 3′: 5′-cyclic monophosphate. This response to PGE 1 is strongly enhanced in cells that were incubated with morphine, methadon, noradrenaline or carbamylcholine for several hours. All these compounds increase the level of cyclic GMP in the cells. As in untreated cells, the effect of PGE 1 can be inhibited by morphine or noradrenaline. The development of the increased response to PGE 1 is dependent on protein synthesis. The increased response to PGE 1 is discussed in connection with morphine tolerance and withdrawal.

Autoradiographic identification and topographical analyses of high affinity serotonin receptor subtypes as a target for the novel putative anxiolytic TVX Q 7821.

TVX Q 7821 is a putative non-benzodiazepine anxiolytic which has a high affinity to 5-HT1 receptors. In this study some of the binding characteristics of the radiolabeled drug using rat brain cryostat sections and the autoradiographic localization of binding sites using the LKB-Ultrofilm technique have been investigated. Parallel experiments have been performed using [3H]serotonin ( [3H]5-HT). Both [3H]TVX Q 7821 and [3H]5-HT bound specifically and in a saturable manner to tissue sections, the Kd values being 6.8 and 3.7 nmol/l, respectively. Quantitative autoradiography using computer-assisted image analysis revealed a mean inhibition by TVX Q 7821 of [3H]5-HT binding of 56% in many brain areas. The inhibition ranged from 80% in the hippocampus and entorhinal area to practically none in the substantia nigra and the dorsal subiculum. Color coded autoradiograms obtained either with [3H]5-HT or [3H]TVX Q 7821 showed a nearly identical pattern of labeling with high receptor densities in the hippocampus, the entorhinal area, the septum, the interpeduncular nucleus and the dorsal raphe. However, in some brain areas striking differences in the intensity of labeling were found. [3H]5-HT but not [3H]TVX Q 7821 bound strongly in the substantia nigra, the dorsal subiculum and the globus pallidus. It is proposed that TVX Q 7821 binds to a subtype of 5-HT1 receptor (the so-called 5-HT1A sites as recently proposed). Thus, the putative anxiolytic TVX Q 7821 may provide a means for the study of the functional role of 5-HT1 receptors.

Quantitative autoradiography of transmitter binding sites with an image analyzer

A computerized image processing system for quantitative receptor autoradiography on tritium-sensitive film is described. The method implements an image analyzer (IBAS 1 + 2) for the acquisition of an image which is evaluated with respect to the regional distribution of binding site densities by a small computer which is connected to a digitizer. Several methodological problems must be considered when using tritium-labelled compounds and an image analyzer. The use of tissue standards and shading procedures, the improvement of signal-to-noise ratio, the calibration of the system which establishes the non-linear relation between grey values and concentration of radioactivity, the contrast enhancement and the quenching problem are discussed. This method permits a quick and precise quantitative analysis of binding site distribution in receptor autoradiography.

Measurements of adenosine 3′:5′‐cyclic monophosphate and membrane potential in neuroblastoma X glioma hybrid cells: Opiates and adrenergic agonists cause effects opposite to those of postaglandin E1

Hybrid cells obtained by fusion of rat glioma and mouse neuroblastoma cells display many properties characteristic of differentiated neurons [ 1,2] . On treatment with N6 -2’0dibutyryl adenosine -3’: 5’cyclic monophosphate (dibutyryl cyclic AMP) they extend long processes [l-4] . In containing choline acetyltransferase of high specific activity [ 1,2,5,6] and dopamine-/3-hydroxylase [3] they have marker enzymes of both cholinergic and adrenergic nerves. They fire action potentials on electrical stimulation or when acetylcholine is applied to their plasma membrane. The response to acetylcholine can be blocked by the cholinergic antagonists d-tubocurarine, atropin or cr-bungarotoxin [ 1,2] . When exposed to prostaglandin El, neuroblastoma [7,8] and hybrid cells [9] increase their intracellular concentration of cyclic AMP. In both cell types this effect can be antagonized by morphine [lo] . The basal level of cyclic AMP in unstimulated cells or the elevated level in cells exposed to a phosphodiesterase inhibitor is lowered in the presence of noradrenaline (NA) [9]. In extension of this work we now report that in the hybrid cells NA at low concentrations also prevents the increase of intracellular levels of cyclic AMP evoked by PGEi . In doing so it is a more potent agent than dopamine, phenylephrine and isoproterenol (order of decreasing potency). The adrenergic a-blocker phentolamine antagonizes the action of NA more effectively than the I(-blocker propranolol. The data suggest the adrenergic receptor to be classified as a-receptor [ 111. Also opiates, in a highly stereospecific

The ontogenetic development of serotonin (5-HT1) receptors in various cortical regions of the rat brain.

SummaryThe distribution of serotonin (5-HT1) receptors in various cortical regions of the rat brain has been examined during ontogenesis by quantitative autoradiography.An increase in binding site density between the first postnatal day and adult age was observed and could be approximated by a sigmoid shaped (logistic) growth curve. A marked heterochrony in the increase of binding site density is found in the 13 analyzed cortical regions. Binding sites develop earlier in neocortex than in allocortical areas. Fifty pereent of the binding site density of adult age is reached in the motor cortex at the 9th postnatal day, followed by the primary somatosensory cortex one day later, by the medial prefrontal cortex on the 12th day, by the fascia dentata on the 14th day and by the CA1-region on the 20th day. A detailed analysis of the frontal, medial prefrontal and hippocampal regions also shows a heterochrony within these regions. Adult values of binding site densities are also reached at different ages in the various cortical regions. The highest receptor densities were observed in the dorsal subiculum, the lowest in the primary somatosensory cortex.

Effect of the 5-HT1A receptor agonist ipsapirone on the local cerebral glucose utilization of the rat hippocampus

Local cerebral glucose utilization (LCGU) was measured in the hippocampus of the rat brain following i.p. injection of the anxiolytic drug and 5-HT1A receptor agonist ipsapirone (TVX Q 7821). Administration of ipsapirone (5 mg/kg) reduced glucose utilization in the various hippocampal areas to variable extent. The most subtle reduction took place in the dorsal subiculum, while the most pronounced decrease was found in sector CA4 of Ammons horn. The degree of LCGU reduction can be related to the 5-HT1A receptor density in the respective areas.

Morphine antagonizes the action of prostaglandin in neuroblastoma cells but not of prostaglandin and noradrenaline in glioma and glioma × fibroblast hybrid cells

The contraction of intestinal muscle evoked by prostaglandin E (PGE) can be inhibited by morphine [l-3]. Experiments from 2 laboratories using different systems demonstrate that morphine may exert its effect by interfering with the stimulation by PGE, of the adenylate cyclase system [4-61. Thus, morphine inhibits the stimulation by PGEr of the formation of adenosine 3’ : 5’cyclic monophosphate (cyclic AMP) in rat brain homogenates [4] and in mouse neuroblastoma and mouse neuroblastoma X rat glioma hybrid cells [ 5,6]. The specificity of the morphine actions is demonstrated by the observation that the inhibitory action of morphine can be prevented by naloxone [4,6], a morphine analog, known to antagonize the action of morphine in vivo [7]. In an extension of the studies mentioned above [4,6], the work presented here was undertaken in order to answer the questions: 1) does morphine interfere with the stimulatory effect of PGEi by competing with PGEi for the same receptor site? 2) Does morphine exert its inhibitory action on all cells in which PGEi stimulates the formation of cyclic AMP? 3) Is the inhibitory effect of morphine also observed in cells in which noradrenaline (NA) is known to increase the intracellular levels of cyclic AMP? The concentration of cyclic AMP in the 3 cell types used for their study is enhanced by different neurohormones: By PGEr in a mouse neuroblastoma line [8,9], by PGEi [lo] or NA [ 11,121 in a rat glioma line and by PGEr or NA in a rat glioma X mouse fibroblast line [lo]. Our results show that the inhibitory action of morphine cannot be overcome by high concentrations

TVX 2706--a new phosphodiesterase inhibitor with antiinflammatory action. Biochemical characterization.

The effects of the anti-inflammatory and analgesic drug 3-ethyl-1-(3-nitrophenyl)-2,4[1H, 3H]-quinazolindione (TVX 2706) on neuronal and glial cell culture systems including neuroblastoma × glioma hybrid cells have been studied. This compound strongly enhances the increase in intracellular levels of cyclic AMP caused by appropriate effectors in all systems tested so far. EC50 values are in the submicromolar range. The effect is apparently neither due to an increased responsiveness of the hybrid cells for an effector like prostaglandin E1 nor to an increased activity of adenylate cyclase, but to an inhibition of both low and high affinity cyclic AMP phosphodiesterases. Half-maximal inhibition of enzyme activity is obtained at 10 μM TVX 2706. The drug is at least equipotent to or more potent than some other common phosphodiesterase inhibitors. Inhibition of phosphodiesterase activity is also observed in homogenates from rat polymorphonuclear leucocytes, where the lowKm-enzyme is preferentially inhibited. TVX 2706 does not interfere with the calmodulin activation of phosphodiesterase. The role of phosphodiesterase inhibition as a possible mechanism of the anti-inflammatory action of TVX 2706 is discussed.