Quantitative autoradiography of angiotensin II AT2 receptors with [I]CGP 42112

Most radiolabeled ligands for angiotensin II (Ang II) receptors do not discriminate between the AT₁ and AT₂ receptor subtypes, which must be distinguished by displacement with selective AT₁ or AT₂ ligands. We compared [¹²⁵I]CGP 42112 with the non-selective agonist [¹²⁵I]Sar¹Angiotensin II. We studied the inferior olive, medial geniculate nucleus and the adrenal medulla, areas rich in AT₂ receptors, using both ligands with quantitative autoradiography and membrane binding techniques. [¹²⁵I]CGP 42112 bound with high affinity (K_d = 0.07−0.3nM, depending on the area studied). [¹²⁵I]CGP 42112 binding was selective for AT₂ receptors, as determined by lack of competition with the AT₁ ligand losartan, and competition by the AT₂ ligands PD 123177 and unlabeled CGP 42112 and the non-selective peptides Ang II and angiotensin III (Ang III). Using [¹²⁵I]CGP binding, we found the same order of potency: CGP 42112 > Ang II = Ang III > PD 123177 using both quantitative autoradiography or membrane binding methods. Our results demonstrate that [¹²⁵I]CGP 42112 is the most selective, highest affinity ligand available for AT₂ receptors. Because of these characteristics, and low non-specific binding, quantitative autoradiography with [¹²⁵I]CGP 42112 is the method of choice to selectively characterize AT₂ receptors, especially in tissues like the brain, with a highly heterogeneous distribution of receptor subtypes.     

Angiotensin AT1 receptors in the preoptic area negatively modulate the cardiovascular and ACTH responses induced in rats by intrapreoptic injection of prostaglandin E2

We previously reported that brain angiotensin II type 2 (AT₂) receptors contribute to the hyperthermia induced by intrahypothalamic (intrapreoptic (i.p.o.)) administration of prostaglandin E₂ (PGE₂) in rats. The present study was carried out to investigate the role of angiotensin II (ANG II) receptors in the cardiovascular and adrenocorticotropic hormone (ACTH) responses induced in rats by i.p.o. injection of PGE₂. PGE₂ (100 ng) produced marked increases in blood pressure, heart rate, and plasma ACTH concentration. These changes were significantly enhanced by i.p.o. treatment with an AT₁-receptor antagonist, losartan, while an AT₂-receptor antagonist, CGP 42112A, had no effect. In contrast, losartan, but not CGP 42112A, reduced the pressor and ACTH responses to i.p.o. injection of a large dose of “exogenous” ANG II (25 ng). These results suggest that while “endogenous” ANG II exerts inhibitory effects on both the cardiovascular and the ACTH responses to i.p.o. PGE₂ by way of preoptic AT₁-receptors, a large dose of exogenous ANG II produces effects opposite to those induced by the endogenous ANG II that is released locally and in small amounts by i.p.o. PGE₂.     

Modulation of net outward current in cultured neurons by angiotensin II: involvement of AT1 and AT2 receptors

In this study we have used whole-cell, voltage-clamp procedures to determine the effects of angiotensin II (AII) on net outward current (I_no) in neurons co-cultured from the hypothalamus and brainstem of 1-day-old rats. I_no is the sum of all inward and outward membrane currents (minus Na⁺, which is blocked by tetrodotoxin) which occur during the repolarization phase of the action potential. We have determined that AII elicits two separate effects on I_no in cultured neurons. AII caused a reversible and concentration (0.1 nM–10 μM)-dependent increase in I_no. This effect is inhibited by the AT₂ receptor-selective antagonists, PD123177 and PD123319 (both 100 nM), but not by the AT₁-selective receptor blocker, DuP753 (Losartan; 100 nM), and so it is mediated by AT₂ receptors. In a smaller number of neurons AII induced a reversible and concentration (0.01 nM–10 μM)-dependent decrease in I_no that was blocked by Losartan (100 nM) but not by PD123177 (100 nM). Thus the decrease in I_no is mediated by AT₁ receptors. Additionally, some neurons displayed both AT₁- and AT₂ receptor-mediated effects on I_no. Our results demonstrate two distinct actions of AII on membrane ionic currents in cultured neurons, effects that are mediated by different AII receptor subtypes.     

[I]4-Aminobenzyl-5′-N-methylcarboxamidoadenosine ([I]AB-MECA) labels multiple adenosine receptor subtypes in rat brain

Adenosine modulates neuronal activity and neurotransmitter release through interaction with cell surface receptors. Four adenosine receptor subtypes, A₁, A_2A, A_2B, and A₃ receptors, have been cloned and characterized. The agonist ligand, [¹²⁵I]AB-MECA ([¹²⁵I]4-aminobenzyl-5′-N-methylcarboxamidoadenosine) has high affinity for recombinant A₁ and A₃ receptors [Olah et al., Mol. Pharmacol., 45 (1994) 978–982]. Rodent A₃ receptors are relatively insensitive to xanthines; inhibition of A₁ receptors with xanthines allows selective detection of A₃ receptors despite the lack of selectivity of the ligand. We studied whether [¹²⁵I]AB-MECA is useful for localization and characterization of A₃ receptors in rat brain. The autoradiographic distribution of total [¹²⁵I]AB-MECA (400 pM) binding closely resembled the pattern of A₁ receptor binding, with highest levels in cerebellum, hippocampus, and thalamus, and moderate levels in cortex and striatum. Drug competition studies confirmed that almost all [¹²⁵I]AB-MECA binding could be attributed to labeling of A₁ receptors. Xanthine amine congener (1 μM) reduced specific [¹²⁵I]AB-MECA binding by >95%, indicating that xanthine-resistant A₃ receptors represent a quantitatively minor subtype. Despite the use of a radioligand with high affinity and high specific activity, the low density of A₃ receptors in rat brain appears insufficient to allow localization, or even consistent detection, of this receptor subtype. In the presence of DPCPX (50 nM, to block A₁ receptors), residual [¹²⁵I]AB-MECA binding to A_2A receptors was observed in the striatum. Thus, [¹²⁵I]AB-MECA labels primarily A₁ and A_2A adenosine receptors in rat brain.     

Identification of extrastriatal dopamine D2 receptors in post mortem human brain with [I]epidepride

The regional distribution of striatal and extrastriatal dopamine D₂ receptors in human brain was studied in vitro with(S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-[¹²⁵I]iodo-2,3-dimethoxybenzamide, [¹²⁵I]epidepride, using post mortem brain specimens from six subjects. Scatchard analysis of the saturation equilibrium binding in twenty-three regions of post mortem brain revealed highest levels of binding in the caudate (16.5 pmol/g tissue) and putamen (16.6 pmol/g tissue) with lower levels seen in the globus pallidus (7.0 pmol/g tissue), nucleus accumbens (7.2 pmol/g tissue), hypothalamus (1.8 pmol/g tissue), pituitary (1.3 pmol/g tissue), substantia innominata (1.0 pmol/g tissue), and amygdala (0.87 pmol/g tissue). Of note was the presence of dopamine D₂ receptors in the four thalamic nuclei studied, i.e. anterior nucleus (1.0 pmol/g tissue), dorsomedial nucleus (0.96 pmol/g tissue), ventral nuclei (0.72 pmol/g tissue), and pulvinar (0.86 pmol/g tissue), at levels comparable to the amygdala (0.87 pmol/g tissue) and considerably higher than levels seen in anterior cingulate (0.26 pmol/g tissue) or anterior hippocampus (0.36 pmol/g tissue). The frontal cortex had very low levels of dopamine D₂ receptors (0.17–0.20 pmol/g tissue) while the inferior and medial temporal cortex had relatively higher levels (0.31–0.46 pmol/g tissue). Inhibition of [¹²⁵I]epidepride binding by a variety of neurotransmitter ligands to striatal, ventral thalamic and inferior temporal cortical homogenates demonstrated that [¹²⁵I]epidepride binding was potently inhibited only by dopamine D₂ ligands. The present study demonstrates that dopamine D₂ receptors are present in basal ganglia, many limbic regions, cortex and in the thalamus. The density of thalamic D₂ receptors is comparable to many limbic regions and is considerably higher than in cortex. Very few frontal lobe D₂ receptors are present in man.     

Localization of AT(2) receptors in the nucleus of the solitary tract of spontaneously hypertensive and Wistar Kyoto rats using [125I] CGP42112: upregulation of a non-angiotensin II binding site following unilateral nodose ganglionectomy

We have examined the binding distribution of a selective AT(2) receptor ligand [125I] CGP42112 in the brain of adult Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). AT(2) receptor localization was also examined in the rat brainstem following unilateral nodose ganglionectomy. Specific [125I] CGP42112 binding was observed in discrete brain regions from both rat strains, including the nucleus of the solitary tract (NTS), and did not differ between WKY and SHR. [125I] CGP42112 binding in the NTS revealed an AT(2) receptor component that was displaceable by PD 123319 and Ang II (50-58%), as well as a non-angiotensin II receptor component (42-49%). Following unilateral nodose ganglionectomy, [125I] CGP42112 binding density on the denervated side of the NTS was increased approximately two-fold in both WKY and SHR. This increased [125I] CGP42112 binding density in the ipsilateral NTS was comprised of a greater non-angiotensin II component than that observed in the sham groups, since only approximately 30% was displaced by PD123319 and angiotensin II. Furthermore, [125I] CGP42112 also revealed high binding density on the denervated side in the dorsal motor nucleus and the nucleus ambiguus in both WKY and SHR. AT(2) receptor immunoreactivity was also visualised in the NTS of sham operated rats, but was not observed in the dorsal motor nucleus or the nucleus ambiguus, nor was it up-regulated following nodose ganglionectomy. These results demonstrate, for the first time, an AT(2) receptor binding site in the NTS, as well as a non-angiotensin II [125I] CGP42112 binding site. These studies also demonstrate that nodose ganglionectomy represents a useful model in which to study a non-angiotensin II [125I] CGP42112 binding site that is up-regulated following degeneration of afferent vagal nerves.     

Adrenalectomy prevents the stress-induced decrease in in vitro [H]Ro15-1788 binding to GABAA benzodiazepine receptors in the mouse

The effect of a single or repeated swim stress on in vivo benzodiazepine receptor binding to various brain regions in adrenalectomized and sham-operated (control) mice was assessed using the benzodiazepine receptor antagonist, [³H]Ro15-1788. In sham-operated mice the binding of [³H]Ro15-1788 to benzodiazepine receptors was reduced in the hippocampus and hypothalamus (single or repeated stress) and cerebral cortex (repeated swim stress) compared to non-stressed mice. In contrast, no alterations in [³H]Ro15-1788 binding were observed in any brain region in adrenalectomized mice after either single or repeated swim stress. These data suggest that an intact hypothalamic-pituitary-adrenal axis is required for the stress-induced decrease in benzodiazepine receptor occupancy measured using the in vivo binding method.     

Species differences in the distribution of central 5-HT1 binding sites: a comparative autoradiographic study between rat and guinea pig

In this study, we compared the localization of central 5-HT₁ binding sites of rat and guinea pig. The 5-HT_1B sites were absent in the guinea pig brain. Good correlations were found between species in the regional distribution of 5-HT₁ sites labelled with [³H]5-HT(r = 0.73), 5-HT_1A sites labelled with [³H]8-OH-DPAT (r = 0.87), and 5-HT_1B versus 5-HT_1D sites labelled with [³H]5-HT in the presence of ipsapirone and DOI (r = 0.76). Despite the overall similarities, species differences were observed in many brain regions. The CA1/CA2 fields of the hippocampus and the dorsal subiculum displayed significantly more 5-HT_1A receptor binding in guinea pig than in rat. Conversely, the 5-HT_1A binding in dorsolateral septum, cingulate cortex and laminae IV-V of the neocortex, was more pronounced in rat. Areas almost exclusively containing 5-HT_1B or 5-HT_1D sites, such as the ventral pallidum, globus pallidus and substantia nigra, expressed markedly more [³H]5-HT binding in rat as compared to guinea pig, while the opposite occurred in claustrum, dorsal endopiriform nucleus, lateral geniculate nucleus, and superficial grey layer of the superior colliculus. The implications of the species differences are illustrated by the binding of [³H]eltoprazine. The distribution of [³H]eltoprazine binding sites showed a good correlation with that of the 5-HT_1B sites in rat (r = 0.89), and with that of the 5-HT_1A sites in guinea pig (r = 0.97). The data give rise to the possibility that differences in the presence and distribution of 5-HT₁ receptor sites are related to species differences in behavioral, neurochemical and physiological responses to drugs with 5-HT₁ receptor affinity.     

Specific bindings of prostaglandin D2, E2 and F2α in postmortem human brain

Binding activities specific for each of [³H]prostaglandin (PG) D₂, E₂ and F_2α were detected in the P₂ fraction of the human brain homogenates. The bindings were time-dependent, saturable and of high affinity;K_dvalues were 30 nM for all the PG bindings. Regional distribution of these binding activities was determined by measuring specific bindings with 10 nM [³H]PG-D₂, [³H]PG-E₂ and [³H]PG-F_2α in the P₂ fractions from 17 brain regions. The PG-D₂ binding activity was high in the hypothalamus, amygdala and hippocampus followed by cerebellar nuclei, thalamus, nucleus accumbens and cerebral cortex. The PG-E₂ binding sites were similarly concentrated in the hypothalamus and the limbic system, but, unlike the PG-D₂ binding, no significant binding of [³H]PG-3₂ was observed in cerebellar nuclei, cerebellar cortex and putamen. Compared with these two PG bindings, PG-F_2α binding activity was low in many areas, but significant binding was detected in the amygdala, cingulate cortex, cerebellar medulla, hippocampus, nucleus accumbens, midbrain and hypothalamus. These results suggest the presence and specific distribution of three distinct types of PG binding activities, i.e. specific binding of PG-D₂, PG-E₂ and PG-F_2α, in the human brain.     

Autoradiographic distribution of α2 adrenoceptors, NAIBS, and 5-HT1A receptors in human brain using [H]idazoxan and [H]rauwolscine

The regional distribution of [³H]idazoxan and [³H]rauwolscine was studied autoradiographically in human brain. [³H]Idazoxan binds with high affinity to α₂ adrenoceptors as well as to non-adrenergic sites (NAIBS). [³H]Rauwolscine, besides binding to α₂ adrenoceptors, also binds to 5-HT_1A receptors. Both radioligands labelled the same population of α₂ adrenoceptors, defined as the epinephrine-displaceable binding component. The highest densities of α₂ adrenoceptors occur in the leptomeninges, cerebral cortex and claustrum; lower densities were visualised in the basal ganglia, thalamus, pons, substantia nigra, cerebellum and medulla oblongata; no α₂ adrenoceptors were detected in amygdala and nucleus ruber. NAIBS were present in all the examined brain areas, with the highest densities found in the basal ganglia and substantia nigra. The finding that certain brain regions, such as the amygdala, contained NAIBS but no detectable α₂ adrenoceptors, suggests that the binding sites are independent from each other. The regional distribution of 5-HT_1A receptors labelled by [³H]rauwolscine is in agreement with previous studies using [³H]8-OH-DPAT.     

Cerebellar CB1 receptor mediation of Δ-THC-induced motor incoordination and its potentiation by ethanol and modulation by the cerebellar adenosinergic A1 receptor in the mouse

The effect of intracerebellar microinfusion of antisense oligodeoxynucleotide to Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and other naturally occurring cannabinoid receptor (CB₁) mRNA on Δ⁹-THC-induced motor impairment was investigated in mice. Δ⁹-THC (15–30 μg/μl; intracerebellar) resulted in a significant motor impairment in a dose-related manner. The intracerebellar pretreatment with antisense oligodeoxynucleotide (3.0 μg/100 nl/12 h; six administrations/mouse) virtually abolished Δ⁹-THC (15 and 25 μg/1 μl; intracerebellar)-induced motor impairment. However, intracerebellar pretreatment with the mismatched oligodeoxynucleotide in exactly the same manner as the antisense was completely ineffective in altering the Δ⁹-THC-induced motor impairment. These results strongly suggest the involvement of CB₁ receptor in the expression of Δ⁹-THC-induced motor impairment. The intracerebellar microinfusion of adenosine A₁-selective agonist, N⁶-cyclohexyladenosine (CHA) (4 ng/100 nl) significantly enhanced Δ⁹-THC-induced motor impairment, suggesting a cerebellar A₁ adenosinergic modulation of motor impairment. A pretreatment with the antisense and the mismatched oligodeoxynucleotide also markedly attenuated and did not alter, respectively, the cerebellar A₁ adenosinergic modulation (enhancement) of Δ⁹-THC-induced motor impairment. There was no change in the normal motor coordination due to intracerebellar pretreatment with antisense and its mismatch, in the presence as well as absence of intracerebellar CHA indicating the selectivity of interactions with Δ⁹-THC. The Δ⁹-THC-induced motor incoordination was also significantly enhanced dose-dependently by systemic (i.p.) ethanol administration suggesting behavioral synergism between the two psychoactive drugs. Pretreatment (intracerebellar) with pertussis toxin (PTX) markedly attenuated Δ⁹-THC- and Δ⁹-THC+CHA-induced motor incoordination suggesting coupling of CB₁ receptor to PTX-sensitive G-protein (G_i/G_o). These data suggested co-modulation by cerebellar cannabinoid and adenosine system of Δ⁹-THC-induced motor impairment. Conversely, the results in the present study also suggested co-modulation by cerebellar adenosine A₁ and CB₁ receptors of ethanol-induced motor impairment, thereby indicating a possible common signal transduction pathway in the expression of motor impairment produced by Δ⁹-THC as well as ethanol.     

Dopamine denervation leads to an increase in the intramembrane interaction between adenosine A2 and dopamine D2 receptors in the neostriatum

We have previously found, in striatal membrane preparations from young (2 months old) rats, that stimulation of adenosine A₂ receptors (with the selective adenosine A₂ agonist CGS 21680) increases the dissociation constants of high- (K_h) and low-affinity (K_l) dopamine D₂ binding sites (labelled with the selective dopamine D₂ antagonist [³H]raclopride) without changing the proportion of high affinity binding sites (R_h). In the present study in striatal preparations from adult (6 months old) rats, it was found that in addition to the increase in both K_h and K_l values, stimulation of adenosine A₂ receptors is associated with an increase in R_h. These result suggest that, in the adult rat, adenosine A₂ stimulation may inhibit the behavioural effects induced by dopamine D₂ stimulation both by decreasing the affinity and the transduction of dopamine D₂ receptors. We have also studied the intramembrane A₂-D₂ receptor interaction in an experimental model of Parkinson's disease, namely in rats with a unilateral 6-OH-dopamine-induced lesion of the nigro-striatal dopamine pathway. It was found that a unilateral dopamine denervation is associated with a higher density of striatal dopamine D₂ receptors in the order of 20%, without any change in their affinity compared with the unlesioned neostriatum. Furthermore, the density (B_max values) of dopamine D₂ receptors in the contralateral neostriatum was significantly higher (about 20%) than in the striatum from native animals. This finding suggests that an unilateral dopamine denervation also induces compensatory long-lasting changes of dopamine D₂ receptors in the contralateral neostriatum. In addition to the hightened sensitivity to dopamine agonists, it is known that the dopamine denervated striatum is more sensitive to adenosine antagonists like methylxanthines. If the adenosine A₂-dopamine D₂ interaction is the main mechanism of action mediating the central effects of methylxanthines, the dopamine denervation might also potentiate this interaction, i.e., dopamine D₂ receptors could be not only more sensitive to dopamine but also to adenosine A₂ receptor activation. Our results support this hypothesis, since membrane preparations from the denervated neostriatum are more sensitive to the effect of CGS 21680 on dopamine D₂ receptors. Thus a low dose of CGS 21680 (3 nM), which is not effective in membrane preparations from the neostriatum of naive animals, is still effective in membranes from the denervated neostriatum. These results underline the potential antiparkinsonian activity of adenosine A₂ antagonists.     

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

The outflow of [³H]5-hydroxytryptamine ([³H]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 [³H]5-HT outflow evoked at 10 Hz was increased up to +30% by CCK₄ 300–1000 nM, the effect being prevented by the CCK_B receptor antagonist GV 150013, 3 nM. The limited sensitivity to CCK₄ seemed to depend on 5-HT auto-receptor feedback because pre-treatment with 100 nM methiothepin enhanced the [³H]5-HT outflow and lowered the CCK₄ threshold concentration from 300 to 30 nM. In addition, pre-treatment with 1 μM bacitracin to inhibit CCK metabolism increased [³H]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 [³H] 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 μM) and NBQX (10 μM) 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.     

GABA, benzodiazepine and histamine-H1 receptors in the guinea pig cerebellum: effects of kainic acid injections studied by autoradiographic methods

By using kainic acid (KA) to perform chemical lesions in the guinea pig cerebellum, we have caused degeneration of Purkinje cells without affecting cell morphology. Near the injection site we found a large decrease in autoradiographically labeled histamine-H1 and benzodiazepine receptors of the molecular layer while those receptors distant from the injection site were unaffected. GABA receptors in the granule cell layer remained uniformly constant even immediately adjacent to the lesion site. This evidence suggests that histamine-H1 and benzodiazepine receptors are present on neuronal elements (possibly on Purkinje cell dendrites) in the molecular layer of the cerebellum and that GABA receptors area associated with the KA-resistant granule cells.     

Autoradiographic mapping of 5-HT1B and 5-HT1D receptors in the post mortem human brain using [H]GR 125743

The distribution of 5-HT_1B and 5-HT_1D receptors in the human post mortem brain was examined using whole hemisphere autoradiography and the radioligand [³H]GR 125743. [³H]GR 125743 binding was highest in the substantia nigra and the globus pallidus. Lower levels were detected in the striatum, with the highest densities in the ventromedial parts. In the amygdala, the hippocampus, the septal region and the hypothalamus, lower [³H]GR 125743 binding was observed, reflecting low densities of 5-HT_1B/1D receptors. In the cerebral cortex, binding was similar in most regions, although restricted parts of the medial occipital cortex were markedly more densely labeled. Binding densities were very low in the cerebellar cortex and in the thalamus. Two methods were used to distinguish between the two receptor subtypes, the first using ketanserin to block 5-HT_1D receptors and the second using SB 224289 to inhibit 5-HT_1B receptor binding. The autoradiograms indicated that in the human brain, the 5-HT_1B receptor is much more abundant than the 5-HT_1D receptor, which seemed to occur only in low amounts mainly in the ventral pallidum. Although [³H]GR 125743 is a suitable radioligand to examine the distribution of 5-HT_1B receptors in the human brain in vitro, the selectivities of ketanserin and SB 224289 are not sufficiently high to give definite evidence for the occurrence of the 5-HT_1D receptor in the human brain.     

Involvement of dopamine D1 and D2 receptors in the ethanol-associated place preference in rats exposed to conditioned fear stress

The present study was designed to investigate: (1) the involvement of dopamine D₁ and D₂ receptors, and (2) the roles of these receptors and endogenous opioid systems (endorphinergic and enkephalinergic systems) in the ethanol-induced place preference in rats exposed to conditioned fear stress using the conditioned place preference paradigm. The administration of ethanol (300 mg/kg, i.p.) induced a significant place preference. The selective D₁ receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H3-benzazepine)hydrochloride (SCH23390; 0.01 and 0.03 mg/kg, s.c.) and the selective D₂ receptor antagonist S(−)-5-(aminosulfonyl)-N-[(1-ethyl-2-pyrrolidinyl)-methyl]-2-methoxybenzamide (sulpiride; 20 and 40 mg/kg, s.c.) significantly attenuated the ethanol-induced place preference. The administration of ethanol (75 mg/kg, i.p.) tended to produce a place preference, but this effect was not significant. SCH23390 (0.03 mg/kg, s.c.) and sulpiride (40 mg/kg, s.c.) significantly attenuated the enhancement of the ethanol (75 mg/kg, i.p.)-induced place preference produced by the μ-opioid receptor agonist morphine (0.1 mg/kg, s.c.). In addition, SCH23390 (0.03 mg/kg, s.c.) also significantly attenuated the enhancement of the ethanol (75 mg/kg, i.p.)-induced place preference produced by the selective δ-opioid receptor agonist 2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aα-octahydroquinolino[2,3,3,-g]isoquinoline (TAN-67; 20 mg/kg, s.c.). On the other hand, sulpiride (40 mg/kg) had no significant effect on the enhancement of the ethanol (75 mg/kg, i.p.)-induced place preference produced by TAN-67. These results suggest that D₁ and D₂ receptors may be involved in the rewarding mechanism of ethanol under psychological stress. In addition, D₁ receptors may participate in the rewarding effect of ethanol modulated by the activation of μ- and δ-opioid receptors, whereas D₂ receptors may participate in the rewarding effect of ethanol modulated by the activation of μ-opioid receptors, but not in that modulated by the activation of δ-opioid receptors.     

[H]Spiperone binds selectively to rat striatal D2 dopamine receptors in vivo: a kinetic and pharmacological analysis

We have determined the kinetic, equilibrium saturation, and pharmacological characteristics of [³H]spiperone ([³H]SPIP) binding to rat brain regional particulate fractions following i.v. injections of [³H]SPIP and compared these parameters to those determined in vitro with traditional ligand-homogenate binding assays. [³H]SPIP binding to rat striatum in vivo and in vitro occurs to a single class of non-interacting binding sites which possess the pharmacological properties of a D₂ dopamine (DA) receptor. The potencies of neuroleptic drugs in inhibiting DA receptor-mediated behaviors correlate with their potencies at displacing striatal [³H]SPIP binding in vivo. While striatum possesses a similar density of [³H]SPIP binding sites in vivo (34 pmol/g) and in vitro (31 pmol/g), binding affinity in vivo is about 200 times lower than in vitro. This difference in binding affinities appears to arise from alterations of [³H]SPIP association and dissociation rate constants brought about by tissue homogenization. The implications of our findings for external imaging of DA receptors and studies of DA receptor function in human brain homogenates are discussed.     

Effects of adrenalectomy and type I or type II glucocorticoid receptor activation on 5-HT1A and 5-HT2 receptor binding and 5-HT transporter mRNA expression in rat brain

We evaluated the effects of adrenalectomy (ADX) and replacement with glucocorticoid receptor agonists on serotonin (5-HT) 5-HT_1A and 5-HT₂ receptor binding in rat brain. 5-HT_1A receptor binding was increased in the CA2–CA4 and the dentate gyrus of the hippocampus 1 week after ADX. This effect was prevented by the systemic administration of aldosterone (10 μg/μl/h) but not by RU28362 (10 μg/μl/h). No significant effect was observed on 5-HT₂ receptor binding in rat cortex. The expression of 5-HT transporter mRNA was unchanged in the raphe nucleus as measured by in situ hybridization.     

Electrophysiological evidence for excitatory 5-HT2 and depressant 5-HT1A receptors on neurones of the rat midbrain tectum

It has been claimed that the aversive behaviour induced by electrical stimulation of the midbrain tectum (MT) has validity as an animal model of panic attack. A great deal of evidence obtained from behavioural studies suggests that 5-HT₂ mechanisms phasically inhibit the substrates of aversion in the MT. In order to test this hypothesis we employed the technique of microiontophoresis of drugs onto neurones of the MT to assess the identity of the receptors mediating the effects of 5-hydroxytryptamine (5-HT). The results obtained show that the majority of 5-HT responsive cells in MT are cells excited by 5-HT (72%). These cells were silent or showed very low spontaneous firing activity, whereas cells depressed by 5-HT showed high spontaneous firing activity at baseline. The 5-HT_1A receptor agonists, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), buspirone and gepirone caused consistent reduction in the firing rate of cells depressed by 5-HT while they did not change the firing activity of cells excited by 5-HT. The excitatory effects induced by 5-HT on MT neurones were clearly attenuated by concomitant application of ketanserin, a highly specific 5-HT₂ antagonist. Excitatory responses to dl-homocysteic acid were not affected by ketanserin. Previous administration of zimelidine, a selective 5-HT uptake inhibitor, caused a significant enhancement of the excitatory effects of 5-HT while similar application of gepirone did not affect the size of the excitatory responses to 5-HT. These results give electrophysiological support to the idea that 5-HT neurotransmission operating through 5-HT₂ receptors may exert a phasic control on functional processes in the MT. It is possible that 5-HT₂ mechanisms in this region may mediate at least part of the therapeutic effects of 5-HT uptake inhibitors in panic disorders.     

The calcium-dependent [H]acetylcholine release from synaptosomes of brown trout (Salmo trutta) optic tectum is inhibited by adenosine A1 receptors: Effects of enucleation on A1 receptor density and cholinergic markers

Presynaptic inhibition is one of the major control mechanisms in the CNS. Previously we reported that A₁ adenosine receptors are highly concentrated in the brain, including optic tectum, of trout and that they inhibited the release of glutamate. The optic tectum is heavily innervated by cholinergic nerve terminals. We have investigated whether A₁ receptors inhibit the presynaptic release of acetylcholine and whether the inhibition is triggered by calcium. The release of [³H]ACh evoked by 30 mM KCl was Ca²⁺ dependent and it was dose-dependently inhibited by the A₁ adenosine receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA) ranging between 10 nM to 100 μM. The maximum of inhibition was reached at 10 μM. The A₁ receptor antagonist 8-cyclopentyltheopylline (CPT, 10 μM), reversed almost completely the inhibition induced by CCPA 10 μM. In Fura-2/AM loaded synaptosomes, K⁺ depolarization raised [Ca²⁺]_i by about 64%. CCPA (10 μM) reduced the K⁺-evoked Ca²⁺ influx increase by about 48% and this effect was completely antagonised by CPT 10 μM. Synaptosome pretreatment with different Ca²⁺ channel blockers differently affected K⁺-evoked Ca²⁺ influx. This was not significantly modified by nifedipine (1 μM, L-type blocker) nor by ω-agatoxin IVA (0.3 μM, P/Q-type blocker), whereas about 50% reduction was shown by 0.5 μM ω-conotoxin GVIA (N-type blocker). Neurochemical parameters associated with cholinergic transmission and the density of A₁ adenosine receptors were measured in the trout optic tectum 12 days after unilateral eye ablation. A significant drop of both acetylcholinesterase (AChE) activity (24%) and choline acetyltransferase (CAT) activity (32%) was observed in deafferentated optic tectum, whereas the high affinity choline uptake did not parallel the decrease in enzyme activity. Eye ablation caused a marked decrease (43%) of A₁ receptor density without changing the affinity. The K⁺-evoked release of [³H]ACh from synaptosomes of deafferentated was not modify as well as the efficacy of 10 μM CCPA in decreasing [³H]ACh release was not apparently modified.